Synthesis and Antibacterial Activity of New Azole, Diazole and Triazole Derivatives Based on p-Aminobenzoic Acid

The p-aminobenzoic acid was applied for the synthesis of substituted 1-phenyl-5-oxopyrrolidine derivatives containing benzimidazole, azole, oxadiazole, triazole, dihydrazone, and dithiosemicarbazide moieties in the structure. All the obtained compounds were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Salmonella enteritidis, Escherichia coli, and Pseudomonas aeruginosa by using MIC and MBC assays. This study showed a good bactericidal activity of γ-amino acid and benzimidazoles derivatives. The antimicrobial activity of the most promising compounds was higher than ampicillin. Furthermore, two benzimidazoles demonstrated good antimicrobial activity against L. monocytogenes (MIC 15.62 µg/mL) that was four times more potent than ampicillin (MIC 65 µg/mL). Further studies are needed to better understand the mechanism of the antimicrobial activity as well as to generate antimicrobial compounds based on the 1-phenyl-5-oxopyrrolidine scaffold.


Introduction
Rapidly growing antimicrobial resistance (AMR) has become a major source of morbidity and mortality worldwide [1]. Increasing AMR among various pathogens has led to fewer treatment options for patients suffering from severe infections caused by drugresistant (DR) pathogens. Moreover, infections caused by DR microorganisms require more extensive treatment, therefore resulting in a longer course of illness and prolonged hospitalization duration [2,3].
The extensive use of various antimicrobials in agriculture and veterinary sectors played a pivotal role in the development of AMR and the selection of highly virulent bacterial strains [4][5][6][7]. The DR pathogens of veterinary origin can further colonize the environment and can be transferred to humans [8,9]. In addition, the genetic determinants encoding AMR phenotypes can be further disseminated via horizontal gene transfer and accumulate in various bacterial species [10,11]. These processes created a vicious cycle that gave rise to multidrug-resistant (MDR) pathogens harboring multiple resistance mechanisms, resulting in bacterial resistance to two and more antimicrobial drugs [12]. To overcome this problem, it is important to develop novel compounds targeting MDR pathogens.
For the synthesis of the target benzoylhydrazine derivative 9, hydrazide 4 was reacted with benzoyl chloride in dichloromethane at reflux for 10 min. The product 9 from the reaction mixture was isolated in 54% yield. The formation of the -CONHNHCOPhfragment was approved by the presence of two singlets at 10.29 (NH) and 10.47 (NH) ppm, and the multiplet was integrated for nine protons of the two aromatic rings in the interval of 7.35-8.08 ppm.
To obtain a compound containing two hydrazinocarbonyl fragments, the reaction was carried out in hydrazine monohydrate using a 17 excess. The target product was obtained in a 54% yield.
The comparison of the spectra of compounds 4 and 10 demonstrated some differences that led to the easy identification of their specific structures. In the 1 H NMR spectrum of compound 4, the singlet at 3.82 ppm ( 13 C, 51.99 ppm), integrated for three protons, The hydrazide functional group can undergo various chemical transformations; using this ability, we thus performed a series of chemical reactions, applying different carbonyl compounds. The reaction of hydrazide 4 with pentane-2,4-dione (2, in refluxing ethanol produced pyrazole derivative 5, and the Paal-Knorr pyrrole synthesis using hexane-2,5-dione (2,5-HD) afforded pyrrole 6. A catalytic amount of glacial acetic acid was used in the reaction. In the 1 H NMR spectrum of 6, the intense singlets at 2.0 and 5.65 ppm were assigned to the protons of two methyl (2-and 5-positions) and two C=CH groups of the pyrrole ring, respectively. The resonances at 103.10 and 126.74 ppm in the 13 C NMR spectrum of compound 6 finally approved the formed pyrrole cycle in the molecule. All NMR spectra of the synthesized compounds are given in the Supplementary Materials.
