Synthesis of 1-(2-Hydroxyphenyl)- and (3,5-Dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives as Promising Scaffolds for the Development of Novel Antimicrobial and Anticancer Agents

Increasing antimicrobial resistance among Gram-positive pathogens and pathogenic fungi remains one of the major public healthcare threats. Therefore, novel antimicrobial candidates and scaffolds are critically needed to overcome resistance in Gram-positive pathogens and drug-resistant fungal pathogens. In this study, we explored 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid and its 3,5-dichloro-2-hydroxyphenyl analogue for their in vitro antimicrobial activity against multidrug-resistant pathogens. The compounds showed structure-dependent antimicrobial activity against Gram-positive pathogens (S. aureus, E. faecalis, C. difficile). Compounds 14 and 24b showed promising activity against vancomycin-intermediate S. aureus strains, and favorable cytotoxic profiles in HSAEC-1 cells, making them attractive scaffolds for further development. 5-Fluorobenzimidazole, having a 3,5-dichloro-2-hydroxyphenyl substituent, was found to be four-fold, and hydrazone, with a thien-2-yl fragment, was two-fold stronger than clindamycin against methicillin resistant S. aureus TCH 1516. Moreover, hydrazone, bearing a 5-nitrothien-2-yl moiety, showed promising activity against three tested multidrug-resistant C. auris isolates representing major genetic lineages (MIC 16 µg/mL) and azole-resistant A. fumigatus strains harboring TR34/L98H mutations in the CYP51A gene. The anticancer activity characterization demonstrated that the 5-fluorobenzimidazole derivative with a 3,5-dichloro-2-hydroxyphenyl substituent showed the highest anticancer activity in an A549 human pulmonary cancer cell culture model. Collectively these results demonstrate that 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives could be further explored for the development of novel candidates targeting Gram-positive pathogens and drug-resistant fungi.


