Synthesis, Molecular Characterization, and Antimicrobial Evaluation of Hydrazones Derived from 2-Hydrazinobenzimidazole

Abstract

Introduction

Hydrazones, due to the structural diversity of their nitrogen atoms, possess both electrophilic and nucleophilic properties, enabling strong hydrogen bonding interactions with enzymes and receptors. This study aimed to synthesize novel hydrazone derivatives and evaluate their antimicrobial potential.

Methods

Hydrazones were synthesized via condensation of 2-hydrazinobenzimidazole with various aldehydes or ketones using citric acid as an eco-friendly catalyst. The (E)-configuration of the products was confirmed through frontier molecular orbital (FMO) calculations. Antimicrobial activities were assessed against selected Gram-positive and Gram-negative bacteria, and fungi. Molecular docking studies were conducted on the most active compounds (3c and 3o) using bacterial and fungal protein targets (2IWC, 2NXW, 1EA1).

Results

Compounds 3c and 3o showed strong antimicrobial activity. Docking studies revealed that both compounds interacted with 2IWC via one H-bond donor to THR531 (3.12 Å), mirroring ampicillin. Against 2NXW, they showed dual H-donor bonding to MET404 with binding energies of –5.96 and –5.72 kcal/mol, comparable to gentamicin. Both also bound ARG326 in 1EA1 with binding energies of –5.97 and –6.0 kcal/mol, similar to nystatin.

Discussion

The comparable binding patterns and energies of compounds 3c and 3o to standard antimicrobial agents suggest that they are promising candidates for further development as broad-spectrum antimicrobial agents.

Introduction

Green chemistry provides essential principles aimed at minimizing environmental and health hazards by designing safer chemical processes and products. A core goal of this approach is the reduction or elimination of toxic substances through the use of benign solvents, renewable feedstocks, and eco-friendly catalysts.1 Among these, citric acid has gained considerable attention as a natural, non-toxic, biodegradable, and cost-effective catalyst extracted from citrus fruits (5–7%).2 It has been successfully applied in the green synthesis of various heterocyclic compounds, including azoles and azines,2 and has facilitated numerous reactions such as Knoevenagel condensation,3,4 Schiff bases,5–7 imines,8 phenolic derivatives,9 and amidoalkyl naphthols.10

In parallel, the pharmaceutical relevance of benzimidazole-based hydrazones has attracted significant research interest due to their broad spectrum of biological activities. These compounds have been reported to possess anti-diabetic,11 anthelmintic,12 antimicrobial, antioxidant,13 anti-inflammatory,14 anticancer,15,16 anticonvulsant,17 antimalarial,18 and antileishmanial19 properties. Furthermore, hydrazones have shown potent inhibitory effects against carbonic anhydrase isoenzymes (I and II),20 making them valuable in rational drug design. Recent advances underscore the therapeutic potential of benzimidazole derivatives, especially in anticancer and antimicrobial research, with new synthetic strategies enhancing their pharmacological relevance.21,22 The hydrazone moiety serves as a versatile pharmacophore capable of binding to various biological receptors,23 while substitution on the phenyl ring enhances biological activity, including anticancer effects24[Figure 1].

Figure 1.

The biological activities of hydrazonobenzimidazole.

Given the increasing demand for sustainable and biologically active compounds, integrating green chemistry protocols with bioactive scaffold design represents a promising research direction. As part of our ongoing work on green synthesis of heterocycles,25–35 we report the citric acid-catalyzed synthesis of benzimidazole-based hydrazones and evaluate their antimicrobial potential. Density Functional Theory (DFT) computations were employed to determine the molecular geometries and electronic properties of the synthesized compounds. Furthermore, molecular docking studies were conducted to gain insights into their interaction with microbial protein targets, supporting their observed biological activities.

Results and Discussion

Chemistry

The synthetic conditions for preparing hydrazonobenzimidazole were accomplished by acid catalyst, especially acetic acid in methanol.26,35 Our study was commenced on a representative reaction of 2-hydrazinobenzimidazole26,35 and benzaldehyde in the presence of (0.1 g) citric acid as an eco-friendly catalyst. The reaction was subjected in various solvents under reflux as shown in Scheme 1 and Table 1.

Scheme 1.

Reaction of 2-hydrazinobenzimidazole and benzaldehyde.

Table 1.

Effect of Solvents on the Product Yield of Compound 3a

Entry	Solvent	Time (h)	Isolated Yield (%)
1	Methanol	3	85
2	Ethanol	3	94
3	DMF	3	84
4	Dioxane	3	82
5	DMSO	3	80

As illustrated in Table 1, ethanol was a superior solvent in terms of the isolated yield of product 3a.

Ethanol was selected due to its moderate polarity and protic nature, which supports effective solvation of both the hydrazinobenzimidazole and carbonyl substrates. Its hydrogen-bonding ability enhances interactions with the citric acid catalyst, improving proton transfer and reaction kinetics. Additionally, ethanol ensures better miscibility of the reactants and facilitates uniform heat distribution during reflux, contributing to higher product yields.

To generalize the scope of our green synthesis protocol, a series of aromatic aldehydes (2a-g) and ketones (2h-j), acetyl heterocycles (2k-n), and isatin (2o) were condensed with 2-hydrazinobenzimidazole (1) in ethanol in the presence of acetic acid or citric acid. The mechanistic pathway was illustrated in Scheme 2 and a comparative yield of the condensation products (3a-o) was depicted in Table 2.

Scheme 2.

Synthesis of hydrazonobenzimidazole (3a-o).

Table 2.

