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Article

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

by
Monika Bertašiūtė
1,
Povilas Kavaliauskas
1,2,3,4,5,
Rita Vaickelionienė
1,*,
Birutė Grybaitė
1,
Vidmantas Petraitis
2,4,5,
Rūta Petraitienė
2,4,
Ethan Naing
2,
Andrew Garcia
2,
Jūratė Šiugždaitė
6,
Raimundas Lelešius
6 and
Vytautas Mickevičius
1,*
1
Department of Organic Chemistry, Kaunas University of Technology, Radvilėnų Rd. 19, 50254 Kaunas, Lithuania
2
Transplantation-Oncology Infectious Diseases Program, Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine of Cornell University, 1300 York Ave., New York, NY 10065, USA
3
Institute for Genome Sciences, School of Medicine, University of Maryland Baltimore School of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, USA
4
Institute of Infectious Diseases and Pathogenic Microbiology, Birštono Str. 38A, 59116 Prienai, Lithuania
5
Biological Research Center, Lithuanian University of Health Sciences, Tilžės St. 18, 47181 Kaunas, Lithuania
6
Department of the Veterinary Pathobiology, Lithuanian University of Health Science, Tilžės St. 18, 47181 Kaunas, Lithuania
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(9), 7966; https://doi.org/10.3390/ijms24097966
Submission received: 29 March 2023 / Revised: 18 April 2023 / Accepted: 24 April 2023 / Published: 27 April 2023
(This article belongs to the Section Molecular Microbiology)
Browse Figures
Versions Notes

Abstract

:
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.

1. Introduction

Infections caused by multidrug-resistant (MDR) Gram-positive bacteria and drug-resistant (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 Gram-positive pathogens, as well as the rising number of cases associated with vancomycin-intermediate 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 incorporation 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 chemotherapy-associated 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.

2. Results and Discussion

2.1. Chemistry

The synthesis was started from the preparation of key carboxylic acid 1a (Supplementary materials, Figures S1 and S2), which was obtained from 2-aminophenol and itaconic acid according to the method described in [27]. The dichloro-substituted derivative 1b was synthesized by treating 1-(2-hydroxyphenyl)-5-pyrrolidine-3-carboxylic acid (1a) with HCl in the presence of hydrogen peroxide (Scheme 1). To expand the library of 3-substituted 1-(2-hydroxy- and 2-hydroxy-3,5-dichlorophenyl)-5-oxopyrrolidines, compounds 1a, b were esterified to the corresponding esters 2a and 2b with methanol in the presence of sulfuric acid as a catalyst. Then, the resulting ester 2a (Supplementary materials, Figures S3 and S4) was reacted with hydrazine monohydrate in refluxing propan-2-ol.
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 1H 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-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxamide (18) or 4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-1-(2-hydroxyphenyl)pyrrolidin-2-one (19) (Supplementary materials, Figures S27–S30).
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 5th 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 1H 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 (1H and 13C) 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 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 (13C NMR, 25–28) are typical to the saturated open-chain carboxylic acids, while in the 13C NMR of cyclized derivatives 21–24, the spectral lines of C=O resonated in the range of 171.89–172.54 ppm. Furthermore, the characteristic closer shifted spectral lines of the carbons of the alkyl chain NHCH2CHCH2CO in the 25–28-series in comparison with the resonances of the corresponding carbons of the cyclized derivatives 21–24 were also observed (Supplementary materials, Figures S33–S62, S65 and S66).

2.2. Novel 1-(2-Hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives Show Gram-Positive Bacteria-Directed Antimicrobial Activity

To understand the antimicrobial properties of novel 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives 1a–28b bearing hydrazone, azole, and azine moieties, libraries of multidrug-resistant bacterial and fungal pathogens were used for the screening assays. The pathogens were selected to represent major WHO-priority bacterial and fungal pathogens harboring genetically defined antimicrobial-resistance determinants [38]. The compounds 1a–28b were screened using the broth microdilution technique with Clinical Laboratory Standard Institute recommendations.
Carboxylic acids 1a, b showed no antibacterial and antifungal activity (MIC > 128 µg/mL), while the transformation of compound 1b to ester 2b resulted in antimicrobial activity against methicillin-resistant S. aureus TCH 1516 (USA 300 lineage) (MIC 64 µg/mL) and New Delhi carbapenemase 1 (NDM-1)-producing A. baumannii AR-0033 (MIC 128 µg/mL) (Table 1).
Compounds 3–6 showed no antibacterial or antifungal activity (MIC > 128 µg/mL), while hydrazone 7 (R = 4-O2NC6H4) showed activity against S. aureus TCH1516 (MIC 64 µg/mL) and C. difficile AR-1067 (MIC 16 µg/mL) (Table 1 and Table 2). Furthermore, the incorporation of the 4-Me2NC6H4 substituent (8) abolished antimicrobial activity against C. difficile AR-1067 (MIC > 128 µg/mL) without affecting antimicrobial activity against S. aureus TCH 1516 (MIC 64 µg/mL). In addition to that, the hydrazone 14-bearing thien-2-yl group showed good activity against S. aureus TCH 1516 (MIC 16 µg/mL) and C. difficile AR-1067 (MIC 32 µg/mL), although no activity was observed against Gram-negative or fungal pathogens (MIC > 128 µg/mL) (Table 1 and Table 2). Interestingly, a 5-nitrothien-2-yl substitution (15) resulted in the loss of antibacterial activity (MIC > 128 µg/mL), although broad-spectrum antifungal activity was observed (Table 2). Compound 15 showed antifungal activity against three tested multidrug-resistant C. auris isolates representing major genetic lineages (MIC 16 µg/mL) (Table 2). Surprisingly, compound 15 showed antifungal activity against azole-resistant A. fumigatus strains with TR34/L98H mutations in the CYP51A gene (Table 2). On the other hand, compound 15 showed significant cytotoxicity in a non-cancerous HSAEC-1 pulmonary cell model, suggesting that the targets modulated by compound 15 are also found in pathogenic fungi (Figure S67). Triazine 20 showed activity against S. aureus TCH 1516 (MIC 32 µg/mL) and P. aeruginosa AR-0064, although no activity was observed against other tested bacterial or fungal pathogens (Table 1).
Among benzimidazoles 21–24a, b, compound 21b (R = 3,5-diCl) showed activity against S. aureus TCH 1516 and C. difficile AR-1067 (MIC 32 and 64 µg/mL). 5-Methyl benzimidazole 22b showed activity against S. aureus TCH 1516 (MIC 64 µg/mL), while 5-chloro benzimidazole 23a showed considerably decreased antibacterial activity against S. aureus TCH 1516 (MIC 128 µg/mL). On the other hand, 5-chloro benzimidazole 23b showed higher antibacterial activity against S. aureus TCH 1516 (MIC 64 µg/mL) in comparison to the 23a analogue. Interestingly, 5-fluoro benzimidazole 24b demonstrated broad-spectrum antibacterial activity against Gram-positive S. aureus TCH 1516 and C. difficile AR-1067 (MIC 8 and 128 µg/mL, respectively), as well Gram-negative NDM-1-producing K. pneumoniae AR-0049 (MIC 128 µg/mL) and P. aeruginosa AR-0064 (MIC 64 µg/mL) (Table 1). Furthermore, compound 24b showed favorable low cytotoxicity in HSAEC-1 cells, making compound 24b an attractive candidate for further hit-to-lead optimization (Figure S67).
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 (Table 1 and Table 2).

2.3. Compounds 14 and 24b Demonstrate Antibacterial Activity against Vancomycin-Intermediate Staphylococcus aureus Strains

After observing the promising antimicrobial activity of novel 3-substituted 1-(2-hydroxyphenyl)-5-oxopyrrolidines against multidrug-resistant S. aureus, we further evaluated if the most promising compounds 14 and 24b are active against vancomycin-intermediate-resistant S. aureus strains with multiple pre-existing resistance mechanisms. To do so, we performed an antimicrobial activity determination of compounds 14 and 24b against five representative strains with the vancomycin-intermediate-resistance phenotype.
Compounds 14 and 24b, bearing thien-2-yl and 5-fluoro benzimidazole substitutions, showed favorable activity against multidrug-resistant S. aureus strains with a vancomycin-intermediate-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)-5-oxopyrrolidines 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.