Introduction
Infections caused by multidrug-resistant (MDR) Gram-positive bacteria and drugresistant (DR) fungi remain a major healthcare problem, with the majority of the cases occurring in critically ill individuals or patients undergoing chemotherapy or solid organ transplantation. Among MDR Gram-positive pathogens, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and Clostridioides difficile (C. difficile) are responsible for the majority of cases. Moreover, bloodstream infections caused by MRSA and other Gram-positive pathogens often have a poor prognosis and result in the death of the patients. Therefore, it is crucial to develop novel compounds targeting Gram-positive pathogens [1].
Vancomycin and other structurally related glycopeptides are the last resort antimicrobials used to treat severe infections caused by Gram-positive pathogens. The resistance to vancomycin was first reported in 1986, and nowadays, resistance is often observed in clinical settings [2,3]. The molecular determinants encoding the resistance to vancomycin are encoded by a transposon located in the plasmids, thus permitting the lateral spread of numerous Gram-positive pathogens. Vancomycin resistance is now being observed in various aerobic and anaerobic Gram-positive pathogens, such as S. aureus, Enterococcus spp., and C. difficile [4,5]. The rapid spread of the resistance determinants among Grampositive pathogens, as well as the rising number of cases associated with vancomycinintermediate or vancomycin-resistant strains, urges the development of novel therapies to restore susceptibility to last-line drugs or provide new candidates selectively targeting drug-resistant pathogens.
Pathogenic fungi are associated with approximately 1.5 million deaths and 1.7 billion superficial infections every year, resulting in a massive economic burden on healthcare [6,7]. Yeast belonging to Candida species (predominantly C. albicans), as well as molds belonging to Aspergillus (predominantly A. fumigatus), are responsible for the majority of invasive fungal infection (IFI) cases worldwide. Various azole antifungal drugs (fluconazole, voriconazole, itraconazole, etc.) are the first line of drugs to treat IFIs caused by Candida spp. and A. fumigatus. Moreover, increasingly drug-resistant Candida species, such as C. auris harboring numerous resistance determinants, are increasingly being isolated from critically ill patients, resulting in a shortage of available treatment options and increased mortality. Furthermore, the emergence of azole-resistant A. fumigatus strains, harboring an azole-resistance (AR) phenotype associated TR34/L98H mutations in the CYP51A gene [8,9], makes these infections caused by AR A. fumigatus extremely lethal and requires various compassionate care or investigational therapeutic options. Therefore, new antifungal drug candidates are critically needed to overcome antifungal resistance in highly drug-resistant Candida species, as well as AR A. fumigatus with TR34/L98H mutations in the CYP51A gene. In addition to that, since fungi are eucaryotic organisms, many targets located in fungal cells overlap with host cellular targets. Therefore, compounds with antifungal activity could be also further explored for their anticancer properties.
Azoles are a diverse and important class of five-membered, nitrogen-containing, heterocyclic organic molecules that may possess other non-carbon atoms, such as oxygen or sulfur, thus making them an extremely structurally versatile class of molecules.
Diversely functionalized azole analogues are considered to be one of the most important frameworks for the development of numerous pharmacologically active compounds with antifungal, antibacterial, antidiabetic, and anticancer activities [10][11][12][13][14][15][16][17][18]. Interestingly, several studies have shown that azole derivatives containing naphthalene show selective and Gram-positive-bacteria-directed antimicrobial activity [19]. Moreover, the conjugation of metal nanoparticles with azole antimicrobial derivatives greatly enhances antibacterial activity against pan-susceptible and drug-resistant S. aureus, suggesting that azole derivatives could be explored as Gram-positive-bacteria-directed antimicrobials [20]. The chemical versatility of azoles and the ability to incorporate numerous substitutions in the core structure makes azoles an attractive scaffold for the development of novel dual active antimicrobial candidates targeting Gram-positive bacterial and fungal pathogens.
The identification of potent pharmacophores is paramount for the development of novel broad-spectrum antimicrobial candidates. As an example, benzimidazole core incor-poration in target structures greatly enhances the pharmacological properties of various molecules due to the formation of fused ring benzimidazole compounds.
Compounds bearing various fused-ring benzimidazole moieties demonstrate promising antimicrobial activities against numerous pathogens [21,22]. Benzimidazole is an electron-rich pharmacophore that can easily accept or donate protons and easily form a variety of weak interactions leading benzimidazole pharmacophores to bind different cellular targets. With that in mind, benzimidazole core-containing compounds have been previously reported to show anticancer activity in different cancer cell culture models [22][23][24]. Therefore, possibly, compounds containing azole and benzimidazole moieties could show potential antifungal and antibacterial activity targeting drug-resistant fungal and bacterial pathogens [25][26][27][28][29][30].
The increasing antimicrobial resistance among Gram-positive bacterial pathogens to last-line antimicrobials urges the identification of novel candidates for further pre-clinical antimicrobial drug development. With growing numbers of cancer and chemotherapyassociated immunosuppression cases, drug-resistant fungal species can often co-infect individuals suffering from infections caused by Gram-positive pathogens. Therefore, the development of novel antimicrobial candidates, targeting multidrug-resistant Gram-positive pathogens and drug-resistant fungi, is critically needed. Our previous studies [31,32] on the development of novel compounds targeting multidrug-resistant pathogens have been reasonably successful in finding effective antimicrobial agents and showed higher bactericidal properties than ampicillin; therefore, we have continued the studies in this field. As the results demonstrate that 5-oxopyrolidine derivatives are attractive cores for the further development of potential candidates targeting multidrug-resistant Gram-positive pathogens and drug-resistant fungi with genetically defined resistance mechanisms, herein, we report the synthesis and bioevaluation of compounds having N-(2-hydroxyphenyl)and N-(3,5-dichloro-2-hydroxyphenyl)-5-oxopyrrolidin-3-yl cores, as well their decyclization products, γ-amino acid derivatives. Their antimicrobial properties were focused on activity against Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Clostridioides difficile, Candida auris, and Aspergillus fumigatus strains. In addition to that, we characterized the in vitro cytotoxic and anticancer properties of novel compounds using the A549 human lung cell culture model.