Hydrazones 7a-c were prepared by the condensation of acid hydrazide 4 with benzaldehyde, 4-methoxybenzaldehyde, and 4-dimethylaminobenzaldehyde in refluxing ethanol (a,c) or a mixture of ethanol and 1,4-dioxane (1:2). In the reaction with itaconic acid, the hydrazide that has the amine group can readily undergo autocatalyzed intramolecular amidation-cyclization reaction to yield a stable 5-membered N-substituted pyrrolidinone cycle. The reaction was carried out in water at reflux for 15 h. Multiplets in the ranges of 2.50-2.90 (COCH 2 ), 3.20-3.40 (CH) 3.56-3.72 (NCH 2 ), and 3.87-4.18 (NCH 2 ) ppm, integrated for 10 protons in total in the 1 H NMR spectrum as well as double sets of the resonances of carbons of the COCH 2 , CH, NCH 2 fragments in the 13 C NMR spectra of compound 8 approve the presence of two pyrrolidinone rings.
For the synthesis of the target benzoylhydrazine derivative 9, hydrazide 4 was reacted with benzoyl chloride in dichloromethane at reflux for 10 min. The product 9 from the reaction mixture was isolated in 54% yield. The formation of the -CONHNHCOPhfragment was approved by the presence of two singlets at 10.29 (NH) and 10.47 (NH) ppm, and the multiplet was integrated for nine protons of the two aromatic rings in the interval of 7.35-8.08 ppm.
To obtain a compound containing two hydrazinocarbonyl fragments, the reaction was carried out in hydrazine monohydrate using a 17 excess. The target product was obtained in a 54% yield.
The comparison of the spectra of compounds 4 and 10 demonstrated some differences that led to the easy identification of their specific structures. In the 1 H NMR spectrum of compound 4, the singlet at 3.82 ppm ( 13 C, 51.99 ppm), integrated for three protons, shows the presence of the methoxy group, and the singlet at 4.35 ppm integrated for two protons proves the presence of the amino group, while in the 1 H NMR of dihydrazide 10, the signal of methoxy group is absent, and the broad singlet at 4.39 ppm is integrated for four protons, which proves the presence of two amino groups in the molecule.
Hydrazones 11a,b were obtained by the condensation of dihydrazide 10 with aromatic aldehydes. The reaction was carried out in the mixture of 2-propanol and 1,4-dioxane (ratio of 1:1.7) at reflux for 12 (a) or 11 (b) h, and products from the reaction mixtures were separated in 77% and 88% yields, respectively.
The synthesized hydrazones 7 and 11 possess amide and azomethine groups in their structures. Based on the experimental and theoretical studies presented in literature [73], it can be stated that due to the presence of the amide fragment and the restricted rotation around the CO−NH bond, the hydrazones exist in DMSO solutions as a mixture of Z/E rotamers in which the Z rotamer predominates. The clearest proof of the existence of conformers produced due to the presence of the CONH fragment was the discovery of the two sets of resonances of the NH group in the low-field region of the 1 H NMR spectra recorded in DMSO-d 6 , where a stronger-field side signal was related to the resonance of the rotamer with the Z structure.
The existence of the mixtures of stereoisomers relative to the CH=N structural fragment of the molecules was also found in the 1 H NMR spectra of the monosubstituted hydrazones 7 and 11. Based on the studies described in the academic literature [73], as well as the data of the spectra of these compounds, we can conclude that the produced mixtures of stereoisomers with the Z-isomer predominated.
The interaction of dihydrazide 10 with phenyl isothiocyanate in refluxing methanol led to the formation of thiosemicarbazide 12, which then under the action of 4% sodium hydroxide at reflux for 6 h and subsequent acidification of the mixture with dilute hydrochloric acid (1:1) to pH 2 afforded heterocyclic compound 13 with two 4-phenyl-5-thioxo-1,2,4triazole moieties in the structure. Resonances in the interval of 9.39-9.75 (4H, 2NHNHCO) as well as 10.12 and 10.40 (2H, 2NH) ppm in the 1 H NMR spectrum of compound 12 is clear evidence for the formation of the -CONHNHCSNH-moiety. Cyclodehydration of this fragment in the presence of a strong base led to the 1,2,4-triazole 13 formation, which was confirmed by the absence of thiosemicarbazide-specific spectral lines and the observation of a decrease and downfield shift of the NH resonances ( 1 H, 13.87 ppm).
