Synthesis and Biological Activity Characterization of Novel 5-Oxopyrrolidine Derivatives with Promising Anticancer and Antimicrobial Activity

The 1-(4-acetamidophenyl)-5-oxopyrrolidine carboxylic acid was applied for synthesizing derivatives bearing azole, diazole, and hydrazone moieties in the molecule. Modification of an acetamide fragment to the free amino group afforded compounds with two functional groups, which enabled to provide a series of 4-substituted-1-(4-substituted phenyl)pyrrolidine-2-ones. The resulted compounds 2 and 4–22 were subjected to the in vitro anticancer and antimicrobial activity determination. The compounds 18–22 exerted the most potent anticancer activity against A549 cells. Furthermore, compound 21 bearing 5-nitrothiophene substituents demonstrated promising and selective antimicrobial activity against multidrug-resistant Staphylococcus aureus strains, including linezolid and tedizolid-resistant S. aureus. These results demonstrate that 5-oxopyrolidine derivatives are attractive scaffolds for the further development of anticancer and antimicrobial compounds targeting multidrug-resistant Gram-positive pathogens.


Introduction
Growing antimicrobial resistance among clinically significant pathogens is considered to be one of the major threats worldwide. Environmental exposure to numerous chemical agents is associated with a growing incidence of cancer. Therefore, it is important to develop novel bioactive molecules that could be further explored as anticancer or antimicrobial agents.
2-Pyrrolidinones are a class of heterocyclic compounds with a vast diversity of derivatives of biological potential. This structural feature is widely spread in many natural products possessing a large range of biological activities. 2-Pyrrolidinone-based Pyrrocidine A is a known antimicrobial compound produced by endophytic fungi Sarocladium zeae [1], (−)-Azaspirene is an angiogenesis inhibitor that is isolated from the fungus Neosartorya sp. [2], feeding deterrent Ypaoamide stimulate glucose uptake [3], Salinosporamide A, aquatic natural product, which is created by the constraining aquatic bacteria Salinispora tropica and Salinispora arenicola, is a potent proteasome inhibitor being studied as a potential anticancer agent [4] and many others. Their significant role is highlighted not only by the wide variety of pharmacological properties [5][6][7][8][9][10] but also by the efficient approved 2 of 17 pharmaceutical products such as Cromakalim, which is a potassium channel-opening vasodilator, Nebracetam, known as a nootropic M1-muscarinic agonist, which induces a rise of intracellular Ca 2+ concentration, a respiratory stimulant Doxapram, as well as Ethosuximide, a medication for prevention and control of the absence or petit mal seizures [11] ( Figure 1). Therefore, the synthesis and evaluation of biological properties of compounds containing this structural element remain a very important area of medicinal chemistry for the discovery and development of efficient therapeutic preparations. Salinispora tropica and Salinispora arenicola, is a potent proteasome inhibitor being studied as a potential anticancer agent [4] and many others. Their significant role is highlighted not only by the wide variety of pharmacological properties [5][6][7][8][9][10] but also by the efficient approved pharmaceutical products such as Cromakalim, which is a potassium channelopening vasodilator, Nebracetam, known as a nootropic M1-muscarinic agonist, which induces a rise of intracellular Ca 2+ concentration, a respiratory stimulant Doxapram, as well as Ethosuximide, a medication for prevention and control of the absence or petit mal seizures [11] (Figure 1). Therefore, the synthesis and evaluation of biological properties of compounds containing this structural element remain a very important area of medicinal chemistry for the discovery and development of efficient therapeutic preparations. As those of 5-oxopyrrolidines, the hydrazones having great biopotential attract more and more researchers involved in the discovery and development of effective pharmaceuticals. The increasing interest in the chemistry of these compounds is related to the fact that such types of compounds are proved to