The reaction resulted in the formation of hydrazide 3 (Supplementary materials, Figures S5 and S6) with an 88.5% yield. To obtain hydrazones 4-15, hydrazide 3 was applied for the condensation with 10 different aromatic aldehydes and thiophene-2-carbaldehyde, as well as its 5-nitro derivative. The reactions were carried out in propan-2-ol at reflux and afforded 1-(2-hydroxyphenyl)-N -substituted-5-oxopyrrolidine-3-carbohydrazides at good to excellent yields (60-90%). Inspection of the 1 H NMR spectra of compounds 4-15 (Supplementary Materials, Figures S7-S22, S63 and S64) revealed two sets of singlets corresponding to the CONH and CH=N protons, confirming the presence of a mixture of Z and E conformational isomers, caused by the restricted rotation around the amide bond, where as it is known [33,34], the Z-form usually predominates. The intense ratio of singlets of the rotamers appeared to be 65:35 for the synthesized hydrazones, while for  To obtain hydrazones 4-15, hydrazide 3 was applied for the condensation with 10 different aromatic aldehydes and thiophene-2-carbaldehyde, as well as its 5-nitro derivative. The reactions were carried out in propan-2-ol at reflux and afforded 1-(2-hydroxyphenyl)-N′-substituted-5-oxopyrrolidine-3-carbohydrazides at good to excellent yields (60-90%). Inspection of the 1 H NMR spectra of compounds 4-15 (Supplementary Materials, Figures S7-S22, S63 and S64) revealed two sets of singlets corresponding to the CONH and CH=N protons, confirming the presence of a mixture of Z and E conformational isomers, caused by the restricted rotation around the amide bond, where as it is known [33,34], the Z-form usually predominates. The intense ratio of singlets of the rotamers appeared to be 65:35 for the synthesized hydrazones, while for compound 13 bearing the 1-naphthyl fragment, the ratio of the Z-form to E-form was found to be 60:40, describing a more stable molecule. Compound 16 was obtained by the reaction of acid hydrazide 3 with 1-(4-aminophenyl)ethan-1-one in propan-2-ol at reflux for 15 h (Supplementary materials, Figures S23 and S24 The 3-methyleneindolin-2-one moiety is a part of naturally occurring compounds and is widely used for the design of biologically active compounds [35,36]. Thus, to incorporate this structural unit into the designed structure, hydrazide 3 was treated with indoline-2,3-dione in refluxing propan-2-ol for 2 h. The product 17 was isolated with a 73% yield (Supplementary materials, Figures S25 and S26).
The target azole 18 and diazole 19 were synthesized via the acid-catalyzed condensation of 3 and hexane-2,5-dione or pentane-2,4-dione, respectively. The presence of drops of glacial acetic acid (18) or hydrochloric acid (19) led to the formation of the target N- A mixture of carbohydrazide 3 and benzil in refluxing glacial acetic acid containing a 10-fold excess of ammonium acetate produced 1,2,4-triazine derivative 20. The spectral and microanalysis data of the compound were in good agreement with the structure (Supplementary materials, Figures S31 and S32).
Next, a series of benzimidazole derivatives variously substituted at 5 th position of the benzimidazole fragment was prepared and identified. The synthesis of compounds 21-24a, b (Scheme 2) was accomplished via the condensation of carboxylic acids 1a, b with the appropriate benzene-1,2-diamine in 6 M hydrochloric acid at reflux for 24 h. The corresponding benzimidazoles 21-24a, b were isolated from the reaction mixtures through the alkalinization of the mixtures with 15% ammonium hydroxide to pH 8. From their 1 H NMR spectra, a characteristic singlet in the range of 10.21-12.58 ppm proved the presence of the NH proton, and an increase in resonances in the aromatic area ( 1 H and 13 C) showed the presence of a new fused aromatic structure in the molecules. Finally, the obtained 5-oxopyrrolidine derivatives 21-24a, b were applied for the preparation of the corresponding γ-amino acids 25-27a, b and 28b using the method described previously [37]. Due to the instability of the pyrrolidinone cycle in strong alkaline medium, the abovementioned compounds 21-24a, b were easily converted into the appropriate butanoic acids 25-28. The comparison of the NMR spectra of study compounds 25-28 with the cleaved pyrrolidinone ring with the spectra of their parent cyclized analogues 21-24 showed the characteristic differences, as for instance, the resonances of the COOHs at approx. 173 ppm ( 13 Figures S25 and S26).
A mixture of carbohydrazide 3 and benzil in refluxing glacial acetic acid containing a 10-fold excess of ammonium acetate produced 1,2,4-triazine derivative 20. The spectral and microanalysis data of the compound were in good agreement with the structure (Supplementary materials, Figures S31 and S32).
Next, a series of benzimidazole derivatives variously substituted at 5 th position of the benzimidazole fragment was prepared and identified. The synthesis of compounds 21-24a, b (Scheme 2) was accomplished via the condensation of carboxylic acids 1a, b with the appropriate benzene-1,2-diamine in 6 M hydrochloric acid at reflux for 24 h. The corresponding benzimidazoles 21-24a, b were isolated from the reaction mixtures through the alkalinization of the mixtures with 15% ammonium hydroxide to pH 8. From their 1 H NMR spectra, a characteristic singlet in the range of 10.21-12.58 ppm proved the presence of the NH proton, and an increase in resonances in the aromatic area ( 1 H and 13 C) showed the presence of a new fused aromatic structure in the molecules. Finally, the obtained 5oxopyrrolidine derivatives 21-24a, b were applied for the preparation of the corresponding γ-amino acids 25-27a, b and 28b using the method described previously [37]. Due to the instability of the pyrrolidinone cycle in strong alkaline medium, the above-mentioned compounds 21-24a, b were easily converted into the appropriate butanoic acids 25-28. The comparison of the NMR spectra of study compounds 25-28 with the cleaved pyrrolidinone ring with the spectra of their parent cyclized analogues 21-24 showed the characteristic differences, as for instance, the resonances of the COOHs at approx. 173 ppm ( 13
Among γ-amino acid derivatives 25-27a, b, 28b, only low antibacterial (25b, MIC 128 µg/mL) activity against Gram-positive S. aureus TCH 1516 and C. difficile AR-1067 and antifungal (26b and 27b) activity against the C. auris AR-381 isolate with the same MIC of 128 µg/mL were observed (Tables 1 and 2). Compounds 14 and 24b, bearing thien-2-yl and 5-fluoro benzimidazole substitutions, showed favorable activity against multidrug-resistant S. aureus strains with a vancomycinintermediate-resistance phenotype and multiple resistance mechanisms ( Table 3). The antibacterial activity of compound 14 (MIC 4-16 µg/mL) was comparable to that of vancomycin (VAN). Compound 24b showed promising antibacterial (MIC 2-8 µg/mL) activity against S. aureus isolates with challenging antimicrobial resistance mechanisms and was comparable to the antimicrobial activity of daptomycin (DAP). Collectively, these results demonstrated that 3-substituted 1-(2-hydroxyphenyl)-5oxopyrrolidines show promising antibacterial activity directed to S. aureus harboring a multidrug-resistant phenotype with emerging multidrug-resistance determinants. Compounds 14 and 24b could be further explored for hit-to-lead optimization or as a scaffold for the development of new compounds with activity against vancomycin-intermediate S. aureus.