Knowing the wide range of applications of the biological properties of benzimidazole derivatives in various fields, including medicine, pharmacy, optics, and others, we decided to synthesize compound 14 to have two benzimidazole moieties in its structure (Scheme 2). Five methods to achieve this goal were used. The reaction conditions and yields of the obtained product are given in Table 1.
as the data of the spectra of these compounds, we can conclude that the produced mixtures of stereoisomers with the Z-isomer predominated.
The interaction of dihydrazide 10 with phenyl isothiocyanate in refluxing methanol led to the formation of thiosemicarbazide 12, which then under the action of 4% sodium hydroxide at reflux for 6 h and subsequent acidification of the mixture with dilute hydrochloric acid (1:1) to pH 2 afforded heterocyclic compound 13 with two 4-phenyl-5-thioxo-1,2,4-triazole moieties in the structure. Resonances in the interval of 9.39-9.75 (4H, 2NHNHCO) as well as 10.12 and 10.40 (2H, 2NH) ppm in the 1 H NMR spectrum of compound 12 is clear evidence for the formation of the -CONHNHCSNH-moiety. Cyclodehydration of this fragment in the presence of a strong base led to the 1,2,4-triazole 13 formation, which was confirmed by the absence of thiosemicarbazide-specific spectral lines and the observation of a decrease and downfield shift of the NH resonances ( 1 H, 13.87 ppm).
Knowing the wide range of applications of the biological properties of benzimidazole derivatives in various fields, including medicine, pharmacy, optics, and others, we decided to synthesize compound 14 to have two benzimidazole moieties in its structure (Scheme 2). Five methods to achieve this goal were used. The reaction conditions and yields of the obtained product are given in Table 1.  The melting of carboxylic acid 2 with benzene-1,2-diamine at 170 • C and then at 230 • C gave the target compound a 51% yield. The condensation of benzene-1,2-diamine with acid 2 in 15% hydrochloric acid (by the Phillips method), at reflux yielded the desired product 14, but the process took 96 h, and the product obtained in only a 8% yield. For this reason, we tried a more modern method using microwaves, where the reaction mixture was exposed to microwave irradiation (140 W) for 15 min under solvent-free conditions. Benzimidazole 14 was obtained in a 40% yield. The reaction in 2-propanol with ammonium chloride as a catalyst did not produce the expected yield of the benzimidazole. The yield of bisbenzimidazole 14 was found to be only 12%. The most efficient method for the preparation of compound 14 appeared to be condensation of dicarboxylic acid 2 with o-phenylenediamine Molecules 2021, 26, 2597 6 of 23 in polyphosphoric acid (PPA) at 120 • C for 6 h. The product was separated in 97% yield. The last-mentioned method was used to obtain methylbenzimidazole derivative 15.
The pyrrolidinone ring of compound 14 was readily decyclized under alkaline hydrolysis conditions by refluxing it in an aqueous 20% sodium hydroxide solution for 2 h. The NMR spectra of the obtained product 16 showed chemical shifts characteristic to the open-chain structure in comparison with the initial compound 14.