2.4. Novel 1-(2-Hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives Demonstrate Structure-Dependent Anticancer Activity

The in vitro anticancer activity of compounds 3-substituted 1-(2-hydroxyphenyl)-5-oxopyrrolidines 1a–28b bearing hydrazone, azole, and azine moieties was determined using viability-based MTT assays. A549 cells were used as a well-described cell culture model to study non-small cell lung adenocarcinoma. The compound treatment-induced cytotoxicity was compared to that of cisplatin (CP), a powerful DNA-binding cytotoxic drug, and the S cycle inhibitor cytosine arabinoside (AraC).
To understand the cytotoxicity of compounds 1a–28b, the A549 cells were exposed to a fixed 100 µM concentration of each compound or cytotoxicity control drugs (CP and AraC) for 24 h. The 3-substituted 1-(2-hydroxyphenyl)-5-oxopyrrolidines 1a–28b demonstrated structure-dependent anticancer activity against A549 cells by affecting the A549 viability by 16.5–101% (Figure 1). The carboxylic acid 1a (R = H) significantly reduced the A549 viability to 63.4% in comparison to that in the untreated controls (p < 0.05). The addition of 3,5-dichloro substitution (1b) greatly enhanced the in vitro anticancer activity by significantly reducing A549 viability to 21.2%. The ester 2a was able to decrease A549 viability (71.3%), although no significant anticancer activity was observed. Interestingly, further transformation of 1b to ester 2b containing a 3,5-dichloro substitution on the phenyl ring was able to significantly (p < 0.001) decrease the anticancer activity against A549 cells (38.3%) (Figure 1).
Hydrazide 3 and hydrazone 4 showed no anticancer activity and failed to significantly affect the A549 viability (101.2 and 101.3%, respectively). The addition of halogen substitutions on the phenyl ring resulted in compounds 5 and 6 with moderate anticancer activity. The hydrazone 5-bearing 4-ClC6H4 substitution significantly decreased A549 viability (65.5%), while chlorine substitution with bromine (4-BrC6H4) (5) slightly decreased anticancer activity (69.9%) (p = 0.0001 and p = 0.001, respectively). The addition of a 4-O2NC6H4 substitution (7) enhanced anticancer activity (57.1%), while the incorporation of a 4-Me2NC6H4 moiety (8) resulted in decreased anticancer activity (73.8%) (p = 0.007) (Figure 1). Moreover, the addition of methoxy groups greatly affected the anticancer activity. The addition of 4-MeOC6H4 (9) or 2,4-di(MeO)C6H3 (10) moieties resulted in the loss of significant anticancer activity against A549 cells (84.8% and 100.9%, respectively). Interestingly, the addition of 2,3,4-tri(MeO)C6H2 (11) or 3,4,5-tri(MeO)C6H2 (12) moieties resulted in the restoration of anticancer activity, since compounds 11 and 12 containing an MeO substitution in the 2, 3, 4- and 3,4,5-positions of the phenyl ring resulted in 74.5% and 76.4% post-treatment viability (p = 0.0095 and p = 0.0254, respectively). On the other hand, the addition of 1-naphthyl (13) or thien-2-yl (14) strongly enhanced the anticancer activity of hydrazones by decreasing the viability to 31.4 and 31.7%, respectively (p < 0.0001). Moreover, further incorporation of a 5-nitrothien-2-yl moiety (15) resulted in the slight loss of anticancer activity (47.7%) (p < 0.001). The incorporation of an aminoacetophenone substitution (16) resulted in decreased anticancer activity (65.1%) (p < 0.001), while the addition of a 2-oxoindolin-3-ylidene fragment (17) resulted in strikingly enhanced anticancer activity (26.2%) (Figure 1).
The pyrrole 18 and triazine 20 derivatives showed promising anticancer activity, while pyrazole 19 was found to decrease it. Compounds 18 and 20 significantly reduced A549 viability to 36.2 and 29.7%, respectively, while compound 19 only reduced the A549 viability to 72.4% (p < 0.05) (Figure 1).
Benzimidazole derivatives 21–24a, b were able to significantly reduce the viability of A549 cells (p < 0.05). Benzimidazole 21a significantly reduced A549 viability by 67.4% (p = 0.003). The 5-methyl analogue 22a reduced the A549 viability to 59.5%, while the addition of the 3,5-dichloro-2-hydroxyphenyl substituent (22b) significantly enhanced the anticancer activity by reducing the viability to 24.5% (p < 0.0001). Benzimidazole 23a, containing a 5-Cl radical, demonstrated reduced anticancer activity (70.3%), while the incorporation of a 3,5-dichloro substitution on the 2-hydroxyphenyl fragment (23b) resulted in the restoration of anticancer activity (49.5%) (p < 0.0001). The 5-fluorobenzimidazole 24a showed weak anticancer activity (87.4%), while the incorporation of a 3,5-dichloro-2-hydroxyphenyl substituent resulted in compound 24b with strikingly increased anticancer activity (16.1%) (p < 0.0001). Notably, the anticancer effect exerted by compound 24b was comparable to that of cytidine arabinoside (AraC, 19.7%) (Figure 1).
Among synthesized γ-amino acid derivatives 25a–28b, compounds were able to significantly decrease A549 viability in comparison to UC (p < 0.05). Compound 25a reduced viability to 76.6% (p < 0.024), while the 3,5-dichloro derivative 25b showed significantly increased anticancer activity (36.0%) (p < 0.0001). Furthermore, the addition of a 5-Me substituent (26a, b) or 5-Cl substituent (27a, b) did not significantly affect anticancer activity in comparison to that of the primary compounds, while the 5-F analogue 28b showed slightly stronger anticancer activity against A549 cells (Figure 1).