be efficient as an analgesic, anti-inflammatory [12], antiviral [13], antimicrobial, [12,14], antitumor [15], anticonvulsant [16] agents show strong antidepressant [17], cardioprotective [18], and antiplatelet [19] properties as well as demonstrate efficiency as antifungal agents [20]. Furthermore, hydrazones can scavenge free radicals, which are the main culprits in different diseases arising from oxidative stress. That includes cardiovascular and Alzheimer's diseases, skin cancer, as well as various inflammation, and oxidative damage to proteins and DNA [21,22]. The hydrazone derivatives appeared to possess antioxidant, antiproliferative, and photoprotective activities and are useful for the prevention of skin cancer and helpful in sunscreen formulations [23,24]. The ongoing drug discovery for effective pharmaceutical agents targeting various cancers and infectious disease agents promotes the assessment of a combination of biologically effective moieties in the molecules, one of which is the model of 2-pyrrolidinone and hydrazone fragments. The effect of such a combination is shown to possess antihypertensive [25], antifungal [26], and antibacterial [27] activities. Our extensive studies [28][29][30][31] confirmed that and extended this investigation. The works focused on the synthesis and evaluation of bioefficacy of 2-pyrrolidinone-based hydrazone derivatives and approved them to be a unique structural moiety for the design of agents with an antibacterial, antioxidant, anticancer, and human carbonic anhydrase inhibition activity.
The assessment for their antimicrobial properties against multidrug-resistant Gramnegative (Klebsiella pneumoniae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) pathogens, and pathogenic fungi (Candida auris, Candida albicans, Aspergillus fumigatus) harboring genetically defined resistance mechanisms revealed bishydrazone with favorable (MIC 2 µg/mL) antibacterial activity against S. aureus, which was independent of the existing antimicrobial resistance phenotype and was comparable to the antimicrobial activity of vancomycin and much higher than that of methicillin and cefoxitin. Furthermore, the antifungal properties appeared to be excellent as the hydrazones possessing a 5-oxopyrrolidine structure showed significantly high MIC (0.9-1.9 µg/mL) against Candida tenuis VKMY-70 and Aspergillus niger VKM F-1119 which As those of 5-oxopyrrolidines, the hydrazones having great biopotential attract more and more researchers involved in the discovery and development of effective pharmaceuticals. The increasing interest in the chemistry of these compounds is related to the fact that such types of compounds are proved to be efficient as an analgesic, anti-inflammatory [12], antiviral [13], antimicrobial, [12,14], antitumor [15], anticonvulsant [16] agents show strong antidepressant [17], cardioprotective [18], and antiplatelet [19] properties as well as demonstrate efficiency as antifungal agents [20]. Furthermore, hydrazones can scavenge free radicals, which are the main culprits in different diseases arising from oxidative stress. That includes cardiovascular and Alzheimer's diseases, skin cancer, as well as various inflammation, and oxidative damage to proteins and DNA [21,22]. The hydrazone derivatives appeared to possess antioxidant, antiproliferative, and photoprotective activities and are useful for the prevention of skin cancer and helpful in sunscreen formulations [23,24]. The ongoing drug discovery for effective pharmaceutical agents targeting various cancers and infectious disease agents promotes the assessment of a combination of biologically effective moieties in the molecules, one of which is the model of 2-pyrrolidinone and hydrazone fragments. The effect of such a combination is shown to possess antihypertensive [25], antifungal [26], and antibacterial [27] activities. Our extensive studies [28][29][30][31] confirmed that and extended this investigation. The works focused on the synthesis and evaluation of bioefficacy of 2-pyrrolidinone-based hydrazone derivatives and approved them to be a unique structural moiety for the design of agents with an antibacterial, antioxidant, anticancer, and human carbonic anhydrase inhibition activity.