Synthesis
Reagents, antibiotics, 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 via 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 (Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts were reported in (d) ppm relative to tetramethylsilane (TMS) with the residual solvent as an internal reference (DMSO-d 6 , δ = 2.50 ppm for 1 H and d = 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 (Perkin-Elmer Inc., Waltham, MA, USA) using KBr pellets. Mass spectra were obtained on a Bruker maXis UHR-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with ESI ionization. Elemental analyses (C, H, N) were conducted using the Elemental Analyzer CE-440 (Exeter Analytical, Inc., Chelmsford, MA, USA); their results were found to be in good agreement (±0.3%) with the calculated values.
The experimental data of 1b are in excellent agreement with those reported in [33].

General procedure of the preparation of esters 2a, b
A mixture of the corresponding carboxylic acid 2a, b (85 mmol), methanol (150 mL), and sulfuric acid (2 mL) was heated at reflux for 2 h and then cooled down and evaporated at reduced pressure. The residue was poured with aqueous 5% sodium carbonate solution (50 mL) and stirred for 5 min and left to cool down. The formed precipitated was filtered off, washed with water, and dried.  13

General procedure for the preparation of hydrazones 4-15
To a hot solution of hydrazide 3 (0.7 g, 3 mmol) in propan-2-ol (15 mL), the corresponding aromatic or non-aromatic aldehyde (4 mmol) was added, and the mixture was heated at reflux for 2 h or 40 min for 14 and then cooled down. The obtained solid was filtered off, washed with propan-2-ol, and dried to give the title compounds 4-15.
Light General method for the preparation of butanoic acids 25-27a, band 28b.
A mixture of the corresponding benzimidazole 21a, b-23a, b, and 24b (2 mmol) and aqueous 20% NaOH solution (10 mL) was heated at reflux for 4 h, then cooled down, and acidified with diluted acetic acid to pH 6. The form precipitate was filtered off, washed with water, and dried to give the title compounds 25-27a, b, and 28b. The products were purified by dissolving them in aqueous 2% sodium hydroxide solution and filtering and acidifying the filtrate with diluted acetic acid to pH 6-7.

Minimal Inhibitory Concentration Determination
The minimal inhibitory concentrations (MICs) of compounds 1a-28b, as well as various antibiotics, were determined according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [41]. The MICs for the compounds and comparator antibiotics were determined according to the testing standard broth microdilution methods described in CLSI document M07-A8 against the libraries of Gram-positive and Gramnegative pathogens, as well as pathogenic fungi. The compounds and antibiotics were dissolved in dimethyl sulfoxide (DMSO) to achieve a final concentration of 30 mg/mL. Series of dilutions were prepared in deep 96-well microplates to achieve 2× assay concentrations (0.5-128 µg/mL) and were then transferred to the assay plates. A standardized inoculum was prepared using a direct colony suspension. Within 15 min of preparation, the adjusted inoculum suspension was diluted in sterile CAMBH to achieve final concentrations of approximately 5 × 10 5 CFU/mL (range, 2 × 10 5 to 8 × 10 5 CFU/mL) in each well. The inoculum was transferred to the assay plates to achieve a 1× assay concentration.
For the anaerobic pathogens (C. difficile), the inoculum was prepared by using anaerobic Sheep Blood agar, and plates were incubated in an anaerobic chamber for 48 h. The inoculum was prepared as described elsewhere, and the plates containing investigational compounds were further incubated in an anaerobic chamber for 24-48 h [42]. Inoculated microdilution plates were incubated at 35 • C for 16 to 20 h in an ambient-air incubator within 15 min of the addition of the inoculum.