3. Materials and Methods

3.1. 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-d6, δ = 2.50 ppm for 1H and d = 39.5 ppm for 13C). 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.
1-(2-Hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid (1a). To a solution of itaconic acid (6.5 g, 50 mmol) in water (16 mL), o-aminophenol (4.91 g, 45 mmol) was added and the mixture was refluxed for 12 h, then cooled down, and the formed precipitate was filtered off, washed with water, and dried.
Pale brown solid, yield 74.4%, m.p. 178–179 °C (from water).
1H NMR (DMSO-d6, 400 MHz), δ: 2.56–2.72 (m, 2H, CH2CO), 3.34–3.42 (m, 1H, CH), 3.78–3.95 (m, 2H, NCH2), 6.80 (t, J = 7.5 Hz, 1H, Harom), 6.90 (d, J = 8.2 Hz, 1H, Harom), 7.10–7.24 (m, 2H, Harom), 9.56 (s, 1H, OH), 12.71 (s, 1H, COOH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.71, 36.23, 50.96 (CH2CO, CH, NCH2), 116.80, 119.18, 125.50, 128.21, 128.30, 152.69 (Carom), 172.20, 174.37 (2C=O) ppm.
IR (KBr), νmax: 1633, 1737 (2CO); 3115 (OH) cm−1.
Calcd. for C11H11NO4, %: C 59.73; H 5.01; N 6.33. Found, %: C 60.09; H 5.14; N 6.54.
1-(3,5-Dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid (1b). To a mixture of acid 1a (13.27 g, 60 mmol), conc. HCl (15 mL) and water (120 mL) 30% H2O2 was added dropwise over 5 min, and the mixture was stirred at 50 °C for 2 h. Then, the mixture was cooled down, and the formed precipitate was filtered off and purified by dissolving it in 5% sodium hydroxide solution, filtering and acidifying the filtrate with hydrochloric acid to pH 1–2.
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.
  • Methyl 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylate (2a). Light grey solid, yield 78.8%, m.p. 128–129 °C (from MeOH).
1H NMR (DMSO-d6, 400 MHz), δ: 2.60–2.73 (m, 2H, CH2CO), 3.45–3.53 (m, 1H, CH), 3.68 (s, 3H, OCH3), 3.80–3.93 (m, 2H, NCH2), 6.80 (t, J = 7.6 Hz, 1H, Harom), 6.89 (d, J = 8.3 Hz, 1H, Harom), 7.09–7.19 (m, 2H, Harom), 9.59 (s, 1H, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.55, 36.00, 50.69 (CH2CO, CH, NCH2), 52.11 (OCH3), 116.74, 119.12, 125.33, 128.22, 128.316, 152.68 (Carom), 171.84, 173.25 (2C=O) ppm.
IR (KBr), νmax: 1656, 1741 (2CO); 3073 (OH) cm−1.
Calcd. for C12H13NO4, %: C 61.27; H 5.57; N 5.95. Found, %: C 61.03; H 5.80; N 6.13.
  • Methyl 1-(3,5-dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylate (2b).
White solid, yield 78.8%, m.p. 149–150 °C (from MeOH).
1H NMR (DMSO-d6, 400 MHz), δ: 2.59–2.71 (m, 2H, CH2CO), 3.41–3.49 (m, 1H, CH), 3.69 (s, 3H, OCH3), 3.81–3.87 (m, 2H, NCH2), 7.27 (s, 1H, Harom), 7.49 (s, 1H, Harom), 9.61 (s, 1H, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.56, 35.98, 50.70 (CH2CO, CH, NCH2), 52.18 (OCH3), 122.38, 122.49, 126.81, 128.13, 128.34, 148.52 (Carom), 172.04, 173.10 (2C=O) ppm.
IR (KBr), νmax: 1659, 1753 (2CO); 3172 (OH) cm−1.
Calcd. for C12H11Cl2NO4, %: C 47.39; H 3.65; N 4.61. Found, %: C 47.44; H 3.61; N 4.66.
  • 1-(2-Hydroxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (3).
A mixture of ester 2 (8.5 g, 35 mmol), hydrazine monohydrate (5 g, 10 mmol), and propan-2-ol (150 mL) was heated at reflux for 2.5 h and then cooled down. The formed precipitate was filtered and washed with propan-2-ol and dried.
Brown solid, yield 88.5%, m.p. 188–189 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: 2.51–2.62 (m, 2H, CH2CO), 3.15–3.26 (m, 1H, CH), 3.66–3.83 (m, 2H, NCH2), 4.32 (s, 2H, NH2), 6.81 (t, J = 7.5 Hz, 1H, Harom), 6.91 (d, J = 8.2 Hz, 1H, Harom), 7.10–7.16 (m, 2H, Harom), 9.29 (s, 1H, NH), 9.58 (s, 1H, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 34.38, 35.59, 51.62 (CH2CO, CH, NCH2), 116.77, 119.18, 125.47, 128.26, 128.36, 152.77 (Carom), 171.77, 172.43 (2C=O) ppm.
IR (KBr), νmax: 1682 (2CO); 2967–3330 (OH, NH, NH2) cm−1.
Calcd. for C11H13N3O3, %: C 56.16; H 5.57; N 17.86. Found, %: C 56.52; H 5.66; N 18.11.
  • 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.
  • N′-benzylidene-1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (4).
White solid, yield 73%, m.p. 224–225 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.62–2.83 (m, 2H, CH2CO), 3.36–3.45 (m, 0.35H, CH), 3.78–4.18 (m, 2H, NCH2, 0.65H, CH), 6.82 (t, J = 7.5 Hz, 1H, NH), 6.87–6.95 (m, 1H, Harom), 7.08–7.19 (m, 2H, Harom), 7.35–7.51 (m, 3H, Harom), 7.62–7.74 (m, 2H, Harom), 8.03, 8,22 (2s, 1H, CH=N), 9.58, 9.60 (2s, 1H, OH), 11.56, 11.61 (2s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.29, 34.15, 34.17, 36.24, 51.08, 51.43 (CH2CO, CH, NCH2), 116.74, 116.79, 119.16, 125.45, 125.55, 126.82, 127.10, 128.22, 128.28, 128.83, 128.87, 129.90, 130.13, 134.14, 143.57, 147.03, 152.66, 152.72, 168.75, 172.22, 172.41, 173.68 (Carom, N=CH, 2C=O) ppm.
IR (KBr), νmax: 1588 (C=N); 1671 (2CO); 2959–3183 (OH, NH) cm−1.
Calcd. for C18H17N3O3, %: C 66.86; H 5.30; N 13.00. Found, %: C 66.92; H 5.23; N 13.08.
  • N′-(4-chlorobenzylidene)-1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (5).
White solid, yield 60%, m.p. 209–210 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.55–2.78 (m, 2H, CH2CO), 3.35–3.42 (m, 0.35H, CH), 3.76–4.18 (m, 2H, NCH2, 0.65H, CH), 6.82 (t, J = 7.5 Hz, 1H, Harom), 6.91 (d, J = 8.0 Hz, 1H, Harom), 7.03–7.26 (m, 2H, Harom), 7.50 (t, J = 8.1 Hz, 2H, Harom), 7.63–7.81 (m, 2H, Harom), 8.01, 8.20 (2s, 1H, CH=N), 9.58, 9.60 (2s, 1H, OH), 11.62, 11.67 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1593 (C=N); 1671 (2CO); 2953–3074 (OH, NH) cm−1.
Calcd. for C18H16ClN3O3, %: C 60.43; H 4.51; N 11.74. Found, %: C 60.35; H 4.46; N 11.73.
  • N′-(4-bromobenzylidene)-1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (6).
White solid, yield 90%, m.p. 217–218 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.59–2.85 (m, 2H, CH2CO), 3.35–3.44 (m, 0.35H, CH), 3.76–4.19 (m, 2H, NCH2, 0.65H, CH), 6.82 (t, J = 7.5 Hz, 1H, Harom), 6.91 (d, J = 8.0 Hz, 1H, Harom), 6.97–7.34 (m, 2H, Harom), 7.38–7.87 (m, 4H, Harom), 8.00, 8.19 (2s, 1H, CH=N); 9.58, 9.59 (2s, 1H, OH), 11.62; 11.68 (2s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.29, 34.10, 34.14, 36.22, 51.01, 51.39 (CH2CO, CH, NCH2), 116.74, 116.78, 119.15, 123.36, 125.44, 125.53, 128.21, 128.27, 128.70, 131.84, 133.44, 142.39, 145.78, 152.64, 152.71,168.83, 172.18, 172.35, 173.74 (Carom, N=CH, 2C=O) ppm.
IR (KBr), νmax: 1592 (C=N); 1671 (2CO); 2954–3076 (OH, NH) cm−1.
Calcd. for C18H16BrN3O3, %: C 53.75; H 4.01; N 10.45. Found, %: C 53.69; H 3.93; N 10.39.
  • 1-(2-Hydroxyphenyl)-N′-(4-nitrobenzylidene)-5-oxopyrrolidine-3-carbohydrazide (7).
Light yellow solid, yield 83.6%, m.p. 210–211 °C (from 2-PrOH).
1H NMR (400 MHz, DMSO-d6), δ: Z/E 65/35, 2.63–2.82 (m, 2H, CH2CO), 3.39–3.48 (m, 0.35H, CH), 3.79–4.19 (m, 2H, NCH2, 0.65H, CH), 6.82 (t, J = 7.6 Hz, 1H, Harom), 6.91 (d, J = 8.2 Hz, 1H, Harom), 7.05–7.22 (m, 2H, Harom),7.88–8.03 (m, 2H, Harom), 8.12 (s, 0.65H, HCN); 8.18–8.37 (m, 2H, Harom, 0.35H, CH=N), 9.61 (s, 1H, OH), 11.85; 11.90 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1586 (C=N); 1673 (2CO); 2926–3079 (OH, NH) cm−1.
Calcd. for C18H16N4O5, %: C 58.69; H 4.38; N 15.21. Found, %: C 58.59; H 4.41; N 15.14.
  • N′-(4-(dimethylamino)benzylidene)-1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (8).
Pale yellow solid, yield 78.1%, m.p. 170–171 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.59–2.80 (m, 2H, CH2CO), 2.95, 2.96 (2s, 6H, N(CH3)2), 3.75–4.10 (m, 2H, NCH2, 1H, CH), 6.73 (t, J = 7.6 Hz, 2H, Harom), 6.82 (t, J = 7.5 Hz, 1H, Harom), 6.86–6.96 (m, 1H, Harom), 7.10–7.19 (m, 2H, Harom), 7.44–7.54 (m, 2H, Harom), 7.89, 8.06 (2s, 1H, CH=N), 9.57, 9.60 (2s, 1H, OH), 11.27, 11.30 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1599 (C=N); 1659 (2CO); 2973–3442 (OH, NH) cm−1.
Calcd. for C20H22N4O3, %: C 65.56; H 6.05; N 15.29. Found, %: C 65.49; H 6.09; N 15.37.