The assessment for their antimicrobial properties against multidrug-resistant Gramnegative (Klebsiella pneumoniae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) pathogens, and pathogenic fungi (Candida auris, Candida albicans, Aspergillus fumigatus) harboring genetically defined resistance mechanisms revealed bishydrazone with favorable (MIC 2 µg/mL) antibacterial activity against S. aureus, which was independent of the existing antimicrobial resistance phenotype and was comparable to the antimicrobial activity of vancomycin and much higher than that of methicillin and cefoxitin. Furthermore, the antifungal properties appeared to be excellent as the hydrazones possessing a 5-oxopyrrolidine structure showed significantly high MIC (0.9-1.9 µg/mL) against Candida tenuis VKMY-70 and Aspergillus niger VKM F-1119 which surpassed Nystatin (7.8 and 15.6 µg/mL, respectively) the antibiotic used to treat various fungal infections [32]. In this paper, we describe the synthesis of 5-oxopyrrolidine derivatives with the acetamide moiety and characterization of their in vitro antimicrobial and anticancer activity. The choice of the acetamide phenyl moiety, in this case, was determined by the wide variety of biological properties of the compounds bearing this fragment [33][34][35][36] as well as the easy deacetylation possibility to obtain compounds with the free amino group.

Synthesis
Above all, our research was focused on the synthesis and verification of antimicrobial and anticancer properties of 5-oxopyrrolidine derivatives. For the solution of this aspiration, the N-(4-aminophenyl)acetamide (1) was chosen and then reacted with an itaconic acid in water at reflux to give 1-(4-acetamidophenyl)-5-oxopyrrolidine-3-carboxylic acid (2) (Scheme 1) as an initial compound for further transformations. Compound 2, when treated with methanol in the presence of a catalytic amount of sulfuric acid in the reaction mixture, afforded methyl ester 3, which without the separation, was subjected to hydrazinolysis to give acid hydrazide 4. To synthesize hydrazones, hydrazide 4 was treated with a 1.5fold excess of the corresponding aromatic aldehyde. The products were obtained in the range of 38-98% yields. The presence of an amide fragment in the molecules of these compounds and the restricted rotation around this bond allowed the formation of the E/Z conformers [37], which presence is clearly demonstrated by the NMR spectra. The 1 H NMR spectra of hydrazones 5-9 showed that the presence of the unsubstituted or 4-substituted phenyl ring causes the formation of the Z and E rotamers in the ratio of 65/35, while when di-or trisubstituted phenyl fragment is attached the ratio of conformers gain the values of 70/30 (10) and 75/25 (11) indicating the growing stability of the Z-form. surpassed Nystatin (7.8 and 15.6 µg/mL, respectively) the antibiotic used to treat various fungal infections [32].
In this paper, we describe the synthesis of 5-oxopyrrolidine derivatives with the acetamide moiety and characterization of their in vitro antimicrobial and anticancer activity. The choice of the acetamide phenyl moiety, in this case, was determined by the wide variety of biological properties of the compounds bearing this fragment [33][34][35][36] as well as the easy deacetylation possibility to obtain compounds with the free amino group.

Synthesis
Above all, our research was focused on the synthesis and verification of antimicrobial and anticancer properties of 5-oxopyrrolidine derivatives. For the solution of this aspiration, the N-(4-aminophenyl)acetamide (1) was chosen and then reacted with an itaconic acid in water at reflux to give 1-(4-acetamidophenyl)-5-oxopyrrolidine-3-carboxylic acid (2) (Scheme 1) as an initial compound for further transformations. Compound 2, when treated with methanol in the presence of a catalytic amount of sulfuric acid in the reaction mixture, afforded methyl ester 3, which without the separation, was subjected to hydrazinolysis to give acid hydrazide 4. To synthesize hydrazones, hydrazide 4 was treated with a 1.5-fold excess of the corresponding aromatic aldehyde. The products were obtained in the range of 38-98% yields. The presence of an amide fragment in the molecules of these compounds and the restricted rotation around this bond allowed the formation of the E/Z conformers [37], which presence is clearly demonstrated by the NMR spectra. The 1 H NMR spectra of hydrazones 5-9 showed that the presence of the unsubstituted or 4-substituted phenyl ring causes the formation of the Z and E rotamers in the ratio of 65/35, while when di-or trisubstituted phenyl fragment is attached the ratio of conformers gain the values of 70/30 (10) and 75/25 (11) indicating the growing stability of the Z-form.  To compare the biological properties of the products ketones (acetone and ethyl methyl ketone) that were used in analog reactions, which gave propan-2-ylidenehydrazine or butan-2-ylidenehydrazine derivatives 12 and 13. Their 1 H NMR spectra showed the presence of conformational and geometric isomers. To interpret their exact structures, detailed and complex spectroscopic studies are required. Whereas this study aimed to synthesize a specific target compound and evaluate its biological properties, a detailed structural analysis was not performed.