A Comparative Yield of Products (3a-o) Under Catalytic Effect (Acetic Acid and Citric Acid)

Compd. No.	R	Ar	Yield %		Ref.
			Citric Acid	Acetic Acid
3a	H	C6H5	94	85	30
3b	H	4-CH3C6H4	92	83	30
3c	H	4-CH3OC6H4	92	82	30
3d	H	4-ClC6H4	90	83	30
3e	H	4-NO2C6H4	90	81	30
3f	H		90	82	-
3g	H		90	80	-
3h	CH3	C6H5	92	81	31
3i	CH3	4-BrC6H4	90	80	31
3j	CH3		88	78	-
3k	CH3		88	79	-
3L	CH3		88	80	-
3m	CH3		88	81	-
3n	CH3		88	82	-
3o			88	80	32

With the optimal conditions, the condensation reactions have been achieved with citric acid catalyst in good to excellent yield (88–94%) relative to acetic acid (78–85%). 4-substituted benzaldehyde with electron-donating or withdrawing groups gave the respective hydrazones (3a-3e) in an excellent yield. Also, dichlorobenzaldehyde with steric hinderance was not affected on the yield percent of the products (3f, 3g). Additionally, the high performance of citric acid catalyst was perceived with aryl (3h-3j), acetyl heterocycles (3k-3n), and isatin (3o) as shown in Table 2.

Although hydrazone formation can proceed without a catalyst, the use of citric acid notably enhances reaction efficiency by increasing yields and reducing reaction times. Its role as a natural, biodegradable, and non-toxic acid aligns with green chemistry principles, offering a sustainable alternative to conventional catalysts. This justifies its necessity in promoting an eco-friendly and efficient synthetic protocol.

The theoretical study using Density Functional Theory (DFT) further supports the experimental results. The FMO analysis showed that the (E)-isomers were more stable than the (Z)-isomers in all phases, as indicated by their lower total electronic energies. Moreover, the HOMO-LUMO energy gaps, ionization potentials, and electrophilicity indices provided insights into the reactivity and stability of the compounds. For instance, smaller HOMO-LUMO gaps indicate higher chemical reactivity, which aligns with the biological activity observed in certain hydrazones. Global descriptors such as softness, hardness, and chemical potential also supported the prediction of the compounds’ behavior during biological interactions. These findings confirm the reliability of the computational models and their relevance in predicting molecular behavior consistent with antimicrobial activity.

Computational Investigations

The optimized structures of compounds 3a-o have been generated and displayed in Gauss View 6.0.16 using DFT with the B3LYP function and 6–31G basis set computations using the Gaussian 09 program. The compounds 3a-o can be formulated in two possible geometrical isomers (Z) and (E) forms (see Figure 2). HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) of the investigated compounds in the gas, aqueous, and ethanol phases were computed and summarized in Tables 3, S1, and S2.

Figure 2.

Geometrical isomers of compounds 3a-o.

Table 3.

Frontier Molecular Orbitals (FMO) Computations of Compounds 3a and 3h

Compd. No.	Optimized Structure	E (Kcal/mol)
		Gas	H2O	EtOH
3a (E)		− 476,738.21	− 476,746.29	− 476,745.89
LUMO		E LUMO (eV)
		Gas	H2O	EtOH
		− 1.46	− 1.42	− 1.42
HOMO		E HOMO (eV)
		Gas	H2O	EtOH
		− 5.19	− 5.32	− 5.32
3a (Z)		E (Kcal/mol)
		Gas	H2O	EtOH
		− 476,734.79	− 476,742.01	− 476,741.66
LUMO		E LUMO (eV)
		Gas	H2O	EtOH
		− 1.42	− 1.37	− 1.37
HOMO		E HOMO (eV)
		Gas	H2O	EtOH
		− 5.24	− 5.45	− 5.44
3h (E)		E (Kcal/mol)
		Gas	H2O	EtOH
		− 501,410.62	− 501,419.09	− 501,418.70
LUMO		E LUMO (eV)
		Gas	H2O	EtOH
		− 1.32	− 1.23	− 1.22
HOMO		E HOMO (eV)
		Gas	H2O	EtOH
		− 5.14	− 5.31	− 5.30
3h (Z)		E (Kcal/mol)
		Gas	H2O	EtOH
		− 501,409.57	− 501,417.02	− 501,416.67
LUMO		E LUMO (eV)
		Gas	H2O	EtOH
		− 1.08	− 0.98	− 0.98
HOMO		E HOMO (eV)
		Gas	H2O	EtOH
		− 5.09	− 5.35	− 5.34

The data in Table 3 supported the superiority of the E-form over Z-form. For example, the electronic energy of 3a (E) in the gas phase = (–476,738.21 Kcal/mol), which is lower than that of 3a (Z) (–476,734.79 Kcal/mol). Similar calculations have been observed in both ethanolic (–476,745.89/–476,741.66 Kcal/mol) and aqueous phases (–476,746.29/–476,742.01 Kcal/mol), respectively.

Additionally, global descriptors such as ionization energy (I), electron affinity (A), global hardness (η), global softness (S), chemical potential (µ), electrophilicity (ω), and nucleophilicity (N) of the synthesized compounds were calculated in three different phases (gas/aqueous/ethanol) as shown in Table 4.

Table 4.

Global Descriptors of Compounds 3a and 3h

No.	I = -EHOMO	A = - ELUMO	η = (EL-EH)/2	S = 1/η	µ = - (I+A)/2	ω= µ2/2η	N = EHOMO-EHOMO TCE
3a (E)	5.19	1.46	1.87	0.54	3.33	2.96	4.22
3a (E) /H2O	5.32	1.42	1.95	0.51	3.37	2.91	4.09
3a (E) /EtOH	5.32	1.42	1.95	0.51	3.37	2.91	4.09
3a (Z)	5.24	1.42	1.91	0.52	3.33	2.90	4.17
3a (Z) /H2O	5.45	1.37	2.04	0.49	3.41	2.85	3.96
3a (Z) /EtOH	5.44	1.37	2.04	0.49	3.41	2.85	3.97
3h (E)	5.14	1.32	1.91	0.52	3.23	2.73	4.27
3h (E) /H2O	5.31	1.23	2.04	0.49	3.27	2.62	4.10
3h (E) /EtOH	5.30	1.22	2.04	0.49	3.26	2.60	4.11
3h (Z)	5.09	1.08	2.01	0.50	3.09	2.37	4.32
3h (Z) /H2O	5.35	0.98	2.19	0.46	3.17	2.29	4.06
3h (Z) /EtOH	5.34	0.98	2.18	0.46	3.16	2.29	4.07

It is worthily mentioned that compounds 3a-o can be represented in three different tautomeric structures as hydrazones (I), conjugated diazines (II) and benzylazo forms (III) as shown in Figure 3. Frontier Molecular Orbitals (FMO) computations of these tautomeric forms were illustrated in Table 5.