  • 1-(2-Hydroxyphenyl)-N′-(4-methoxybenzylidene)-5-oxopyrrolidine-3-carbohydrazide (9).
Light pink solid, yield 78%, m.p. 157–158 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.58–2.87 (m, 2H, CH2CO), 3.36–3.41 (m, 0.35H, CH), 3.78, 3.80 (2s, 3H, OCH3), 3.82–4.15 (m, 2H, NCH2, 0.65H, CH), 6.82 (t, J = 7.5 Hz, 1H, Harom), 6.89–7.20 (m, 5H, Harom), 7.63 (t, J = 8.8 Hz, 1H, Harom), 7.97, 8.15 (2s, 1H, CH=N), 9.58, 9.60 (2s, 1H, OH), 11.43, 11.48 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1592 (C=N); 1668 (2CO); 2952–3473 (OH, NH) cm−1.
Calcd. for C19H19N3O4, %: C 64.58; H 5.42; N 11.89. Found, %: C 64.50; H 5.48; N 11.81.
  • 1-(2-Hydroxyphenyl)-N′-(2,4-dimethoxybenzylidene)-5-oxopyrrolidine-3-carbohydrazide (10).
White solid, yield 66.6%, m.p. 149–150 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.58–2.80 (m, 2H, CH2CO), 3.28–3.34 (m, 0.35H, CH), 3.80, 3.81, 3.83, 3.85 (4s, 6H, 2OCH3), 3.84–4.14 (m, 2H, NCH2, 0.65H, CH), 6.55–6.66 (m, 2H, Harom), 6.76–7.96 (m, 2H, Harom), 7.05–7.22 (m, 2H, Harom), 7.74 (d, J = 8.5 Hz, 1H, Harom), 8.27, 8.46 (2s, 1H, CH=N), 9.58 (s, 1H, OH), 11.37, 11.46 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1589 (C=N); 1670 (2CO); 2841–3182 (OH, NH) cm−1.
Calcd. for C20H21N3O5, %: C 62.65; H 5.52; N 10.96. Found, %: C 62.58; H 5.52; N 10.89.
  • 1-(2-Hydroxyphenyl)-N′-(2,3,4-trimethoxybenzylidene)-5-oxopyrrolidine-3-carbohydrazide (11).
White solid, yield 87.2%, m.p. 234–235 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35 2.61–2.80 (m, 2H, CH2CO), 3.35–3.41 (m, 0.35H, CH), 3.76, 3.77, 3.81, 3.82, 3.84 (5s, 9H, 3OCH3), 3.86–4.15 (m, 2H, NCH2, 0.65H, CH), 6.78–6.95 (m, 3H, Harom), 7.09–7.19 (m, 2H, Harom), 7.56 (dd, J = 8.8, 5.6 Hz, 1H, Harom), 8.21, 8.37 (2s, 1H, CH=N), 9.57, 9.59 (2s, 1H, OH), 11.43, 11.55 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1590 (C=N); 1669 (2CO); 2948–3322 (OH, NH) cm−1.
Calcd. for C21H23N3O6, %: C 61.01; H 5.61; N 10.16. Found, %: C 60.97; H 5.68; N 10.12.
  • 1-(2-Hydroxyphenyl)-N′-(3,4,5-trimethoxybenzylidene)-5-oxopyrrolidine-3-carbohydrazide (12).
Light grey solid, yield 82.3%, m.p. 166–167 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35 2.61–2.84 (m, 2H, CH2CO), 3.37–3.42 (m, 0.35H, CH), 3.68, 3.70 (2s, 3H, OCH3), 33.80, 3.82, (2s, 6H, 2OCH3), 3.83–4.16 (m, 2H, NCH2, 0.65H, CH), 6.82 (q, J = 7.2 Hz, 1H, Harom), 6.87–6.94 (m, 1H, Harom), 6.98, 7.01 (2s, 2H, Harom), 7.08–7.20 (m, 2H, Harom), 7.93, 8.13 (2s, 1H, CH=N), 9.57, 9.61 (2s, 1H, OH), 11.58, 11.59 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1580 (C=N); 1663 (CO); 2940–3540 (OH, NH) cm−1.
Calcd. for C21H23N3O6, %: C 61.01; H 5.61; N 10.16. Found, %: C 61.11; H 5.68; N 10.09.
  • 1-(2-Hydroxyphenyl)-N′-(naphth-1-ylmethylene)-5-oxopyrrolidine-3-carbohydrazide (13).
White solid, yield 83.1%, m.p. 184–186 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.58–2.94 (m, 2H, CH2CO), 3.39–3.51 (m, 0.4H, CH), 3.76–4.16 (m, 2H, NCH2), 4.16–4.32 (m, 0.6H, CH), 6.83 (q, J = 7.3 Hz, 1H, Harom), 6.92 (t, J = 8.0 Hz, 1H, Harom), 7.00–7.30 (m, 2H, Harom), 7.44–8.20 (m, 6H, Harom), 8.59, 8.87 (2d, J = 8.5 Hz, 1H, Harom), 8.73, 8.83 (2s, 1H, CH=N), 9.60 (s, 1H, OH), 11.61, 11.73 (2s, 1H, NH) ppm.
IR (KBr), νmax: 1587 (C=N); 1665 (2CO); 2969–3176 (OH, NH) cm−1.
Calcd. for C22H19N3O3, %: C 70.76; H 5.13; N 11.25. Found, %: C 70.70; H 5.19; N 11.18.
  • 1-(2-Hydroxyphenyl)-5-oxo-N′-(thien-2-ylmethylene)pyrrolidine-3-carbohydrazide (14).
Light yellow solid, yield 85%, m.p. 224–225 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35 2.60–2.81 (m, 2H, CH2CO), 3.34–3.41 (m, 0.35H, CH), 3.73–4.08 (m, 2H, NCH2, 0,65H, CH), 6.77–6.96 (m, 2H, Harom), 7.01–7.25 (m, 3H, Harom), 7.41, 7.46 (2d, J = 3.2 Hz, 1H, Harom), 7.61, 7.66 (2d, J = 5.0 Hz, 1H, Harom), 8.19, 8.42 (2s, 1H, CH=N), 9.58, 9.59 (2s, 1H, OH), 11.54, 11.56 (2s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.14, 34.12, 34.24, 35.22, 51.05,51.42 (CH2CO, CH, NCH2),116.73, 116.77, 119.16, 125.44, 125.50, 127.86, 127.98, 128.26, 128.43, 129.01, 130.38, 13,108, 138.57, 142.20, 152.71, 168.59, 172.34, 173.26 (Carom, N=CH, 2C=O) ppm.
IR (KBr), νmax: 1589 (C=N); 1670 (2CO); 2883–3097 (OH, NH) cm−1.
HRMS (ESI) for C16H15N3O3S + H+, calcd. 330.0912, found 330.0908 [M + H+].
Calcd. for C16H15N3O3S, %: C 58.35; H 4.59; N 12.76. Found, %: C 58.49; H 4.62; N 12.79.
  • 1-(2-Hydroxyphenyl)-5-oxo-N′-(5-nitrothien-2-ylmethylene)pyrrolidine-3-carbohydrazide (15).
White solid, yield 82.9%, m.p. 172–173 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35 2.62–2.81 (m, 2H, CH2CO), 3.38–3.45 (m, 0.35H, CH), 3.75–4.10 (m, 2H, NCH2, 0.65H, CH), 6.82 (t, J = 7.5 Hz, 1H, Harom), 6.91 (d, J = 8.1 Hz, 1H, Harom), 7.04–7.26 (m, 2H, Harom), 7.04–7.26 (m, 2H, Harom), 7.52, 7.56 (2d, J = 4.2 Hz, 1H, Harom), 8.11 (dd, J = 6.6, 4.5 Hz, 1H, Harom), 8.19, 8.47 (2s, 1H, CH=N), 9.58, 9.60 (2s, 1H, OH), 11.96 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.98, 34.05, 36.28, 50.93, 51.24 (CH2CO, CH, NCH2), 116.73, 116.79, 119.17, 125.46, 128.29, 129.20, 130.65, 136.83. 140.56, 146.53, 146.58, 150.53, 150.88, 152.72, 169.25, 172.06, 172.22, 173.92 (Carom, N=CH, 2C=O) ppm.
IR (KBr), νmax: 1565 (C=N); 1663 (2CO); 2965–3183 (OH, NH) cm−1.
HRMS (ESI) for C16H14N4O5S + Na+, calcd. 397.0583, found 397.0577 [M + Na+].
Calcd. For C16H14N4O5S, %: C 51.33; H 3.77; N 14.97. Found, %: C 51.40; H 3.85; N 14.89.
  • N′-(1-(4-aminophenyl)ethylidene)-1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (16)
A mixture of hydrazide 3 (1.18 g, 5 mmol), 1-(4-aminophenyl)ethan-1-one (0.68 g, 5 mmol) and propan-2-ol (15 mL) was heated at reflux for 15 h, then cooled down, and the formed precipitate was filtered of, washed with propan-2-ol, and dried.
White solid, yield 72.2%, m.p. 226–227 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.15, 2.17 (2s, 3H, CH3), 2.59–2.81 (m, 2H, CH2CO), 3.52–3.61 (m, 0.35H, CH), 3.72–4.19 (m, 2H, NCH2, 0.65H, CH), 5.43, 5.47 (2s, 2H, NH2), 6.55 (d, J = 8.3 Hz, 2H, Harom), 6.74–7.00 (m, 2H, Harom), 7.03–7.24 (m, 2H, Harom), 7.34–7.66 (m, 2H, Harom), 9.57, 9.60 (2s, 1H, OH), 10.34, 10.47 (2s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: Z/E 65/35, 13.24, 13.81 (CH3), 33.39, 34.42, 36.03 (CH2CO, CH), 51.29, 51.84 (NCH2), 113.11, 113.26, 116.72, 116.79, 119.15, 125.02, 125.22, 125.51, 125.57, 127.19, 127.61, 128.19, 128.27, 148.98, 150.01, 150.27, 152.64, 152.72, 153.78 (Carom), 168.80, 172.43, 172.57, 174.02 (C=O) ppm.
IR (KBr), νmax: 1591 (C=N); 1645; 1666 (CO); 2960–3464 (OH, NH) cm−1.
Calcd. for C19H20N4O3, %: C 64.76; H 5.72; N 15.90. Found, %: C 64.69; H 5.80; N 15.82.
  • 1-(2-Hydroxyphenyl)-5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide (17).
White solid, yield 72.6%, m.p. 196–197 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: Z/E 65/35, 2.65–2.84 (m, 2H, CH2CO), 3.83–4.37 (m, 2H, NCH2, 1H, CH), 6.82 (t, J = 7.6 Hz, 1H, Harom), 6.86–6.96 (m, 2H, Harom), 6.98–7.18 (m, 3H, Harom), 7.38 (t, J = 7.9 Hz, 1H, Harom), 8,07, 8.13 (2s, 1H, Harom), 9.60 (s, 1H, OH), 10.81 (s, 1H, NH), 11.35 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 33.22, 34.63 (CH2CO, CH), 51.12 (NCH2), 110.63, 115.21, 116.76, 119.17, 121.71, 125.46, 126.09, 126.41, 128.32, 132.60, 132.96, 143.85, 152.75, 164.58, 172.11 (Carom, 3C=O) ppm.
IR (KBr), νmax: 1601 (C=N); 1694, 1716, 1732 (3CO); 3023–3380 (OH, NH) cm−1.
Calcd. for C19H16N4O4, %: C 62.63; H 4.43; N 15.38. Found, %: C 62.57; H 4.51; N 15.29.
  • 1-(2-Hydroxyphenyl)-N-(2,5-dimethyl-1H-pyrrol-1-yl)-5-oxopyrrolidine-3-carboxamide (18).
To a solution of hydrazide 3 (0.7 g, 3 mmol) in propan-2-ol (15 mL), hexane-2,5-dione (1.10 g, 9.5 mmol) and acetic acid (3 drops) were added, and the mixture was refluxed for 4 h. After completion of the reaction (TLC), the cooled mixture was diluted with water (25 mL) and stirred for 15 min. The formed precipitate was filtered of, washed with propan-2-ol, and dried.