The target azoles 14 and 15 were easily obtained from the hydrazide 4 and the appropriate aliphatic diketone. When reacting with pentane-2,4-dione (2,4-PD) in propan-2-ol with the addition of a catalytic amount of hydrochloric acid, the 3,5-dimethylpyrazole 14 was prepared, while the condensation with hexane-2,5-dione (2,5-HD) at the same conditions but using acetic acid as a catalyst instead the 2,5-dimethylpyrrole derivative 15 was formed. The structures were confirmed by their spectral data. In the 1 H NMR spectrum of 14, the protons belonging to the CH of the pyrazole cycle gave a singlet at 6.23 ppm, and protons of two methyl groups of the pyrazole ring gave two singlets in up-field of the spectrum, at 2.21 and 2.49 ppm. The 13 C NMR spectrum showed resonances at 111.59, 13.56, and 14.07 ppm, respectively. A structure of pyrrole 15 was approved by the presence of characteristic peaks at 1.99, 5.65, and 10.90, 10.91 ppm, which were assigned to the protons of the methyl groups, the pyrrole CH, and the amide group. The 13 C NMR spectrum showed characteristic spectral lines at 10.96 and 103.11 ppm, which were assigned to the carbons of the methyl groups and CH-CH fragment of the pyrrole cycle.
To obtain the compound with the free amino group, the deacylation reaction of the synthesized compound 2 was performed in refluxing dilute hydrochloric acid, followed by transferring the resulting amino hydrochloride into the base form 16 with sodium acetate. The 1 H NMR spectrum of compound 16 revealed a singlet at 7.65 ppm integrated for 2 protons, which is characteristic of the amine group attached to the phenyl ring in the molecule.
To compare the chemical properties of the compounds, the hydrazide 17 bearing amine, and hydrazide functional groups were prepared. A refluxing mixture of acid 16 and hydrazine monohydrate in toluene gave the target hydrazide 17 over 16 h. When interpreting the 1 H NMR spectrum of compound 17, the broad singlets belonging to 2NH 2 and NH groups, as expected, were at 5.57 and 9.29 ppm, respectively.
The functional groups present in compounds 16 and 17 were applied to obtain variously substituted derivatives 18-22 (Scheme 2). Thus, acid 16 was condensed with ophenylenediamine in refluxing diluted hydrochloric acid (1:1) for 36 h. The target benzimidazole 18 was separated by the addition of sodium acetate to an aqueous solution of the formed precipitate. The NMR spectra showed good agreement with the expected structure. To compare the biological properties of the products ketones (acetone and ethyl methyl ketone) that were used in analog reactions, which gave propan-2-ylidenehydrazine or butan-2-ylidenehydrazine derivatives 12 and 13. Their 1 H NMR spectra showed the presence of conformational and geometric isomers. To interpret their exact structures, detailed and complex spectroscopic studies are required. Whereas this study aimed to synthesize a specific target compound and evaluate its biological properties, a detailed structural analysis was not performed.