Figure 3.

Tautomeric forms of compounds 3a-o.

Table 5.

(FMO) Computations of the Tautomeric Forms (I, II, and III) of 3a and 3h

Compd. (3a)		E (I)	E (II)	E (III)
Optimized structure
E (Kcal/mol)	Gas	− 476,738.21	− 476,737.53	− 476,723.28
	H2O	− 476,746.29	− 476,745.10	− 476,730.62
	EtOH	− 476,745.89	− 476,744.72	− 476,730.24
LUMO
E LUMO (eV)	Gas	− 1.46	− 1.01	− 2.35
	H2O	− 1.42	− 1.23	− 2.45
	EtOH	− 1.42	− 1.22	− 2.45
HOMO
EHOMO (eV)	Gas	− 5.19	− 4.88	− 6.24
	H2O	− 5.32	− 5.03	− 6.34
	EtOH	− 5.32	− 5.03	− 6.34
Compd. (3h)		E (I)	E (II)	E (III)
Optimized structure
E (Kcal/mol)	Gas	− 501,410.62	− 501,410.88	− 501,398.75
	H2O	− 501,419.09	− 501,418.06	− 501,406.05
	EtOH	− 501,418.70	− 501,417.69	− 501,404.54
LUMO
E LUMO (eV)	Gas	− 1.32	− 0.89	− 2.15
	H2O	− 1.23	− 1.09	− 2.32
	EtOH	− 1.22	− 1.08	− 2.26
HOMO
E HOMO (eV)	Gas	− 5.14	− 4.84	− 6.18
	H2O	− 5.31	− 4.98	− 6.35
	EtOH	− 5.30	− 4.97	− 6.31

The data in Table 5 pointed out that the electronic energies of tautomeric forms (I, II, and III) of compound 3a in the gas phase were (–476,738.21, –476,737.53, and –476,723.28 Kcal/mol), respectively, that revealing the superior stability of hydrazone tautomer (I) over conjugated azine tautomer (II) (∆E = 0.68 Kcal/mol) and benzylazo tautomer (III) (∆E = 14.93 Kcal/mol). Similarly, the electronic energies of compound 3h (I, II, and III) in the gas phase were (–501,410.62, –501,410.88, –501,398.75 Kcal/mol), respectively. In the same manner, the electronic energy of (I) is lower than those of (II) and (III) forms in both aqueous and ethanolic phases for compounds 3a and 3h (Table 5).

To confirm the stability of hydrazone form (I), the global descriptors of three tautomeric forms (I, II, and III) of compounds 3a and 3h were computed in three different phases (gas, aqueous and ethanolic) as summarized in Table S3.

The in vitro Antimicrobial Screening

The synthesized compounds 3a-o were evaluated in vitro for their antimicrobial activity against two Gram-positive bacteria: Bacillus subtilis (BS, ATCC 6633) and Staphylococcus aureus (SA, ATCC 6538), and two Gram-negative bacteria: Pseudomonas aeruginosa (PA, ATCC 90274) and Klebsiella pneumoniae (KP, ATCC 13883). Additionally, one yeast, Candida albicans (CA, ATCC 10221), and one fungus, Aspergillus fumigatus (AF, ATCC 14109), were also tested. The standard drugs used for comparison were Gentamicin (Gram-positive bacteria), Ampicillin (Gram-negative bacteria) and Nystatin (fungi). The antimicrobial activity was assessed by measuring the inhibition zone diameter (IZD) in mm/mg of sample at a concentration of 30 µg/mL for each compound. The results were summarized in Table 6.

Table 6.

Preliminary Antimicrobial Activity for the Synthesized Compounds 3a-o

Compd.	Pathogenic Microorganisms mm
	Gram Positive Bacteria		Gram Negative Bacteria		Yeast	Fungi
	Bacillus subtilis	Staphylococcus aureus	Pseudomonas aeruginosa	Klebsiella pneumoniae	Candida albicans	Aspergillus fumigatus
3a	26.5 ± 0.9	25.3 ± 0.8	20.1 ± 0.8	NA	22.4 ± 0.8	NA
3b	27.0 ± 0.8	28.2 ± 0.6	22.8 ± 0.9	29.8 ± 0.9	27.7 ± 0.7	NA
3c	29.2 ± 0.7	31.7 ± 0.9	26.3 ± 0.7	32.2 ± 1.0	29.8 ± 0.7	21.3 ± 0.5
3d	25.2 ± 0.8	27.1 ± 0.8	18.4 ± 0.7	23.3 ± 0.7	22.3 ± 0.7	NA
3e	NA	22.3 ± 0.9	NA	21.9 ± 0.8	23.2 ± 0.8	NA
3f	NA	20.5 ± 0.8	NA	NA	24.8 ± 0.9	NA
3g	23.4 ± 0.9	19.7 ± 0.5	21.0 ± 0.7	NA	27.6 ± 0.7	NA
3h	29.0 ± 1.0	29.4 ± 0.9	25.6 ± 0.5	29.2 ± 0.8	27.5 ± 0.9	15.6 ± 0.8
3i	23.2 ± 0.9	26.1 ± 0.9	21.0 ± 0.8	NA	24.6 ± 0.8	NA
3j	27.6 ± 0.9	29.4 ± 0.7	21.2 ± 0.9	29.8 ± 0.7	20.9 ± 0.8	19.4 ± 0.7
3k	29.6 ± 0.7	30.9 ± 0.9	21.8 ± 0.7	28.5 ± 0.8	28.4 ± 0.9	17.7 ± 0.8
3L	28.3 ± 1.3	29.7 ± 0.9	21.9 ± 0.6	27.8 ± 1.1	26.6 ± 0.8	18.6 ± 0.9
3m	29.0 ± 0.8	30.0 ± 0.9	21.9 ± 0.6	27.6 ± 0.8	25.0 ± 0.7	20.5 ± 0.7
3n	28.3 ± 0.8	31.0 ± 0.8	22.7 ± 0.8	27.8 ± 0.6	25.3 ± 0.7	21.7 ± 0.8
3o	29.6 ± 0.7	31.9 ± 0.7	23.7 ± 0.6	28.5 ± 0.9	25.9 ± 0.6	25.0 ± 0.9
Ampicillin	29.7 ± 0.9	30.2 ± 0.8	–	–	–	–
Gentamicin	–	–	29.8 ± 0.8	28.3 ± 0.5	–	–
Nystatin	–	–	–	–	29.4 ± 0.8	33.4 ± 0.9

Notes: Values are mean inhibition zone diameter (mm) ± standard deviation of three replicates. Mean zone of inhibition measured in millimeters, generated by several pathogenic bacteria. Control for Bacteria was Gentamycin and for fungi was Fluconazole.