The esterification of amino acid 16 with methanol in the presence of a catalytic amount of sulfuric acid was unsuccessful when the action of a strong acid led to the cyclization of the butanoic fragment to the initial pyrrolidinone ring. Therefore, to prepare acid hydrazide, we had to choose another synthesis route. Butanoic acid hydrazide 17 was obtained directly from pyrrolidinone derivatives 14 and its decyclized product-butanoic acid 16. The interaction of butanoic acid 16 with hydrazine monohydrate proceeded successfully under mild conditions, i.e., heating the reaction mixture in 2-propanol at reflux for 20 h afforded acid hydrazide 17. However, to obtain it from the pyrrolidinone derivative 14, tightened reaction conditions were needed. Therefore, the reaction was carried out in excess hydrazine monohydrate at reflux for 6 h. The 1 H and 13 C NMR spectra of 17 confirmed the open-chain compound. In the NMR spectra of 17, the triplet at 6.34 ppm was ascribed to the NH proton, the singlets at 4.43 (NH 2 , 1 H) and 9.12 (NHNH 2 , 1 H), and the spectral line at 169.90 (C=O, 13 C) approved the formed acid hydrazide moiety.
The condensation of hydrazide 17 with hexane-2,5-dione was investigated. As expected, the reaction in 2-propanol at reflux, with the presence of a catalytic amount of hydrochloric acid, afforded 2,5-dimethylpyrrole derivative 18. The singlets at 1.62 and 1.95 (CH 3 ) and the doublets at 5.52, 5.57 (C=CH) ppm in the 1 H NMR spectrum, in addition to resonances at 10.50, 11.00, and 102.76, 102.81 ppm of the corresponding groups in the 13 C NMR spectrum, prove the presence of the 2,5-dimethylpyrrole fragment.
Benzimidazoles play an important role in modern medicinal chemistry by being an important pharmacophore. With this notion, it is important to develop a large amount of structurally diverse benzimidazoles with potential medicinal properties.
For this purpose, the functionalization of benzimidazole 14 was performed (Scheme 3). The functionalization at nitrogen is perhaps the most common, and therefore it was chosen for the investigation. Initially, N-substituted benzimidazole derivative 19 was synthesized by the alkylation of bisbenzimidazole 14 with ethyl chloroacetate in acetone in the presence of potassium carbonate and a catalytic amount of TBAI (tetrabutylammonium iodide). The reaction was carried out at reflux for 20 h. The obtained ethyl ester 19 further was applied for the preparation of hydrazide 20. The structures of the synthesized compounds 19 and 20 were determined by spectral methods and chemical transformations.
The refluxing of 19 with hydrazine monohydrate in 1,4-dioxane for 18 h led to the formation of compound 20 containing two 2-hydrazinyl-2-oxoethyl moieties, whose presence is confirmed by the singlets at 4. 49  Carbonyl compounds are frequently used as derivatization agents. The condensation of acid hydrazide with diketone pentane-2,4-dione gave 3,5-dimethylpyrazole derivatives 21 a 82% yield. The reaction of 21 was performed for 5 h in refluxing 2-propanol and in the presence of a catalytic amount of hydrochloric acid. The reaction product was isolated from the reaction mixture by diluting it with water. The data of the 1 H and 13 C NMR spectroscopic techniques and elemental analysis confirmed the proposed structures of the synthesized pyrazole 21.
Oxadiazole derivative 22 was obtained by the ring closure reaction of the hydrazide 20 with carbon disulfide in alkaline medium obtained by using potassium hydroxide, which was dissolved in methanol, and then CS 2 was added dropwise to the cooled solution. After a thorough stirring for 15 min, the required amount of hydrazide was added, and the obtained reaction mixture was refluxed for 12 h. The acidifying of the aqueous solution of the reaction mixture with hydrochloric acid to pH 1 afforded the desired derivative 22 with two 1,3,4-oxadiazole moieties in the molecule. The signals in the NMR spectra of the compound were an exact match of the protons and carbon atoms of the obtained structure.  Carbonyl compounds are frequently used as derivatization agents. The condensation of acid hydrazide with diketone pentane-2,4-dione gave 3,5-dimethylpyrazole derivatives 21 a 82% yield. The reaction of 21 was performed for 5 h in refluxing 2-propanol and in the presence of a catalytic amount of hydrochloric acid. The reaction product was isolated from the reaction mixture by diluting it with water. The data of the 1 H and 13 C NMR spectroscopic techniques and elemental analysis confirmed the proposed structures of the synthesized pyrazole 21.