The target azoles 14 and 15 were easily obtained from the hydrazide 4 and the appropriate aliphatic diketone. When reacting with pentane-2,4-dione (2,4-PD) in propan-2ol with the addition of a catalytic amount of hydrochloric acid, the 3,5-dimethylpyrazole 14 was prepared, while the condensation with hexane-2,5-dione (2,5-HD) at the same conditions but using acetic acid as a catalyst instead the 2,5-dimethylpyrrole derivative 15 was formed. The structures were confirmed by their spectral data. In the 1 H NMR spectrum of 14, the protons belonging to the CH of the pyrazole cycle gave a singlet at 6.23 ppm, and protons of two methyl groups of the pyrazole ring gave two singlets in up-field of the spectrum, at 2.21 and 2.49 ppm. The 13 C NMR spectrum showed resonances at 111.59, 13.56, and 14.07 ppm, respectively. A structure of pyrrole 15 was approved by the presence of characteristic peaks at 1.99, 5.65, and 10.90, 10.91 ppm, which were assigned to the protons of the methyl groups, the pyrrole CH, and the amide group. The 13 C NMR spectrum showed characteristic spectral lines at 10.96 and 103.11 ppm, which were assigned to the carbons of the methyl groups and CH-CH fragment of the pyrrole cycle.
To obtain the compound with the free amino group, the deacylation reaction of the synthesized compound 2 was performed in refluxing dilute hydrochloric acid, followed by transferring the resulting amino hydrochloride into the base form 16 with sodium acetate. The 1 H NMR spectrum of compound 16 revealed a singlet at 7.65 ppm integrated for 2 protons, which is characteristic of the amine group attached to the phenyl ring in the molecule. To compare the chemical properties of the compounds, the hydrazide 17 bearing amine, and hydrazide functional groups were prepared. A refluxing mixture of acid 16 and hydrazine monohydrate in toluene gave the target hydrazide 17 over 16 h. When interpreting the 1 H NMR spectrum of compound 17, the broad singlets belonging to 2NH2 and NH groups, as expected, were at 5.57 and 9.29 ppm, respectively.
The functional groups present in compounds 16 and 17 were applied to obtain variously substituted derivatives 18-22 (Scheme 2). Thus, acid 16 was condensed with o-phenylenediamine in refluxing diluted hydrochloric acid (1:1) for 36 h. The target benzimid- singlets at 1.95 (2CH 3 ) and 5.78 (CH-CH pyr ) ppm in the 1 H NMR spectrum. The 13 C NMR spectrum showed peaks at 12.86 and 105.79 ppm, respectively, which are characteristic of the 2,5-dimethylpyrrole cycle.
To verify the hydrazide 17 and to evaluate the impact of substituents on the biological properties of compounds, condensation reactions with carbonyl compounds (heteroaromatic aldehydes and aliphatic diketone) were carried out. The reactions with 2thiophenecarboxaldehyde and its 5-nitro analog in an aqueous propanolic (15:1) medium in the presence of hydrochloric acid as a catalyst leading to the formation of the appropriate hydrazones 20 and 21. The 1 H NMR spectra of these compounds exhibited four down-field singlet signals at 11.55, 11.57, 11.59, and 11.61 (20) and two ones at 11.99 and 12.02 (21) ppm attributed to the protons of the NH. The azomethine CH=N protons resonated between 8.20-8.85 (20) and 8.47-9.01 (21) ppm.
Compound 22 bearing two 2,5-dimethylpyrrole cycles was prepared from compound 17 by the condensation reaction with a 4-fold excess of hexane-2,5-dione in methanol with the addition of a catalytic amount of glacial acetic acid. The reaction at reflux for 4 h resulted in the formation of a desired product 22. The presence of amine and hydrazide groups afforded an asymmetric bis(pyrrole) molecule. The 1 H NMR spectrum fully confirmed the formed structure: two singlets at 1.96 and 2.01 ppm each integrated for six protons were attributed for 4CH 3 , and two singlets at 5.65 and 5.79 ppm where each integrated for two protons were ascribed to 2CH-CH pyr and only one signal at 10.94 ppm was assigned to NH exhibiting that one pyrrole is attached directly to the phenyl ring, and another one is inserted in the molecule via amide moiety.