Abbreviation: NA, No activity.

The results presented in Table 6 revealed varying levels of antimicrobial efficacy across the tested microorganisms. The synthesized compounds exhibited diverse degrees of activity against Gram-positive and Gram-negative bacteria. Among the tested compounds, 3c displayed exceptional activity against Gram-positive bacteria, with inhibition zone diameters (IZDs) of 31.7 ± 0.9 mm against S. aureus and 29.2 ± 0.7 mm against B. subtilis, closely approximating the activity of the standard ampicillin (30.2 ± 0.8 mm and 29.7 ± 0.9 mm, respectively). Similarly, compounds 3h, 3j, 3k, 3L, 3m, 3n, and 3o demonstrated strong activity against both Gram-positive strains, with IZDs ranging from 27.6 to 31.9 mm.

Against Gram-negative bacteria, 3c exhibited superior activity, particularly against K. pneumoniae, with an IZD of 32.2 ± 1.0 mm, surpassing the efficacy of Gentamicin (28.3 ± 0.5 mm). Other compounds, including 3b, 3h, and 3j–3o, also displayed considerable activity against K. pneumoniae, with IZDs between 27.6 and 29.8 mm. Notably, 3c and 3h demonstrated significant activity against P. aeruginosa, with IZDs of 26.3 ± 0.7 mm and 25.6 ± 0.5 mm, respectively. These results underscore the potential of these compounds as effective agents against Gram-negative bacterial infections.

The antifungal activity of the compounds was evaluated against C. albicans and A. fumigatus. Most compounds showed promising activity against C. albicans, with 3c exhibiting the highest activity (IZD: 29.8 ± 0.7 mm), surpassing the standard nystatin (29.4 ± 0.8 mm). Compounds 3b, 3h, 3k, and 3L also demonstrated strong activity against C. albicans, with IZDs ranging from 26.6 to 28.4 mm. For A. fumigatus, moderate activity was observed. Compound 3o exhibited the highest activity, with an IZD of 25.0 ± 0.9 mm, although it was less effective than nystatin (33.4 ± 0.9 mm). Compounds 3c, 3h, 3j, 3k, 3L, 3m, and 3n demonstrated moderate activity against A. fumigatus, with IZDs ranging from 15.6 to 21.7 mm.

Structure-Activity Relationship (SAR) of Synthesized Hydrazones (3a–o)

The antimicrobial activity of the synthesized hydrazones (3a–o) is strongly influenced by specific structural modifications. Electron-donating groups, such as –OCH₃ (in 3c) and –CH₃ (in 3b), significantly enhanced antimicrobial potency compared to electron-withdrawing substituents. This improvement likely stems from increased electron density on the aromatic ring, which facilitates stronger hydrogen bonding and π–π interactions with microbial targets. Conversely, compounds with strong electron-withdrawing groups exhibited reduced activity, underscoring the advantage of electron-rich systems in promoting target engagement.

Heteroaromatic substitutions further boosted efficacy, with derivatives containing thiophene, pyridine, indole, or coumarin (3k–3n) showing markedly improved activity. The planarity and heteroatom interactions of these moieties are proposed to enhance both target affinity and membrane permeability. Lipophilicity also played a critical role: bulkier, more lipophilic compounds (eg, 3j with naphthyl and 3o with isatin) demonstrated superior activity, likely due to improved penetration of microbial lipid bilayers, as supported by their stronger docking scores. Notably, steric hindrance was well-tolerated, as evidenced by the retained activity of dichloro-substituted derivatives (3f, 3g), suggesting flexibility for further structural optimization.

The most potent compounds (3c and 3o) exhibited a consistent correlation between their electronic properties, binding interactions, and biological activity. Their small HOMO-LUMO gaps (indicating favorable charge transfer) and strong hydrogen-bonding interactions with key microbial residues align with their exceptional in vitro performance. Collectively, these findings highlight that antimicrobial potency in hydrazones can be maximized by incorporating electron-donating groups, heterocyclic rings, and lipophilic features—strategies that synergistically improve target binding, membrane penetration, and overall bioavailability.

Molecular Docking

The molecular docking evaluation was performed using the Molecular Operating Environment (MOE) software. The interaction between the selected proteins (PDB: 2IWC, 2NXW, and 1EA1) is utilized to assess the antimicrobial activity of the most promising hydrazone derivatives (3c and 3o) using Ampicillin, Gentamicin, and Nystatin as benchmark antibiotics (essential antimicrobial references). A comparison study between the ligands and antimicrobial references depended on the number of hydrogen bond interactions, the binding energy score, and the distance apart between the ligand/reference antibiotic and crucial amino acid residues in the protein. The investigation results for the interaction between antimicrobial references (Ampicillin/Gentamicin/Nystatin) or ligands (3c and 3o) with different proteins (2IWC as a gram-positive bacterial strain, 2NXW as a gram-negative bacterial strain and 1EA1 as antifungal strain) were shown in Table 7.

Table 7.