Oxadiazole derivative 22 was obtained by the ring closure reaction of the hydrazide 20 with carbon disulfide in alkaline medium obtained by using potassium hydroxide, which was dissolved in methanol, and then CS2 was added dropwise to the cooled solution. After a thorough stirring for 15 min, the required amount of hydrazide was added, and the obtained reaction mixture was refluxed for 12 h. The acidifying of the aqueous solution of the reaction mixture with hydrochloric acid to pH 1 afforded the desired derivative 22 with two 1,3,4-oxadiazole moieties in the molecule. The signals in the NMR Derivative 23 with two thiosemicarbazide moieties in the molecule was obtained in the reaction of hydrazide 20 with phenyl isothiocyanate. The reaction was performed in methanol at reflux for 6 h, and the obtained hydrazinecarbothioamine 23 was then applied for the synthesis of triazolethione using a method of cyclization in basic conditions. Cyclization was performed in an aqueous 4% sodium hydroxide solution in order to prevent 5-oxopyrrolidine ring breakage with the subsequent acidification of the reaction mixture with dilute hydrochloric acid (1:1) to pH 2. However, the process failed, and efforts to separate the cyclized product were unsuccessful. The spectral data (NMR, IR, and elemental analysis) of thiosemicarbazide 23 were in full agreement with the proposed structure. The multiplet in the range of 9.48-10.04 (4NH) ppm and the singlets at 10.51 and 10.58 (2NH) ppm in the 1 H NMR spectrum for 23 as well as additional peaks in the interval of 7.08-7.95 ppm prove the presence of the CONHNHCSNHPh fragment.
The hydrazones 24 were prepared by the condensation of hydrazide 20 with the corresponding aromatic aldehyde (benzaldehyde (a), 4-nitro-(b), 4-fluoro-(c), 3-chloro-(d) and 2,3-dimethoxybenzaldehyde (e)) in a molar ration of 1:7.5. The reactions were carried out by heating the mixtures at reflux for 5 h, except in case b when the reaction proceeded for 8 h. The products were separated in good to excellent yields in the range of 77%-94%.
When 1 H-NMR spectra of compounds 24 were observed, two sets of signals of the protons at different ppm were seen. This is because of the compounds, which have an arylidene hydrazide structure. The restricted rotation around the CO−NH bond causes the formation of a mixture of Z/E rotamers, whereas the presence of a double bond of CH=N influences the formation of geometric isomers, which are clearly visible in the spectra of these compounds [74]. The ratio in each case was calculated by using 1 H-NMR data, and they are as follows: 0.75:0.25 for a-c, e and 0.8:0.2 for d.

The Antimicrobial Activity of the Synthesized Compounds
In this study, we aimed to synthesize a series of novel benzimidazole derivates bearing ethyl ester, hydrazide, 3,5-dimethylpyrazole, 2,5-dimethylpyrrole, oxadiazole, thiosemicarbazide, and hydrazone moieties and to investigate their in vitro antimicrobial properties against a series of Gram-positive and Gram-negative bacterial pathogens.
The antimicrobial properties of synthesized compounds 3-24 were investigated against Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella enteritidis (Supplementary Table S1). The minimal inhibitory concentration (MIC) was evaluated by the broth dilution method, while the minimal bactericidal concentration (MBC) was determined by plating.
The results obtained in this study showed that all tested methyl derivatives 3-9, dihydrazide 10, and its derivatives 12 and 13 demonstrated moderate antimicrobial activity on all tested bacteria strains (Table 2). Moreover, hydrazones 11a,b did not demonstrate antimicrobial activity on neither Gram-positive nor Gram-negative bacteria. Hydrazone 11b harboring 4-chlorobenzylidene fragment demonstrated weak but selective bactericidal activity on S. aureus (250 µg/mL) but not on other Gram-positive or Gram-negative organisms ( Table 2).