The Anticancer Activity of 5-Oxopyrrolidine Derivatives 2 and 4-22
To characterize the biological activity of compounds 2 and 4-22, we determined the anticancer properties of novel 5-oxopyrrolidine derivatives using well established A549 human lung adenocarcinoma model. To better understand the toxicity of the novel compounds, we also used HSAEC-1 KT human small airway epithelial cells that served as non-cancerous cells derived from the pulmonary environment. We exposed the A549 and HSAEC1-KT cells with a fixed 100 µM concentration of each compound for 24 h and evaluated the post-treatment viability using an MTT assay. The compound-mediated cytotoxicity was compared with cisplatin (CP), a standard chemotherapeutic drug used for lung cancer treatment.
The compounds exhibited the structure-depended anticancer activity on A549 cells. Carboxylic acid 2 that was generated from starting compound 1 showed weak anticancer activity and resulted in 78-86% post-treatment viability ( Figure 2). The compound conversion to acid hydrazide 4 did not enhance the anticancer activity. Notably, the conversion of hydrazide 4 to hydrazone greatly improved the anticancer activity in a structure-dependent manner ( Figure 2). The incorporation of phenyl ring (compound 5) did not significantly affect the anticancer activity in comparison to compound 4. Furthermore, 4-chlorophenyl and 4-bromophenyl substitutions (6 and 7, respectively) enhanced the anticancer activity by reducing the A549 viability to 64 and 61%, respectively. Furthermore, besides enhanced anticancer activity, compound 6 exhibited increased cytotoxicity towards non-cancerous HSAEC1-KT cells ( Figure 3). Interestingly, 4-dimethylamino phenyl substitution showed the most potent anticancer activity (8), which was significantly higher than compound 4 (p < 0.05), while incorporation 4-methoxy group in the phenyl ring ameliorated the anticancer activity (compound 9). Compounds 6 and 7 reduced the A549 viability, although no statistically significant effect was observed when the anticancer activity of 6 and 7 were compared with starting compound 4. Furthermore, di-and trimethoxy substitutions in the phenyl ring (compounds 10 and 11) resulted in a significant loss of anticancer activity (p < 0.05) in comparison to compound 8 ( Figure 2). trimethoxy substitutions in the phenyl ring (compounds 10 and 11) resulted in a significant loss of anticancer activity (p < 0.05) in comparison to compound 8 ( Figure 2).  Among azole derivatives, 2,5-dimethylpyrrole derivative 15 exerted more potent activity than 3,5-dimethylpyrazole 14 by reducing the A549 viability to 66%. Compound 15 exhibited noticeable cytotoxic activity towards non-cancerous cells, suggesting that the 2,5-dimethylpyrrole moiety is important for conferring the cytotoxic activity in normal and cancerous cells. Compounds containing free amino group, except 16, and their derivatives bearing various structural substitutions showed more potent anticancer activity than those with an acetylamino fragment in the molecules with no significant cytotoxic effect on non-cancerous cells (Figures 2 and 3). Bis hydrazone 20, containing 2-thienyl fragments and its analog 21 with two 5-nitrothienyl moieties in the structure, demonstrated the highest anticancer activity amongst all tested 5-oxopyrrolidine derivatives and had favorable low cytotoxic properties on non-cancerous cells (Figures 2 and 3).
Finally, the structure-activity relationship study of the investigated hydrazones 5-11, 20, 21 has shown (Figure 2) that the anticancer activity of the hydrazones 5-11 with trimethoxy substitutions in the phenyl ring (compounds 10 and 11) resulted in a significant loss of anticancer activity (p < 0.05) in comparison to compound 8 ( Figure 2).  Among azole derivatives, 2,5-dimethylpyrrole derivative 15 exerted more potent activity than 3,5-dimethylpyrazole 14 by reducing the A549 viability to 66%. Compound 15 exhibited noticeable cytotoxic activity towards non-cancerous cells, suggesting that the 2,5-dimethylpyrrole moiety is important for conferring the cytotoxic activity in normal and cancerous cells. Compounds containing free amino group, except 16, and their derivatives bearing various structural substitutions showed more potent anticancer activity than those with an acetylamino fragment in the molecules with no significant cytotoxic effect on non-cancerous cells (Figures 2 and 3). Bis hydrazone 20, containing 2-thienyl fragments and its analog 21 with two 5-nitrothienyl moieties in the structure, demonstrated the highest anticancer activity amongst all tested 5-oxopyrrolidine derivatives and had favorable low cytotoxic properties on non-cancerous cells (Figures 2 and 3).