Ligand-Protein Interaction of the Antibiotics and Compounds (3c, 3o) with Proteins

Compd.	Protein	Ligand–Protein Interactions/Type of Interactions	Binding Energy (Kcal/mol)	Root Mean Square Deviation RMSD	Bond Distance (Å)
Ampicillin	2IWC	THR 531 (A) / H-donor THR 531 (A) / H-acceptor ASN 441 (A) / H-acceptor ILE 533 (A) / H-acceptor SER 439 (A) / H-acceptor ASN 478 (A) / pi-H	− 6.07	1.39	3.30 2.98 3.04 3.16 3.40 3.6
3c	2IWC	THR 531 (A) H-donor ASN 441 (A) H-donor ASN 441 (A) H-acceptor ILE 533 (A) pi-H	− 5.63	1.09	2.99 3.35 3.13 4.03
3o	2IWC	THR 531 (A) H-donor GLY 530 (A) H-acceptor TYR 438 (A) pi-H ASN 441 (A) pi-H	− 5.24	1.93	2.94 3.34 3.77 3.85
Gentamicin	2NXW	MET 434 (A) / H-donor MET 404 (A) / H-donor MET 404 (A) / H-donor ALA 402 (A) / H-donor	− 5.18	1.55	4.41 3.96 4.17 2.94
3c	2NXW	MET 404 (A) / H-donor MET 404 (A) / H-donor TRP 459 (A) / pi-H	− 5.96	1.21	3.24 3.53 4.81
3o	2NXW	MET 404 (A) H-donor LEU 462 (A) H-acceptor TRP 459 (A) pi-pi	− 5.26	0.98	3.46 3.32 3.92
Nystatin	1EA1	HIS 392 (A) / H-donor SER 261 (A) / H-donor PRO 386 (A) / H-donor TYR 76 (A) / H-acceptor ARG 326 (A) / H-acceptor	− 4.32	2.4	2.51 2.55 2.72 2.72 2.79
3c	1EA1	ARG 326 (A) H-acceptor ALA 256 (A) pi-H	− 6.16	1.21	2.87 4.38
3o	1EA1	ARG 326 (A) H-acceptor ARG 95 (A) pi-cation ARG 95 (A) pi-cation ARG 96 (A) pi-cation ARG 96 (A) pi-cation	− 5.75	0.95	3.44 4.65 4.14 3.99 3.17

Ampicillin had a binding energy score = –6.07 Kcal/mol (RMSD = 1.39) with 2IWC protein (Table 7). It exhibited five H-bond interactions (one H-donor and four H-acceptors). The H-acceptor interactions with THR531, ASN441, ILE533, and SER439 with 2.98, 3.04, 3.16, and 3.40 Å, respectively, in addition to an H-donor bond with THR531 with 3.32 Å. Compounds 3c and 3o interacted with 2IWC through binding energy scores of −5.63 and −5.24 Kcal/mol, respectively. 3c as a model example displayed three H-bonds (two H-bond donors with side chain amino acid THR531 and main chain amino acid ASN441) and one H-bond acceptor main chain amino acid ASN441. The investigated candidates bounded to the same amino acid (S) as the reference antibiotic ampicillin. Thus, the inhibition efficacy of the tested compounds against gram-positive bacteria is confirmed by these interactions (Tables 7 and 8).

Table 8.

2D and 3D of Ligand’s Interaction (3c and 3o) with 2IWC, 2NXW, and 1EA1 Proteins

No.	Protein	2D	3D
3c	2IWC
3o	2IWC
3c	2NXW
3o	2NXW
3c	1EA1
3o	1EA1

Similarly, the interaction of gentamicin with 2NXW protein (Table 7) was substantiated via four H-donor bonds with amino acids MET404, MET434, and ALA402, with binding energy (−5.18 Kcal/mol) and RMSD = 1.55 Å. Compounds 3c as a model example bonded with 2NXW protein via two H-donor bonds with the main chain amino acid MET404 and one pi-H bond with the main chain amino acid TRP459, that reflected the inhibition potency of these compounds towards gram-negative bacterial strain (Tables 8). Additionally, binding of nystatin with 1EA1, as a fungal protein, was achieved (Table 7) via three H-donor bonds with the amino acids HIS392, SER261, and PRO386, and two H-acceptor bonds with TYR76 and ARG326 amino acids with binding energies = −4.32 Kcal/mol. Both compounds 3c and 3o were consistently connected to the same amino acid residue (ARG326) in 1EA1 protein with binding energies of −6.16 and −5.75 Kcal/mol, respectively, that supported the biological activity of the tested compounds (3c and 3o) against C. albicans and A. fumigatus (Tables 7 and 8).

The 2D and 3D images of the ligand’s interaction (3c and 3o) with 2IWC, 2NXW, and 1EA1 proteins were compiled in Table 8.

Experimental

Materials and Instruments

A digital melting point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK) was used to measure the melting points (uncorrected) of products 3a-o. Shimadzu FTIR 8101 PC infrared spectrophotometer (Shimadzu, Tokyo, Japan) has recorded IR spectra in potassium bromide discs. Recording of ¹H-NMR (at 300 MHz) and ¹³C-NMR (at 75 MHz) spectra was manipulated on A Varian Mercury VXR-300 spectrometer using tetramethyl silane as an internal standard. The samples were dissolved in deuterated dimethylsulfoxide (DMSO-d₆). GCMS-Q1000-EX Shimadzu spectrometer measured Mass spectra at an ionizing voltage of 70 eV. A German-made Elementarvario LIII CHNS analyzer was used to measure the elemental analyses of the synthesized products.

General Procedure for the Reactions of 2-Hydrazonobenzimidazole (1) and Carbonyl Compounds

A mixture of 2-hydrazonobenzimidazole (1) [0.148 g, 1 mmol] and carbonyl compounds 2a-o (1 mmol of each) in ethanol (20 mL) and few drops of acetic acid (or 0.1 g citric acid) was refluxed for 3 hours (checked by TLC until complete disappearance of starting materials). After evaporation of excess ethanol under reduced pressure, the collected precipitate was filtered and recrystallized from the appropriate solvent to give pure products 3a-o.