Diester 3 and pyrazole derivative 5 exhibited good bactericidal activity (MIC and MBC of 31.25 µg/mL) against B. cereus. Interestingly, compounds 3 and 5 did not show a good antimicrobial activity against other Gram-positive organisms, suggesting the possible presence of B. cereus-specific targets of compound 5 ( Table 2).
Benzimidazole 14, methylbenzimidazole 15, γ-amino acid 16, and oxadiazole 22 demonstrated broad-spectrum antimicrobial activity that targets both Gram-negative and Gram-positive microorganisms ( Table 2). Compound 16 demonstrated the highest antimicrobial activity against all tested strains, suggesting the important role of γ-amino acid moiety for biological activity.
Furthermore, in this study, the bactericidal activity of hydrazide 17 and diester 19 bactericidal activity on S. aureus was comparable to ampicillin (62.5 µg/mL). The incorporation of γ-amino acid moiety in compound 16 and 4-(nitrobenzylidene)hydrazinyl fragment in 24b resulted in a compound with good activity against Gram-negative organisms (MIC and MBC at 31.25 µg/mL). In this study, compound 16 bearing γ-amino acid moiety demonstrated the most potent antimicrobial activity against a broad spectrum of microorganisms, demonstrating the importance of the abovementioned fragment as an antibacterial pharmacophore.
Interestingly, benzimidazoles 14 and 15 were an exception in this assay and had an exclusive activity against L. monocytogenes, although no activity was observed when tested against S. aureus or B. cereus. Compound 14 demonstrated slightly better, near-MIC bactericidal activity (MBC 15.62 µg/mL) ( Table 2). Benzimidazole 15 bearing 5(6)-methyl moiety showed one dilution higher MBC (31.25 µg/mL), suggesting that the 5(6)-methyl moiety is important for bactericidal activity against L. monocytogenes.
The investigations of structure-activity based relationships revealed some evident facts that changes in the 1-phenyl-5-oxopyrrolidine backbone by an incorporation of benzimidazole moieties greatly affect the biological properties of the compounds. The data presented in Table 2 demonstrate that benzimidazole 14 shows broad-spectrum antimicrobial activity, which was most evident when tested against L. monocytogenes. The antimicrobial activity of compound bearing a nitro group (24b) in the benzene ring was confirmed in this study when stronger antibacterial properties against the E. coli and P. aeruginosa strains were seen in comparison to other hydrazones 24. The results generated during this study are expected to be a foundation for the development of novel 1-phenyl-5-oxopyrrolidine-based antimicrobials. Further studies are needed to better understand the mechanism of antimicrobial activity as well as to generate more potent antimicrobial compounds based on the 1-phenyl-5-oxopyrrolidine nucleus.

Synthesis
Reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The reaction course and purity of the synthesized compounds were monitored by TLC using aluminum plates precoated with Silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). Melting points were determined with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and were uncorrected. NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer. Chemical shifts were reported in (δ) ppm relative to tetramethylsilane (TMS) with the residual solvent as internal reference ([D6]DMSO, δ = 2.50 ppm for 1 H and δ = 39.5 ppm for 13 C). Data were reported as follows: chemical shift, multiplicity, coupling constant (Hz), integration, and assignment. IR spectra (ν, cm −1 ) were recorded on a Perkin-Elmer Spectrum BX FT-IR spectrometer using KBr pellets. Mass spectra were obtained on a Bruker maXis UHRTOF mass spectrometer with ESI ionization. Elemental analyses (C, H, N) were conducted using the Elemental Analyzer CE-440; their results were found to be in good agreement (±0.3%) with the calculated values.