Light yellow solid, yield 94.4%, m.p. 197–198 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: 1.98, 2.01 (2s, 6H, 2CH3), 2.71 (d, J = 8.4 Hz, 2H, CH2CO), 3.46–3.58 (m, 1H, CH), 3.80–4.03 (m, 2H, NCH2), 5.66 (s, 2H, Hpyrr), 6.83 (t, J = 7.6 Hz, 1H, Harom), 6.92 (d, J = 8.3 Hz, 1H, Harom), 7.08–7.19 (m, 2H, Harom), 9.62 (s, 1H, OH), 10.87 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 10.93, 10.97 (2CH3); 33.76, 35.31 (CH2CO, CH), 51.42 (NCH2), 103.10, 116.72, 119.18, 125.35, 126.75, 126.78, 128.37, 152.77 (Carom), 171.87, 172.06 (2C=O) ppm.
IR (KBr), νmax: 1666 (2CO); 2978–3272 (OH, NH) cm−1.
Calcd. for C17H19N3O3, %: C 65.16; H 6.11; N 13.41. Found, %: C 65.24; H 6.02; N 13.37.
  • 1-(2-Hydroxyphenyl)-4-(3,5-dimethyl-1H-pyrazol-1-carbonyl)pyrrolidin-2-one (19).
To a solution of hydrazide 3 (0.7 g, 3 mmol) in propan-2-ol (15 mL), pentane-2,4-dione (0.6 g, 6 mmol) and hydrochloric acid (3 drops) were added, and the mixture was refluxed for 2.5 h. After completion of the reaction (TLC), the cooled mixture was diluted with water (25 mL) and stirred for 15 min. The formed precipitate was filtered of, washed with propan-2-ol, and dried.
White solid, yield 68.3%, m.p. 139–140 °C (from 2-PrOH).
1H NMR (DMSO-d6, 400 MHz), δ: 2.19 (s, 3H, CH3), 2.50 (s, 3H, CH3, overlaps with the signal of the DMSO-d6), 2.64–2.86 (m, 2H, CH2CO); 3.82–4.09 (m, 2H, NCH2), 4.15–4.85 (m, 1H, CH), 6.22 (s, 1H, Hpyr), 6.81 (t, J = 7.6 Hz, 1H, Harom), 6.90 (d, J = 7.9 Hz, 1H, Harom), 7.08–7.21 (m, 2H, Harom), 9.59 (s, 1H, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 13.55, 14.08 (CH3), 33.46, 36.74 (CH2CO, CH), 51.21 (NCH2), 111.52 (CHpyr), 116.72, 119.14, 125.33, 128.32, 143.82, 152.07, 152.70 (Carom, Cpyr), 171.84, 172.60 (2C=O) ppm.
IR (KBr), νmax: 1584 (C=N); 1660; 1725 (CO); 2932–3426 (OH) cm−1.
Calcd. for C16H17N3O3, %: C 64.20; H 5.72; N 14.04. Found, %: C 64.11; H 5.80; N 13.97.
  • 1-(2-Hydroxyphenyl)-4-(5,6-diphenyl-1,2,4-triazin-3-yl)pyrrolidine-2-one (20)
A mixture of hydrazide 3 (2.35 g, 10 mmol), 1,2-diphenylethane-1,2-dione (2.10 g, 10 mmol), ammonium acetate (7.71 g, 100 mmol), and glacial acetic acid (40 mL) was heated at reflux for 24 h, then cooled down, and diluted with water (60 mL). The obtained oily mass was washed with hot water (2 × 60 mL), then poured with aqueous 5% hydrochloric acid solution (50 mL), and refluxed for 5 min. After cooling, the formed precipitate was filtered off, washed with water, and dried. To purify the solid, the crystalline product was dissolved in acetone (10 mL), and the solution was slowly poured into hexane (100 mL) in a thin stream. The formed solid was filtered off and washed with hexane.
Light brown solid, yield 58.8%, m.p. 163–164 °C (acetone:hexane, 1:10).
1H NMR (DMSO-d6, 400 MHz), δ: 3.03 (d, J = 8.2 Hz, 2H, CH2CO), 4.15 (dd, J = 9.2, 5.4 Hz, 1H, NCH2), 4.25–4.38 (m, 1H, NCH2, 1H, CH), 6.83 (t, J = 7.6 Hz, 1H, Harom), 6.93 (d, J = 8.1 Hz, 1H, Harom), 7.14 (t, J = 7.8 Hz, 1H, Harom), 7.22 (d, J = 7.7 Hz, 1H, Harom), 7.37–7.55 (m, 10H, Harom), 9.63 (s, 1H, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 35.96, 38.15 (CH2CO, CH), 53.22 (NCH2), 116.81, 119.13, 125.69, 128.20, 128.42, 128.53, 129.33, 129.47, 129.74, 130.68, 135.39, 135.46, 152.71, 155.87, 156.05, 167.07 (Carom), 172.51 (C=O) ppm.
IR (KBr), νmax: 1585 (C=N); 1656 (CO); 3067 (OH) cm−1.
Calcd. for C25H20N4O2, %: C 73.51; H 4.94; N 13.72. Found, %: C 73.44; H 5.02; N 13.68.
  • General procedure for the preparation of benzimidazoles 21–24a, b.
A mixture of acid 1a or 1b (10 mmol), the corresponding o-phenylenediamine (30 mmol), and 6 N hydrochloric acid (30 mL) was refluxed for 24 h, then cooled down, and treated with aqueous 15% ammonium hydroxide solution to pH 8. The formed precipitate was filtered off, washed with water, and dried. The target benzimidazoles 21–24a, b were recrystallized from propan-2-ol.
  • 4-(1H-benzo[d]imidazol-2-yl)-1-(2-hydroxyphenyl)pyrrolidine-2-one (21a).
Dark yellow solid, yield 97.6%, m.p. 214–215 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.62–3.08 (m, 2H, CH2CO), 3.77–4.28 (m, 3H, NCH2, CH), 6.84 (t, J = 7.5 Hz, 1H, Harom), 6.95 (d, J = 8.3 Hz, 1H, Harom), 7.15–7.22 (m, 4H, Harom), 7.50–7.58 (m, 2H, Harom), 10.49 (s, 1H, OH), 12.58 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 32.12, 36.67 (CH2CO, CH), 53.25 (NCH2), 114.73, 115.16, 116.97, 119.03, 121.89, 125.23, 128.48, 128.61, 153.37, 155.96 (Carom), 172.26 (C=O) ppm.
IR (KBr), νmax: 1598 (C=N); 1675 (CO); 2561–3175 (OH, NH) cm−1.
Calcd. for C17H15N3O2, %: C 69.61; H 5.15; N 14.33. Found, %: C 69.66; H 5.21; N 14.37.
  • 4-(1H-benzo[d]imidazol-2-yl)-1-(3,5-dichloro-2-hydroxyphenyl)pyrrolidin-2-one (21b).
Light brown solid, yield 94.1%, m.p. 231–232 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.76–2.86 (m, 1H, CH2CO), 3.01–3.11 (m, 1H, CH2CO), 4.06–4.34 (m, 3H, NCH2, CH), 7.32–7.44 (m, 3H, Harom), 7.46–7.62 (m, 1H, Harom), 7.64–7.72 (m, 2H, Harom), 13.08 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 32.08, 36.27 (CH2CO, CH), 53.28 (NCH2), 114.25, 121.91, 122.27, 123.99, 127.34, 127.42, 128.59, 134.21, 140.01, 149.52, 155.36 (Carom), 172.06 (C=O) ppm.
IR (KBr), νmax: 1578 (C=N); 1680 (CO); 3065–3180 (OH, NH) cm−1.
Calcd. for C17H13Cl2N3O2, %: C 56.37; H 3.62; N 11.60. Found, %: C 56.32; H 3.67; N 11.55.
  • 4-(5-Methyl-1H-benzo[d]imidazol-2-yl)-1-(2-hydroxyphenyl)pyrrolidine-2-one (22a).
White solid, yield 90.8%, m.p. 229–230 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.42 (s, 3H, CH3), 2.72 (dd, J = 16,4, 4.8 Hz, 1H, CH2CO), 3.00 (dd, J = 16.4, 8.0 Hz, 1H, CH2CO), 3.94–4.19 (m, 3H, NCH2, CH), 6.83 (t, J = 7.5 Hz, 1H, Harom), 6.95 (d, J = 8.3 Hz, 1H, Harom), 7.06 (d, J = 8.2 Hz, 1H, Harom), 7.11–7.25 (m, 2H, Harom), 7.36 (s, 1H, Harom), 7.45 (d, J = 8.1 Hz, 1H, Harom), 9.26 (br. s, 1H, OH), 11.36 (br. s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 21.23 (CH3), 31.91, 36.59 (CH2CO, CH), 53.13 (NCH2), 114.00, 114.46, 116.98, 119.01, 123.77, 125.16, 128.50, 128.63, 131.64, 135.60, 137.12, 153.41, 155.39 (Carom), 172.11 (C=O) ppm.
IR (KBr), νmax: 1597 (C=N); 1672 (CO); 2917–3121 (OH, NH) cm−1.
Calcd. for C18H17N3O2, %: C 70.34; H 5.58; N 13.67. Found, %: C 70.26; H 5.61; N 13.61.
  • 1-(3,5-Dichloro-2-hydroxyphenyl)-4-(5-methyl-1H-benzo[d]imidazol-2-yl)pyrrolidin-2-one (22b).
Light yellow solid, yield 88.9%, m.p. 238–239 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.47 (s, 3H, CH3), 2.85–3.09 (m, 2H, CH2CO), 3.98–4.48 (m, 3H, NCH2, CH), 7.02–7.90 (m, 5H, Harom), 13.37 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 21.15 (CH3), 30.50, 35.99 (CH2CO, CH), 52.44 (NCH2), 113.50, 113.71, 122.01, 122.31, 126.14, 127.28, 127.48, 128.58, 131.03, 132.98, 134.49, 149.37, 154.39 (Carom), 171.89 (C=O) ppm.
IR (KBr), νmax: 1573 (C=N); 1684 (CO); 3055–3240 (OH, NH) cm−1.
Calcd. for C18H15Cl2N3O2, %: C 57.46; H 4.02; N 11.17. Found, %: C 57.52; H 3.98; N 11.19.
  • 4-(5-Chloro-1H-benzo[d]imidazol-2-yl)-1-(2-hydroxyphenyl)pyrrolidine-2-one (23a).
Brown solid, yield 96.8%, m.p. 159–160 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.74–3.07 (m, 2H, CH2CO), 3.97–4.19 (m, 3H, NCH2, CH), 6.83 (t, J = 7.5 Hz, 1H, Harom), 6.95 (d, J = 8.0 Hz, 1H, Harom), 7.13–7.20 (m, 2H, Harom), 7.23–7.29 (m, 1H, Harom), 7.59 (d, J = 8.6 Hz, 1H, Harom), 7.64 (s, 1H, Harom), 9.39 (br. s, 1H, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 31.79, 36.15 (CH2CO, CH), 52.98 (NCH2), 114.47, 115.81, 116.88, 119.09, 122.61, 124.92, 125.25, 126.72, 128.41, 128.51, 135.94, 138.32, 152.79, 153.10, 156.97 (Carom), 172.05 (C=O) ppm.
IR (KBr), νmax: 1600 (C=N); 1675 (CO); 2564–3098 (OH, NH) cm−1.