2-(2-Benzylidenehydrazinyl)-1H-Benzo[d]imidazole (3a)

Yellow powder, mp = 293–295°C, [lit. mp = 291°C]36 (EtOH/DMF); IR (KBr) υ = 3432 (NH), 2923 (CH-), 1600 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 7.09–7.51 (m, 9H, Ar-H), 8.33 (s, 1H, CH=N), 10.72 (s, 1H, NH), 12.53 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 113.1, 114.4, 122.5, 123.9, 126.2, 127.8, 137.1, 141.5, 143.0, 149.7, 150.1, 158.1 (Ar-C) ppm; MS, m/z (%) 236 (M⁺, 31), 197 (61), 155 (54), 116 (100), 89 (32). Anal. Calcd. For C₁₄H₁₂N₄ (236.11): C, 71.17; H, 5.12; N, 23.71. Found: C, 71.29; H, 5.28; N, 23.91%.

2-[2-(4-Methylbenzylidene)hydrazinyl]-1H-Benzo[d]imidazole (3b)36

Yellow powder, mp = 275–277°C, [lit. mp = 277°C] (DMF); IR (KBr) υ = 3430 (NH), 2919 (CH-), 1602 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.38 (s, 3H, CH₃), 7.10–7.52 (m, 8H, Ar-H), 8.31 (s, 1H, CH=N), 10.74 (s, 1H, NH), 12.53 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 20.4 (CH₃), 114.4, 115.1, 122.3, 123.4, 126.9, 128.8, 131.4, 132.4, 136.2, 140.8, 144.4, 150.8 (Ar-C) ppm; MS, m/z (%) 250 (M⁺, 43), 136 (57), 121 (100), 95 (25). Anal. Calcd. For C₁₅H₁₄N₄ (250.12): C, 71.98; H, 5.64; N, 22.38. Found: C, 72.09; H, 5.73; N, 22.21%.

2-[2-(4-Methoxybenzylidene)hydrazinyl]-1H-Benzo[D]imidazole (3c)36

Yellow powder, mp = 215–217°C, [lit. mp = 212°C] (EtOH); IR (KBr) υ = 3412, 3204 (NH), 30212 (CH=), 2960, 2914 (CH-), 1602 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 3.69 (s, 3H, OCH₃), 7.11–7.56 (m, 8H, Ar-H), 8.33 (s, 1H, CH=N), 10.68 (s, 1H, NH), 12.52 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 56.2 (OCH₃), 114.3, 121.5, 128.4, 128.8, 129.3, 129.6, 129.8, 130.0, 146.4, 148.5, 152.3, 158.0 (Ar-C) ppm; MS, m/z (%) 266 (M⁺, 52), 158 (85), 142 (100), 138 (89), 89 (86), 75 (43). Anal. Calcd. For C₁₅H₁₄N₄O (266.12): C, 67.65; H, 5.30; N, 21.04. Found: C, 67.49; H, 5.18; N, 20.91%.

2-[2-(4-Chlorobenzylidene)hydrazinyl]-1H-Benzo[D]imidazole (3d)36

Yellow powder, mp = 273–275°C, [lit. mp = 270°C] (DMF); IR (KBr) υ = 3426, 3246 (NH), 3024 (CH=), 2959 (CH-), 1596 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 7.18–7.92 (m, 8H, Ar-H), 8.72 (s, 1H, CH=N), 10.70 (s, 1H, NH), 12.51 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 115.1, 115.8, 122.3, 123.4, 128.9, 129.2, 130.2, 132.4, 135.2, 142.8, 148.4, 160.8 (Ar-C) ppm; MS, m/z (%) 270 (M⁺, 40), 188 (46),150 (89), 75 (100). Anal. Calcd. For C₁₄H₁₁ClN₄ (270.07): C, 62.11; H, 4.10; N, 20.70. Found: C, 62.19; H, 4.17; N, 20.55%.

2-[2-(4-Nitrobenzylidene)hydrazinyl]-1H-Benzo[D]imidazole (3e)36

Yellow powder, mp = 287–289°C, [lit. mp = 283°C] (DMF); IR (KBr) υ = 3451 (NH), 3013 (CH=), 1594 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 7.10–7.89 (m, 8H, Ar-H), 8.62 (s, 1H, CH=N), 10.28 (s, 1H, NH), 12.53 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 115.2, 116.1, 123.4, 124.3, 125.2, 125.9, 132.2, 136.4, 139.2, 142.1, 150.4, 160.8 (Ar-C) ppm; MS, m/z (%) 281 (M⁺, 20), 203 (52), 187 (53), 102 (100), 75 (80). Anal. Calcd. For C₁₄H₁₁N₅O₂ (281.09): C, 59.78; H, 3.94; N, 24.90. Found: C, 59.55; H, 4.07; N, 24.85%.

2-[2-(2,4-Dichlorobenzylidene)hydrazinyl]-1H-Benzo[D]imidazole (3f)

Yellow powder, mp = 247–249°C (EtOH); IR (KBr) υ = 3433, 3203 (NH), 2958, 2918 (CH-), 1598 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 7.10–7.89 (m, 7H, Ar-H), 8.72 (s, 1H, CH=N), 10.28 (s, 1H, NH), 12.53 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 114.8, 115.3, 122.4, 123.4, 123.9, 127.5, 128.2, 129.3, 130.2, 131.4, 131.9, 132.8, 138.4, 160.7 (Ar-C) ppm; MS, m/z (%) 304 (M⁺, 31), 279 (36), 231 (49), 154 (56), 127 (100), 77 (39). Anal. Calcd. For C₁₄H₁₀Cl₂N₄ (304.03): C, 55.10; H, 3.30; N, 18.36. Found: C, 55.19; H, 3.19; N, 18.25%.

2-[2-(2,6-Dichlorobenzylidene)hydrazinyl]-1H-Benzo[D]imidazole (3g)

Yellow powder, mp = 271–273°C (DMF); IR (KBr) υ = 3425 (NH), 2915 (CH-), 1603 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 7.11–7.89 (m, 7H, Ar-H), 8.72 (s, 1H, CH=N), 10.29 (s, 1H, NH), 12.53 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 114.8, 115.2, 123.4, 123.9, 124.6, 127.5, 128.2, 129.3, 130.2, 131.4, 131.9, 133.8, 138.4, 160.6 (Ar-C) ppm; MS, m/z (%) 304 (M⁺, 62), 251 (60), 194 (36), 173 (100), 133 (63), 73 (92). Anal. Calcd. For C₁₄H₁₀Cl₂N₄ (304.03): C, 55.10; H, 3.30; N, 18.36. Found: C, 55.20; H, 3.19; N, 18.18%.