4-((4-(1H-benzimidazol-2-yl)phenyl)amino)-3-(1H-benzimidazol-2-yl)butanehydrazide
Method B: A mixture of pyrrolidinone 14 (1.97 g, 5 mmol) and hydrazine monohydrate (20 g, 400 mmol) was heated at reflux for 6 h and then was cooled to room temperature, diluted with 2-propanol (30 mL), and filtered off. The filtrate was evaporated under reduced pressure; the residue was poured with water and stirred for 10 min. The obtained solid was filtered off, washed with plenty of water, and recrystallized from water to give the title compound 14 (light brown solid, yield 1.34 g, 63%, m. p. 166-167 • C). 1 (18): To a mixture of hydrazide 17 (2.13 g, 5 mmol) and hexane-2,5-dione (3.42 g, 30 mmol) in 2-propanol (50 mL), conc. hydrochloric acid (2.5 mL) was added dropwise; the mixture was heated at reflux for 4 h, then cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was poured with water (30 mL) and stirred for 10 min. The obtained solid was filtered off, washed with water, and recrystallized from methanol to give the title compound 18 (white solid, yield 1.79 g, 71%, m. p. 227-228 • C). 1 Table S1). Each test organism was subcultured on Tryptic Soy Agar (TSA) at 37 • C, for 24 h. After incubation, the representative colonies were suspended in 5 mL of Tryptic Soy Broth (TBS) and further incubated at 37 • C for 24 h to initiate the liquid culture. The bacterial cultures were normalized using a spectrophotometer (OD 600 nm ), and the final inoculum (1 × 10 7 CFU/mL) was achieved by diluting the culture with fresh TSB.

Preparation of the Test Compounds
The test compounds 3-24 were dissolved in hybridoma grade DMSO to achieve a 50 mg/mL stock solution. The stock solution was further diluted in TSB supplemented with 1% of DMSO to produce the series of dilutions (0, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL). Ampicillin was dissolved in sterile deionized water and the series of dilutions (0, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL) were prepared as described above.

Evaluation of Minimal Inhibitory Concentration
The minimal inhibitory concentration (MIC) of the compounds 3-24 and ampicillin were determined by the broth dilution method as described by Balouiri et al. [75] with brief modifications. The tubes containing diluted compounds in TSB were inoculated with normalized bacterial inoculum (100 µL) to achieve a final bacterial concentration of 1 × 10 6 CFU/mL. The inoculated tubes were incubated at 37 • C for 24 h. After incubation, the turbidity was evaluated visually and MIC was estimated. The MIC was defined as the lowest concentration of the test compound that inhibits the visual growth of the test organism.

Determination of Minimal Bactericidal Concentration
The minimal bactericidal concentration (MBC) was determined as described by Parvekar et al. [76]. After a MIC evaluation of the novel compounds and ampicillin, aliquots of 100 µL were taken from tubes without growth and plated on TSA. The plates were incubated at 37 • C for 48 h. After incubation, the plates were evaluated, and the minimal bactericidal concentration (MBC) was estimated. The MBC was defined as the lowest concentration of the test compound that fully suppresses the growth of the test organism.

Conclusions
In this study, the chemical transformations of p-aminobenzoic acid were carried out, and a series of 1-phenyl-5-oxopyrrolidine derivatives with hydrazone, pyrazole, thiosemicarbazide, triazole, oxadiazole fragments were obtained. A convenient and efficient method for the synthesis of benzimidazoles by heating reagents in polyphosphoric acid was proposed.
The synthesized compounds were evaluated for their antibacterial activity against a panel of clinically relevant Gram-positive and Gram-negative pathogens. The antimicrobial activity evaluation revealed that the γ-amino acid derivative 16 bearing two benzimidazole fragments demonstrated the strongest broad-spectrum bactericidal activity on both Grampositive and Gram-negative organisms. The antimicrobial activity of compound 16 was notably greater than that of ampicillin. Furthermore, benzimidazoles 14 and 15 showed promising, broad-spectrum antibacterial activity against tested pathogens, with notably good bactericidal activity against L. monocytogenes. Collectively, these results demonstrated that the 5-oxopyrrolidine 14 could be further explored as a potential pharmacophore in the development of novel antimicrobials targeting clinically significant bacterial pathogens. Further studies are needed to better understand the safety, tolerability, and in vivo activity of the most promising compounds.