Calcd. for C17H14ClN3O2, %: C 62.30; H 4.31; N 10.82. Found, %: C 62.22; H 4.40; N 10.90.
  • 4-(5-Chloro-1H-benzo[d]imidazol-2-yl)-1-(3,5-dichloro-2-hydroxyphenyl)pyrrolidin-2-one (23b).
Light grey solid, yield 93.4%, m.p. 191–192 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.98–3.02 (m, 2H, CH2CO), 4.12–4.18 (m, 2H, NCH2), 4.40–4.48 (m, 1H, CH), 7.22 (s, 1H, Harom), 7.40–7.43 (m, 1H, Harom), 7.45–7.50 (m, 2H, Harom), 7.71–7.78 (m, 1H, Harom), 9.25 (br. s, 1H, OH), 12.47 (br. s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 30.45, 35.80 (CH2CO, CH), 52.41 (NCH2), 113.87, 115.60, 122.14, 122.33, 125.01, 127.26, 126.56, 128.58, 138.64, 140.15, 149.23, 156.11 (Carom), 171.87 (C=O) ppm.
IR (KBr), νmax: 1606 (C=N); 1702 (CO); 3054–3298 (OH, NH) cm−1.
Calcd. for C17H12Cl3N3O2, %: C 51.48; H 3.05; N 10.59. Found, %: C 51.51; H 3.09; N 10.62.
  • 4-(5-Fluoro-1H-benzo[d]imidazol-2-yl)-1-(2-hydroxyphenyl)pyrrolidine-2-one (24a).
Grey solid, yield 87%, m.p. 159–160 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.73–2.77 (m, 1H, CH2CO), 2.82–3.00 (m, 1H, CH2CO), 3.98–4.16 (m, 3H, NCH2, CH), 6.83 (t, J = 7.5 Hz, 1H, Harom), 6.91–7.22 (m, 4H, Harom), 7.34 (s, 1H, Harom), 7.46–7.62 (m, 1H, Harom), 10.16, 10.19 (2s, 1H, OH), 12.72 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 32.09, 36.41 (CH2CO, CH), 53.18 (NCH2), 109.58, 111.91, 112.03, 116.91, 119.09, 125.31, 128.40, 128.52, 153.16, 156.80, 157.67, 158.64 (Carom), 172.26 (C=O) ppm.
IR (KBr), νmax: 1600 (C=N); 1675 (CO); 2564–3098 (OH, NH) cm−1.
Calcd. for C17H14FN3O2, %: C 65.59; H 4.53; N 13.50. Found, %: C 65.64; H 4.54; N 13.46.
  • 1-(3,5-Dichloro-2-hydroxyphenyl)-4-(5-fluoro-1H-benzo[d]imidazol-2-yl)pyrrolidin-2-one (24b).
Dark yellow solid, yield 89.5%, m.p. 183–184 °C.
1H NMR (DMSO-d6, 400 MHz), δ: 2.57–2.68 (m, 1H, CH2CO), 2.98–3.08 (m, 1H, CH2CO), 3.93–4.20 (m, 3H, NCH2, CH), 7.01–7.15 (m, 1H, Harom), 7.24–7.47 (m, 2H, Harom), 7.48–7.64 (m, 2H, Harom), 12.40 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 32.32, 36.78 (CH2CO, CH), 52.96 (NCH2), 110.08, 110.34, 121.84, 122.18, 127.40, 127.46, 128.54, 149.61, 157.67, 158.59 (d, JC–F = 235.3 Hz) (Carom), 172.54 (C=O) ppm.
IR (KBr), νmax: 1601 (C=N); 1705 (CO); 3051–3290 (OH, NH) cm−1.
HRMS (ESI) for C17H12Cl2FN3O2 + H+, calcd. 380.0369, found 380.0365 [M + H+].
Calcd. for C17H12Cl2FN3O2, %: C 53.71; H 3.18; N 5.00. Found, %: C 53.66; H 3.15; N 4.96.
  • General method for the preparation of butanoic acids 25–27a, b and 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.
  • 3-(1H-benzo[d]imidazol-2-yl)-4-((2-hydroxyphenyl)amino)butanoic acid (25a).
Dark brown solid, yield 92%, m.p. 185 °C (decomp.).
1H NMR (DMSO-d6, 400 MHz), δ: 2.72–3.02 (m, 2H, CH2CO), 3.26–3.76 (m, 3H, NHCH2, CH), 6.28–6.75 (m, 2H, Harom), 7.09–7.17 (m, 4H, Harom), 7.48–7.58 (m, 2H, Harom), 11.70 (br. s, 3H, NH, 2OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 35.66, 36.88 (CH2CO, CH), 46.60 (NHCH2), 109.79, 113.49, 114.68, 115.96, 117.00, 118.98, 119.72, 121.19, 121.84, 128.49, 128.61, 136.99, 144.08, 153.46, 156.43 (Carom), 173.55 (C=O) ppm.
IR (KBr), νmax: 1579 (C=N); 1673 (CO); 2927–3369 (OH, NH) cm−1.
Calcd. for C17H17N3O3, %: C 65.58; H 5.50; N 13.50. Found, %: C 65.53; H 5.54; N 13.56.
  • 3-(1H-benzo[d]imidazol-2-yl)-4-((3,5-dichloro-2-hydroxyphenyl)amino)butanoic acid (25b).
Brown solid, yield 92.9%, m.p. 199 °C (decomp.).
1H NMR (DMSO-d6, 400 MHz), δ: 2.71–2.99 (m, 2H, CH2CO), 3.26–3.74 (m, 3H, NHCH2, CH), 5.62 (s, 1H, NH), 6.25–6.75 (m, 2H, Harom), 7.09–7.18 (m, 2H, Harom), 7.49–7.53 (m, 2H, Harom), 12.30 (br. s, 2H, NH, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 35.01, 35.78 (CH2CO, CH), 46.21 (NHCH2), 107.78, 114.56, 120.46, 121.33, 124.76, 138.47, 140.63, 155.70 (Carom), 173.14 (C=O) ppm.
IR (KBr), νmax: 1607 (C=N); 1711 (CO); 3062–3298 (OH, NH) cm−1.
Calcd. for C17H15Cl2N3O3, %: C 53.70; H 3.98; N 11.05. Found, %: C 53.78; H 4.01; N 11.06.
  • 4-((2-Hydroxyphenyl)amino)-3-(5-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid (26a).
Light orange solid, yield 96.4%, m.p. 160 °C (decomp.).
1H NMR (DMSO-d6, 400 MHz), δ: 2.39, 2.41 (2s, 3H, CH3), 2.64–3.02 (m, 2H, CH2CO), 3.19–3.75 (m, 3H, NHCH2, CH), 4.83 (br. s, 1H, NH), 6.30–7.57 (m, 7H, Harom), 9.22 (br. s, 1H, OH), 12.27 (br. s, 2H, NH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 21.28 (CH3), 35.41, 36.91 (CH2CO, CH), 46.63 (NHCH2), 109.77, 113.49, 116.02, 117.02, 119.00, 119.73, 122.62, 123.28, 125.19, 128.52, 128.65, 130.33, 136.92, 144.06, 153.49, 155.61 (Carom), 173.17 (C=O) ppm.
IR (KBr), νmax: 1579 (C=N); 1673 (CO); 2920–3147 (OH, NH) cm−1.
Calcd. for C18H19N3O3, %: C 66.45; H 5.89; N 12.91. Found, %: C 66.39; H 5.83; N 12.97.
  • 4-((3,5-Dichloro-2-hydroxyphenyl)amino)-3-(5-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid (26b).
Grey solid, yield 93.7%, m.p. 177 °C (decomp.).
1H NMR (DMSO-d6, 400 MHz), δ: 2.37 (s, 3H, CH3), 2.50–2.59 (m, 2H, CH2CO), 3.19–3.55 (m, 2H, NHCH2), 3.56–3.76 (m, 1H, CH), 5.92 (br. s, 1H, NH), 6.24–6.70 (m, 2H, Harom), 6.90, 7.33 (2d, 2H, J = 8.5 Hz, Harom), 7.24 (s, 1H, Harom), 9.55 (br. s, 2H, NH, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 23.19 (CH3), 33.43, 36.14 (CH2CO, CH), 46.84 (NHCH2), 107.08, 114.45, 119.86, 122.25, 129.83, 141.57, 157.20 (Carom), 173.97 (C=O) ppm.
IR (KBr), νmax: 1579 (C=N); 1689 (CO); 3104–3378(OH, NH) cm−1.
Calcd. for C18H18Cl2FN3O3, %: C 54.84; H 4.35; N 10.66. Found, %: C 54.89; H 4.30; N 10.70.
  • 3-(5-Chloro-1H-benzo[d]imidazol-2-yl)-4-((2-hydroxyphenyl)amino)butanoic acid (27a).
Brown solid, yield 89.9%, m.p. 169 °C (decomp.).
1H NMR (DMSO-d6, 400 MHz), δ: 2.72–3.01 (m, 2H, CH2CO), 3.25–3.49 (m, 3H, NHCH2), 3.52–3.74 (m, 1H, CH), 4.85 (s, 1H, NH), 6.24–6.70 (m, 3H, Harom), 6.95–7.25 (m, 2H, Harom), 7.49–77.52 (m, 2H, Harom), 9.20, 12,41 (2br. s, 2H, NH, OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 35.47, 35.81 (CH2CO, CH), 46.59 (NHCH2), 109.75, 113.49, 116.04, 119.09, 119.70, 121.22, 121.44, 121.46, 125.64, 128.37, 128.49, 131.19, 136.82, 144.04, 153.09, 157.66 (Carom), 173.08 (C=O) ppm.
IR (KBr), νmax: 1576 (C=N); 1681 (CO); 3069–3360 (OH, NH) cm−1.
Calcd. for C17H16ClN3O3, %: C 59.05; H 4.66; N 12.15. Found, %: C 59.10; H 4.62; N 12.09.
  • 3-(5-Chloro-1H-benzo[d]imidazol-2-yl)-4-((3,5-dichloro-2-hydroxyphenyl)amino)butanoic acid (27b).
Pale grey solid, yield 92.5%, m.p. 187 °C (decomp.).
1H NMR (DMSO-d6, 400 MHz), δ: 2.52–2.71 (m, 2H, CH2CO), 3.32–3.53 (m, 3H, NHCH2), 3.61–3.74 (m, 1H, CH), 5.86 (br. s, 1H, NH), 6.50 (s, 2H, Harom), 7.10, 7.45 (2d, 2H, J = 8.5 Hz, Harom), 7.52 (s, 1H, Harom), 10.47 (br. s, 3H, NH, 2OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 36.16, 39.00 (CH2CO, CH), 46.62 (NHCH2), 107.36, 114.50, 115.48, 120.26, 121.10, 122.74, 125.32, 140.76, 141.25 158.97 (Carom), 173.158 (C=O) ppm.
IR (KBr), νmax: 1589 (C=N); 1706 (CO); 3097–3391 (OH, NH) cm−1.
Calcd. for C17H14Cl3N3O3, %: C 49.24; H 3.40; N 10.13. Found, %: C 49.19; H 3.35; N 10.17.
  • 4-((3,5-Dichloro-2-hydroxyphenyl)amino)-3-(5-fluoro-1H-benzo[d]imidazol-2-yl)butanoic acid (28b).
Light brown solid, yield 94.3%, m.p. 182 °C (decomp.).
1H NMR (DMSO-d6, 400 MHz), δ: 2.59–2.97 (m, 2H, CH2CO), 3.27–3.75 (m, 3H, NHCH2, CH), 5.62 (s, 1H, NH), 6.58–6.63 (m, 1H, Harom), 7.11–7.28 (m, 2H, Harom), 7.43–7.09 (m, 2H, Harom),11.90 (br. s, 3H, NH, 2OH) ppm.
13C NMR (DMSO-d6, 101 MHz), δ: 35.10, 35.67 (CH2CO, CH), 46.19 (NHCH2), 107.74, 109.11, 109.36, 114.54, 120.45, 122.24, 124.75, 127.40, 127.49, 128.52, 138.44, 140.60, 157.11, 157.75, 158.28 (d, JC-F = 234.1 Hz) (Carom), 173.11 (C=O) ppm.
IR (KBr), νmax: 1587 (C=N); 1704 (CO); 3088–3385 (OH, NH) cm−1.
HRMS (ESI) for C17H14Cl2FN3O3 + H+, calcd. 398.0474, found 398.0472 [M + H+].
Calcd. for C17H14Cl2FN3O3, %: C 51.28; H 3.54; N 10.55. Found, %: C 51.23; H 3.52; N 10.57.