2-[2-(1-Phenylethylidene)hydrazinyl]-1H-Benzo[D]imidazole (3h)37

Yellow powder, mp = 237–239°C (EtOH); IR (KBr) υ = 3432 (NH), 3035 (CH=), 2912 (CH-), 1599 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.46 (s, 3H, CH₃), 7.09–7.52 (m, 9H, Ar-H), 10.52 (s, 1H, NH), 12.45 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 18.4 (CH₃), 115.4, 116.1, 123.3, 123.7, 124.9, 128.1, 128.4, 130.4, 135.2, 136.8, 141.4, 160.8 (Ar-C) ppm; MS, m/z (%) 250 (M⁺, 70), 192 (48), 160 (96), 112 (100), 76 (82). Anal. Calcd. For C₁₅H₁₄N₄ (250.12): C, 71.98; H, 5.64; N, 22.38. Found: C, 72.03; H, 5.71; N, 22.28%.

2-[2-(1-(4-Bromophenyl)ethylidene)hydrazinyl]-1H-Benzo[D]imidazole (3i)37

Yellow powder, mp = 250–252°C (EtOH); IR (KBr) υ = 3385 (NH), 1585 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.47 (s, 3H, CH₃), 7.10–7.74 (m, 8H, Ar-H), 10.54 (s, 1H, NH), 12.49 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 18.6 (CH₃), 115.4, 116.2, 123.3, 123.7, 124.9, 128.1, 128.6, 130.4, 134.9, 136.6, 141.4, 160.7 (Ar-C) ppm; MS, m/z (%) 328 (M⁺, 37), 197 (67), 138 (69), 126 (100), 116 (87), 77 (48). Anal. Calcd. For C₁₅H₁₃BrN₄ (328.03): C, 54.73; H, 3.98; N, 17.02. Found: C, 54.81; H, 3.77; N, 16.88%.

2-[2-(1-(Naphthalen-2-Yl)ethylidene)hydrazinyl]-1H-Benzo[D]imidazole (3j)

Yellow powder, mp = 288–290°C, (DMF); IR (KBr) υ = 3414, 3204 (NH), 2915 (CH-), 1601 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.46 (s, 3H, CH₃), 7.11–8.45 (m, 11H, Ar-H), 10.62 (s, 1H, NH), 12.53 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 18.8 (CH₃), 114.9, 115.1, 122.8, 123.1, 126.4, 126.8, 127.2, 127.6, 128.3, 128.6, 129.2, 129.8, 131.9, 133.2, 134.2, 135.8, 142.4, 158.1 (Ar-C) ppm; MS, m/z (%) 300 (M⁺, 100), 247 (19), 219 (15), 140 (33). Anal. Calcd. For C₁₉H₁₆N₄ (300.14): C, 75.98; H, 5.37; N, 18.65. Found: C, 76.09; H, 5.48; N, 18.81%.

2-[2-(1-(Thiophen-2-Yl)ethylidene)hydrazinyl]-1H-Benzo[D]imidazole (3k)

Yellow powder, mp = 244–246°C, (EtOH); IR (KBr) υ = 3433 (NH), 2921 (CH-), 1596 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.47 (s, 3H, CH₃), 7.11–7.63 (m, 7H, Ar-H), 10.60 (s, 1H, NH), 12.54 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 18.7 (CH₃), 114.9, 115.2, 123.1, 124.4, 124.8, 125.2, 127.6, 128.1, 134.4, 135.8, 142.4, 158.7 (Ar-C) ppm; MS, m/z (%) 256 (M⁺, 71), 199 (39), 184 (100), 127 (66), 77 (68). Anal. Calcd. For C₁₃H₁₂N₄S (256.08): C, 60.92; H, 4.72; N, 21.86; S, 12.51. Found: C, 61.09; H, 4.68; N, 21.88; S, 12.36%.

2-[2-(1-(Pyridin-3-Yl)ethylidene)hydrazinyl]-1H-Benzo[D]imidazole (3L)

Yellow powder, mp = 263–265°C (EtOH/DMF); IR (KBr) υ = 3387 (NH), 1592 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.43 (s, 3H, CH₃), 7.09–8.67 (m, 8H, Ar-H), 10.58 (s, 1H, NH), 12.42 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 19.3 (CH₃), 115.2, 115.9, 122.3, 123.7, 123.9, 126.1, 132.4, 133.2, 134.2, 136.8, 141.4, 145.2, 160.4 (Ar-C) ppm; MS, m/z (%) 251 (M⁺, 65), 204 (45), 123 (44), 106 (98), 91 (100). Anal. Calcd. For C₁₄H₁₃N₅ (251.12): C, 66.92; H, 5.21; N, 27.87. Found: C, 67.03; H, 5.33; N, 27.91%.

2-[2-(1-(1H-Indol-3-Yl)ethylidene)hydrazinyl]-1H-Benzo[D]imidazole (3m)

Yellow powder, mp = 257–259°C (EtOH/DMF); IR (KBr) υ = 3413, 3259, 3207 (NH), 3009 (CH=), 2968 (CH-), 1591 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.41 (s, 3H, CH₃), 7.08–8.26 (m, 9H, Ar-H), 10.54 (s, 1H, NH), 11.87 (s, 1H, NH), 12.49 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 18.7 (CH₃), 112.2, 113.4, 114.3, 115.7, 120.9, 121.1, 123.4, 123.9, 127.8, 128.1, 133.2, 134.2, 135.8, 137.4, 141.2, 160.4 (Ar-C) ppm; MS, m/z (%) 288 (M⁺, 32), 132 (20), 119 (25), 106 (100), 91 (70). Anal. Calcd. For C₁₇H₁₅N₅ (289.13): C, 70.57; H, 5.23; N, 24.20. Found: C, 70.33; H, 5.11; N, 24.11%.