In Vitro Cytotoxic Activity Determination
The viability of A549 and HSAEC1-KT cells after the treatment with compounds or cisplatin, which served as the cytotoxicity control, was evaluated by using a commercial MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay (ThermoFisher Scientific, Waltham, MA, USA). Briefly, cells were plated in the flat-bottomed 96-well microplates (1 × 10 4 cells/well) and incubated overnight to facilitate the attachment. The test compounds were dissolved in hybridoma-grade DMSO (Sigma-Aldrich, St. Louis, MO, USA) and then further serially diluted in cell culture media containing 0.25% DMSO to achieve 100 µM for each compound.
Subsequently, the media from the cells were aspirated, and the compounds were added to the microplates. The cells were incubated at 37 • C, with 5% CO 2 , for 24 h. After incubation, 10 µL of Vybrant ® MTT Cell Proliferation Reagent (ThermoFisher Scientific) was added, and cells were further incubated for 4 h. After incubation, the media were aspirated, and the resulting formazan was solubilized through the addition of 100 µL of DMSO. The absorbance was then measured at 570 nm using a microplate reader (Multiscan, ThermoFisher Scientific). The following formula was used to calculate the % of A549 viability: ([AE − AB]/[AC − AB]) × 100%. AE, AC, and AB were defined as the absorbance of experimental samples, untreated samples, and blank controls, respectively. The experiments were performed in triplicate.

Statistical Analysis
The results are expressed as the mean ± standard deviation (SD). Statistical analyses were performed with Prism (GraphPad Software, version 9, San Diego, CA, USA), using the Kruskal-Wallis test and two-way ANOVA. p < 0.05 was accepted as significant.

Conclusions
In the present study, a series of 1-(2-hydroxyphenyl)-and (3,5-dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acids derivatives was synthesized, characterized, and evaluated for their antimicrobial activity using representative multidrug-resistant bacterial pathogens with emerging and genetically defined resistance mechanisms. In addition to that, the in vitro cytotoxic properties were characterized using A549 human lung cell culture models.
The results revealed that the 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives showed selective, Gram-positive bacteria-directed antimicrobial activity. The incorporation of a 2-thienyl fragment in the hydrazone structure significantly enhanced antibacterial activity against methicillin-resistant S. aureus TCH 1516 (USA 300 lineage) (MIC 16 µg/L) and C. difficile AR-1067 (MIC 32 µg/mL), although no activity was observed against Gram-negative or fungal pathogens (MIC > 128 µg/mL). This suggests that the 2-thienyl fragment plays an important role in the mechanism of action of these compounds against Gram-positive pathogens. A dechlorinated derivative with a 5-fluorobenzimidazole moiety resulted in an increase in the antimicrobial activity spectrum. A compound containing a 5-fluorobenzimidazole moiety showed 1-fold higher antimicrobial activity against S. aureus TCH 1516 (MIC of 8 µg/mL), as well as Gram-negative pathogens, except for A. baumannii. Interestingly, the replacement of 2-thienyl with a 5-nitro-2-thienyl fragment in hydrazone strongly increased the antifungal activity of the compounds against drug-resistant Candida and Aspergillus isolates. These results demonstrated that 1-(2-hydroxyphenyl)and (3,5-dichloro-2-hydroxyphenyl-5-oxopyrrolidine-3-carboxylic acid derivatives could be further explored as a promising scaffold for the discovery of antimicrobial candidates targeting multidrug-resistant Gram-positive pathogens and drug-resistant fungi. Further studies are needed to better understand the cellular targets, pharmacological properties, and safety of these compounds. Finally, the in vitro anticancer activity characterization showed that compounds demonstrated structure-depended anticancer activity against A549 cells. Among all tested compounds, 1-(3,5-dichloro-2-hydroxyphenyl)-4-(5-fluoro-1H-benzo[d]imidazol-2-yl)pyrrolidin-2-one showed the highest cytotoxic properties, making it as an attractive candidate for further anticancer compound development.

Conflicts of Interest:
The authors declare no conflict of interest.