3.2. Bacterial Strains and Culture Conditions

The multidrug-resistant and genetically defined isolates were obtained from the ARisolate bank at Centre for Disease Control (CDC, United States). S. aureus TCH 1516 (USA300) was obtained from the American Type Culture Collection and maintained as a laboratory strain [39,40] that was used for in vitro and in vivo pharmacological screening. Prior to the study, all strains were maintained in commercial cryopreservation systems at −80 °C. Bacterial strains were subcultured on Columbia Sheep Blood agar or Tryptic-Soy agar (Becton Dickenson, Franklin Lakes, NJ, USA), while fungi were propagated on Potato-Dextrose agar (Becton Dickenson, Franklin Lakes, NJ, USA). Unless otherwise specified, all antibacterial susceptibility studies were performed in Cation-Adjusted Mueller–Hinton broth (CAMBH) for liquid cultures (Liofilchem, Via Szia, Italy) or MOPS/RPMI media for fungal assays (Becton Dickenson, Franklin Lakes, NJ, USA).

3.3. 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 Gram-negative 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 × 105 CFU/mL (range, 2 × 105 to 8 × 105 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.

3.4. Cell Lines and Culture Conditions

The non-cancerous HSAEC1-KT cells (CRL-4050) were kindly provided by Dr. Arryn Craney (Orlando Health, Orlando, FL, USA) and were maintained in complete SAGM culture media (Lonza, Moristown, NJ, USA) containing growth supplements. The non-small cell human lung carcinoma A549 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (10% FBS) (Gibco, Waltham, MA, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin (P/S). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Cells were fed every 2–3 days (for A549) or every 3 days (HSAEC1-KT) and subcultured upon reaching 70–80% confluence.

3.5. 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 × 104 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% CO2, 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.

3.6. 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.

4. 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/1422-0067/24/9/7966/s1.

Author Contributions

Conceptualization, V.M.; methodology, P.K., R.V., B.G., V.P., J.Š. and R.L.; software, P.K., E.N., A.G.; validation, M.B., P.K., R.V., B.G., V.P., A.G., J.Š. and R.L.; formal analysis, R.V.; investigation, M.B., P.K., R.V., B.G., V.P., R.P., E.N., A.G. and R.L.; resources, R.V. and P.K.; data curation, P.K., R.P. and B.G.; writing—original draft preparation, R.V. and P.K.; writing—review and editing, R.V., B.G., V.P., E.N., A.G., P.K. and R.L.; visualization, P.K. and R.V.; supervision, V.M. and J.Š.; project administration, V.M. and J. Š. All authors have read and agreed to the published version of the manuscript.