3-[1-(2-(1H-Benzo[D]imidazol-2-Yl)hydrazono)ethyl]-2H-Chromen-2-One (3n)

Yellow powder, mp = 271–273°C (DMF); IR (KBr) υ = 3419 (NH), 2926 (CH-), 1721 (C=O), 1606 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 2.47 (s, 3H, CH₃), 7.09–8.57 (m, 9H, Ar-H), 11.19 (s, 1H, NH), 12.48 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 18.7 (CH₃), 115.2, 115.9, 118.3, 121.9, 122.4, 123.3, 125.8, 126.1, 128.5, 131.2, 133.4, 135.2, 141.4, 149.2, 153.1, 158.4, 160.5 (Ar-C) ppm; MS, m/z (%) 318 (M⁺, 43), 144 (37), 126 (100), 78 (78), 64 (85). Anal. Calcd. For C₁₈H₁₄N₄O₂ (318.11): C, 67.92; H, 4.43; N, 17.60. Found: C, 67.83; H, 4.32; N, 17.41%.

3-[2-(1H-Benzo[D]imidazol-2-Yl)hydrazono]indolin-2-One (3o)38

Yellow powder, mp = 305–307°C [lit. mp = 320°C] (DMF); IR (KBr) υ = 3419, 3201 (NH), 3076 (CH=), 2917 (CH-), 1680 (C=O), 1602 (C=N) cm^{−1; 1}H NMR (DMSO-d₆) δ: 7.03–7.81 (m, 8H, Ar-H), 9.51 (s, 1H, NH), 10.99 (s, 1H, NH), 12.48 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆): δ: 115.5, 116.7, 123.9, 128.8, 128.9, 129.4, 129.5, 129.7, 130.0, 136.3, 140.8, 146.2, 151.4, 155.7 (Ar-C), 170.9 (C=O) ppm; MS, m/z (%) 277 (M⁺, 100), 198 (36), 182 (86), 153 (66), 75 (95). Anal. Calcd. For C₁₅H₁₁N₅O (277.10): C, 64.97; H, 4.00; N, 25.26. Found: C, 64.85; H, 3.91; N, 25.09%.

DFT Calculations

All computational calculations were performed using the Gaussian 09W software package.39,40 The molecular geometry of the studied compounds was optimized using the density functional theory B3LYP method, by implementing the standard 6–31G(d,p) basis set. The visualization of the optimized structures was performed using GaussView 6.0.1.41–44

Frontier Molecular Orbitals and Global Reactivity Indexes

The Frontier Molecular Orbitals and the global descriptors of the investigated compounds were achieved under the same calculation level. Popular qualitative chemical concepts derived from conceptual DFT were measured to define the reactivity as well as the stability of a system45–51 (see Electronic supplementary files).

Antimicrobial Activity

The antimicrobial activity of the prepared compounds was tested using strains from the RCMB culture collection at Al-Azhar University, Egypt. The well-diffusion method assessed activity against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae), and filamentous fungi (Aspergillus gillus, Candida albicans). Microbial cultures (10⁸ cells/mL for bacteria, 10⁵ cells/mL for fungi) were spread on agar plates, and 100 μL of the compound solution (10 mg/mL) was added to 6 mm wells. Plates were incubated at 37°C for bacteria/yeast (24–48 h) and 28°C for fungi (48 h). Inhibition zones were measured, with larger zones indicating higher antimicrobial activity.52 DMSO served as a negative control, showing no inhibition, while ampicillin, gentamycin and nystatin were used as positive controls for bacteria and fungi, respectively.

Docking Methodology

The docking studies were carried out on Dell Inspiron 3847 [Intel(R) Core(TM) i7-4790 CPU @ 3.60GHz, 16GB of DIMM DDR3-1333/1600 memory, Windows 10 home (64 Bit)]. Molecular Operating Environment (MOE) package version 2022.02 (Chemical Computing Group, Inc. Molecular Operating Environment was used for performing docking studies).

Receptor Preparation

Docking procedures were performed using Crystal Structure of Mecr1 Extracellular Antibiotic-Sensor with open form-penicillin G (PDB: 2IWC), Phenylpyruvate Decarboxylase with Thiamine diphosphate (PDB: 2NXW), and CYP51 with Fluconazole (PDB: 1EA1) was obtained from protein data bank (PDB). The structures were first repaired, and then appropriately protonated in the presence of ligands using the Protonate 3D, energy minimized with force field: amber10: EHT until it reached an RMS (root mean square) gradient of 0.00001 kcal/mol/Å.

Ligand Preparation

Compounds 3c and 3o were built in ChemBioDraw Ultra 14.0 and sketched in MOE.

Docking Procedure

The MOE 2022.02 procedure’s standard protocol was applied in this work. The Alpha Triangle placement determines the positions by randomly superposing ligand atom triplets alpha sphere dummies in the receptor site to create poses. The output database dock file, which was arranged according to the final score function (S), or the score of the final stage that was not set to zero, featured distinct poses for each ligand. The database browser was used to visually inspect the different poses of each ligand, and the best poses were chosen.

Conclusion

A green synthetic approach was applied to prepare a series of benzimidazole-based hydrazones using citric acid as an acid catalyst. These hydrazones 3a–o were formulated as (E) configuration. The synthesized compounds, particularly 3c and 3o, exhibit promising antimicrobial activities against a range of pathogenic microorganisms. Their broad-spectrum efficacy, coupled with performance comparable to standard antimicrobial agents, underscores their potential as valuable additions to the arsenal against microbial infections. Molecular docking results suggest that specific structural features, such as electron-donating groups on aromatic rings (3c) and the presence of a heterocyclic moiety (3o), significantly influence antimicrobial efficacy. Increased lipophilicity also appears to enhance cell wall penetration and target binding. Future work may explore in vivo biological testing and structural optimization for enhanced selectivity and potency. Potential applications include the development of new antimicrobial agents with environmentally friendly synthetic profiles. Limitations of this study include the absence of in vivo data and the need for broader antimicrobial screening to fully establish the therapeutic potential of these compounds.

Funding Statement

This work was funded by the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R80), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Disclosure

The authors declare no conflicts of interest in this work.