Funding

The Doctoral Fund of Kaunas University of Technology No. A-410, approved 26 June 2019.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Ijms 24 07966 sch001 550
Scheme 1. Synthesis of compounds 1–20. 2a R = H; 2b R = Cl; 4 Ar = C6H5, 5 Ar = 4-ClC6H4, 6 Ar = 4-BrC6H4, 7 Ar = 4-O2NC6H4, 8 Ar = 4-Me2NC6H4, 9 Ar = 4-MeOC6H4, 10 Ar = 2,4-di(MeO)C6H3, 11 Ar = 2,3,4-tri(MeO)C6H2, 12 Ar = 3,4,5-tri(MeO)C6H2, 13 Ar = 1-naphthyl, 14 Ar = thien-2-yl, 15 Ar = 5-nitrothien-2-yl. Reagents and conditions: i 6M HCl, H2O, H2O2, 25 °C, 1 h; ii MeOH, H2SO4, Δ, 2 h; 5% Na2CO3; iii N2H4·H2O, 2-PrOH, Δ, 2.5 h; iv ArCHO or CarbCHO, 2-PrOH, Δ, 2 h (for 4–13, 15) or 40 min (for 14) h; v 4′-aminoacetophenone, 2-PrOH, Δ, 15 h; vi isatin, 2-PrOH, Δ, 2 h; vii hexane-2,5-dione, 2-PrOH, AcOH, Δ, 4 h, H2O; viii pentane-2,4-dione, HCl, Δ, 2.5 h, H2O; ix 1,2-diphenyl-1,2-ethanedione, AcOH, NH4OAc, Δ, 24 h, H2O; 5% HCl, Δ, 5 min.
Scheme 1. Synthesis of compounds 1–20. 2a R = H; 2b R = Cl; 4 Ar = C6H5, 5 Ar = 4-ClC6H4, 6 Ar = 4-BrC6H4, 7 Ar = 4-O2NC6H4, 8 Ar = 4-Me2NC6H4, 9 Ar = 4-MeOC6H4, 10 Ar = 2,4-di(MeO)C6H3, 11 Ar = 2,3,4-tri(MeO)C6H2, 12 Ar = 3,4,5-tri(MeO)C6H2, 13 Ar = 1-naphthyl, 14 Ar = thien-2-yl, 15 Ar = 5-nitrothien-2-yl. Reagents and conditions: i 6M HCl, H2O, H2O2, 25 °C, 1 h; ii MeOH, H2SO4, Δ, 2 h; 5% Na2CO3; iii N2H4·H2O, 2-PrOH, Δ, 2.5 h; iv ArCHO or CarbCHO, 2-PrOH, Δ, 2 h (for 4–13, 15) or 40 min (for 14) h; v 4′-aminoacetophenone, 2-PrOH, Δ, 15 h; vi isatin, 2-PrOH, Δ, 2 h; vii hexane-2,5-dione, 2-PrOH, AcOH, Δ, 4 h, H2O; viii pentane-2,4-dione, HCl, Δ, 2.5 h, H2O; ix 1,2-diphenyl-1,2-ethanedione, AcOH, NH4OAc, Δ, 24 h, H2O; 5% HCl, Δ, 5 min.
Ijms 24 07966 sch001
Ijms 24 07966 sch002 550
Scheme 2. Synthesis of benzimidazole derivatives 21–28. a R = H, b R = Cl; 21, 25 R1 = H, 22, 26 R1 = Me, 23, 27 R1 = Cl, 24, 28 R1 = F. Reagents and conditions: x the corresponding benzene-1,2-diamine, 6N HCl, Δ, 24 h; 15% NH4OH to pH 8; xi 20% NaOH, Δ, 4 h; diluted AcOH to pH 6.
Scheme 2. Synthesis of benzimidazole derivatives 21–28. a R = H, b R = Cl; 21, 25 R1 = H, 22, 26 R1 = Me, 23, 27 R1 = Cl, 24, 28 R1 = F. Reagents and conditions: x the corresponding benzene-1,2-diamine, 6N HCl, Δ, 24 h; 15% NH4OH to pH 8; xi 20% NaOH, Δ, 4 h; diluted AcOH to pH 6.
Ijms 24 07966 sch002
Ijms 24 07966 g001 550
Figure 1. The viability of A549 non-small cell lung cancer cells after 24 h treatment with compounds 1a–28b and reference drugs: cisplatin (CP) and cytosine arabinoside (AraC) at a fixed concentration of 100 µM. The post-treatment viability was evaluated via MTT assays, and the % viability was calculated from the untreated controls. Data shown are the mean ± SD values from three separate experiments for each group.
Figure 1. The viability of A549 non-small cell lung cancer cells after 24 h treatment with compounds 1a–28b and reference drugs: cisplatin (CP) and cytosine arabinoside (AraC) at a fixed concentration of 100 µM. The post-treatment viability was evaluated via MTT assays, and the % viability was calculated from the untreated controls. Data shown are the mean ± SD values from three separate experiments for each group.
Ijms 24 07966 g001
Table
Table 1. The in vitro antimicrobial activity of novel 1,3-disubstituted 5-oxopyrrolidines 1a–28b bearing hydrazone, azole, and azine moieties against representative multidrug-resistant bacterial pathogens with genetically defined resistance mechanisms. The antimicrobial activity is expressed as the average of the minimal inhibitory concentration (MIC) for each compound or control antibiotics determined from triplicate experiments.
Table 1. The in vitro antimicrobial activity of novel 1,3-disubstituted 5-oxopyrrolidines 1a–28b bearing hydrazone, azole, and azine moieties against representative multidrug-resistant bacterial pathogens with genetically defined resistance mechanisms. The antimicrobial activity is expressed as the average of the minimal inhibitory concentration (MIC) for each compound or control antibiotics determined from triplicate experiments.
CompoundMinimal Inhibitory Concentration (MIC, µg/mL)
S. aureus
TCH 1516		A. baumannii
AR-0033		K. pneumoniae
AR-0049		P. aeruginosa
AR-0064		C. difficile
AR-1067
1a>128>128>128>128>128
1b>128>128>128>128>128
2a>128>128>128>128>128
2b64128>128>128>128
3>128>128>128>128>128
4>128>128>128>128>128
5>128>128>128>128>128
6>128>128>128>128>128
764>128>128>12816
864>128>128>128>128
9128>128>128>128>128
10>128>128>128>128>128
11>128>128>128>128>128
12>128>128>128>128>128
13>128>128>128>128>128
1416>128>128>12832
15>128>128>128>128>128
16>128>128>128>128>128
17>128>128>128>128>128
18>128>128>128>128>128
19>128>128>128>128>128
2032>128>128128128
21a>128>128>128>128>128
21b32>128>128>12864
22a>128>128>128>128>128
22b64>128>128>128>128
23a128>128>128>128>128
23b64>128>128>128>128
24a>128>128>128>128>128
24b8>12812864128
25a>128>128>128>128>128
25b128>128>128>128128
26a>128>128>128>128>128
26b>128>128>128>128>128
27a>128>128>128>128>128
27b>128>128>128>128>128
28b>128>128>128>128>128
Clindamycin32N/AN/AN/A32
MetronidazoleN/AN/AN/AN/A2
Vancomycin2N/AN/AN/A2
MeropenemN/A321616N/A
Table
Table 2. The in vitro antimicrobial activity of novel 1,3-disubstituted 5-oxopyrrolidines 1a–28b bearing hydrazone, azole, and azine moieties against representative multidrug-resistant fungal pathogens. The antimicrobial activity is expressed as the average of the minimal inhibitory concentration (MIC) for each compound or control antibiotics determined from triplicate experiments.
Table 2. The in vitro antimicrobial activity of novel 1,3-disubstituted 5-oxopyrrolidines 1a–28b bearing hydrazone, azole, and azine moieties against representative multidrug-resistant fungal pathogens. The antimicrobial activity is expressed as the average of the minimal inhibitory concentration (MIC) for each compound or control antibiotics determined from triplicate experiments.
CompoundsMinimal Inhibitory Concentration (MIC, µg/mL)
C. auris
AR-381		C. auris
AR-382		C. auris
AR-383		A. fumigatus
AR-731		A. fumigatus
AR-732		A. fumigatus
AR-733
1a>128>128>128>128>128>128
1b>128>128>128>128>128>128
2a>128>128>128>128>128>128
2b>128>128>128>128>128>128
3>128>128>128>128>128>128
4>128>128>128>128>128>128
5>128>128>128>128>128>128
6>128>128>128>128>128>128
7>128>128>128>128>128>128
8>128>128>128>128>128>128
9>128>128>128>128>128>128
10>128>128>128>128>128>128
11>128>128>128>128>128>128
12>128>128>128>128>128>128
13>128>128>128>128>128>128
14>128>128>128>128>128>128
15161616326432
16>128>128>128>128>128>128
17>128>128>128>128>128>128
18>128>128>128>128>128>128
19>128>128>128>128>128>128
20>128>128>128>128>128>128
21a>128>128>128>128>128>128
21b>128>128>128>128>128>128
22a>128128128>128>128>128
22b>128>128>128>128>128>128
23a>128>128>128>128>128>128
23b>128>128>128>128>128>128
24a>128>128>128>128>128>128
24b>128>128>128>128>128>128
25a>128>128>128>128>128>128
25b>128>128>128>128>128>128
26a>128>128>128>128>128>128
26b128>128>128>128>128>128
27a>128>128>128>128>128>128
27b128>128>128>128>128>128
28b>128>128>128>128>128>128
Fluconazole816128N/AN/AN/A
Flucytosine2<0.5<0.5N/AN/AN/A
Voriconazole<0.5<0.5<0.5212
Table
Table 3. The antimicrobial activity of compounds 14 and 24b against multidrug resistant S. aureus strains with a vancomycin-intermediate-resistance phenotype. The antimicrobial activity is expressed as the average of minimal inhibitory concentration (MIC) for the selected compounds or control antibiotics determined from triplicate experiments.
Table 3. The antimicrobial activity of compounds 14 and 24b against multidrug resistant S. aureus strains with a vancomycin-intermediate-resistance phenotype. The antimicrobial activity is expressed as the average of minimal inhibitory concentration (MIC) for the selected compounds or control antibiotics determined from triplicate experiments.
Bacterial StrainResistance MechanismsAntimicrobial Activity (MIC, µg/mL)
1424bVANDAP
S. aureus AR-215aph-STPH, DHA1, erm(A), mecA, spc, tet(38)16842
S. aureus AR-216aph(3′)-III, aph-STPH, fosB, mecA, mph(C), sat-4A8884
S. aureus AR-217aph-STPH, blaI, dfrG, fosB, mecA, Z16442
S. aureus AR-218aph(3′)-III, aph-STPH, DHA1, erm(A), mecA, norA, spc, tet(38), tet(K)8442
S. aureus AR-219aac(6′)-aph(2″), aadD, aph-STPH, DHA1, erm(A), mecA, norA, spc, tet(38), tet(K)4284
S. aureus AR-220aph-STPH, DHA1, erm(A), mecA, norA, spc, tet(38)16842
Abbreviations: VAN—vancomycin, DAP—daptomycin.
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Bertašiūtė, M.; Kavaliauskas, P.; Vaickelionienė, R.; Grybaitė, B.; Petraitis, V.; Petraitienė, R.; Naing, E.; Garcia, A.; Šiugždaitė, J.; Lelešius, R.; et al. 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. Int. J. Mol. Sci. 2023, 24, 7966. https://doi.org/10.3390/ijms24097966

AMA Style

Bertašiūtė M, Kavaliauskas P, Vaickelionienė R, Grybaitė B, Petraitis V, Petraitienė R, Naing E, Garcia A, Šiugždaitė J, Lelešius R, et al. 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. International Journal of Molecular Sciences. 2023; 24(9):7966. https://doi.org/10.3390/ijms24097966

Chicago/Turabian Style

Bertašiūtė, Monika, Povilas Kavaliauskas, Rita Vaickelionienė, Birutė Grybaitė, Vidmantas Petraitis, Rūta Petraitienė, Ethan Naing, Andrew Garcia, Jūratė Šiugždaitė, Raimundas Lelešius, and et al. 2023. "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" International Journal of Molecular Sciences 24, no. 9: 7966. https://doi.org/10.3390/ijms24097966

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