Finally, the structure-activity relationship study of the investigated hydrazones 5-11, 20, 21 has shown (Figure 2) that the anticancer activity of the hydrazones 5-11 with Hydrazones 12 and 13 showed weak to no anticancer activity on A549 cells as well as weak cytotoxic activity on HSAEC1-KT cells (Figure 3).
Among azole derivatives, 2,5-dimethylpyrrole derivative 15 exerted more potent activity than 3,5-dimethylpyrazole 14 by reducing the A549 viability to 66%. Compound 15 exhibited noticeable cytotoxic activity towards non-cancerous cells, suggesting that the 2,5-dimethylpyrrole moiety is important for conferring the cytotoxic activity in normal and cancerous cells. Compounds containing free amino group, except 16, and their derivatives bearing various structural substitutions showed more potent anticancer activity than those with an acetylamino fragment in the molecules with no significant cytotoxic effect on non-cancerous cells (Figures 2 and 3). Bis hydrazone 20, containing 2-thienyl fragments and its analog 21 with two 5-nitrothienyl moieties in the structure, demonstrated the highest anticancer activity amongst all tested 5-oxopyrrolidine derivatives and had favorable low cytotoxic properties on non-cancerous cells (Figures 2 and 3).
Finally, the structure-activity relationship study of the investigated hydrazones 5-11, 20, 21 has shown (Figure 2) that the anticancer activity of the hydrazones 5-11 with aromatic moieties is lower compared to hydrazones 20, 21 containing heterocyclic fragments. The most active compounds of aromatic hydrazones were compounds containing dimethylamino-, chloro-, and bromo-substituents in the aromatic ring. As can be seen from the study data, compound 16 exhibited low anticancer activity, but when its functional groups are modified to fragments of benzimidazole (18) or dimethylpyrrole (19), their anticancer activity increases strongly. Therefore, in the future, it would be worth expanding such modifications in search of new compounds with high anticancer activity.
These results suggest that the 5-oxopyrrolidine derivatives can suppress the viability in the A549 human lung cell adenocarcinoma model in a structure-depended manner. In addition to that, 5-oxopyrrolidine derivatives obtained from compounds containing the free amino group exert the most promising anticancer activity demonstrating the importance of the free amino group in the search for anticancer agents with low cytotoxicity toward non-cancerous cells.
After demonstrating that compound 21 exerts the in vitro antibacterial activity against S. aureus, we decided to check whether the S. aureus-directed activity depends on the pre-existing S. aureus resistance mechanisms. We have screened compound 21 using vancomycin-intermediate and oxazolidines (linezolid/tedizolid) resistant strains and compared the MIC values with clinically approved drugs.
The compound 21 demonstrated favorable activity (MIC 1-8 µg/mL) against multidrugresistant and vancomycin-intermediate Staphylococcus aureus isolates harboring major multidrug-resistance determining mechanisms (Table 1). On the other hand, higher MIC values (4-64 µg/mL) were observed when linezolid/tedizolid-resistant strains were used for the assays ( Table 2). Many nitro groups containing compounds have enhanced antimicrobial activity under anaerobic conditions. As an example, FDA approved drug metronidazole has excellent activity against anaerobic bacteria, while little to no activity against aerobes. After demonstrating that the nitro group containing compound 21 shows favorable activity against Gram-positive pathogens, we evaluated if the nitro group could confer enhanced activity against anaerobes. To do so, we used representative anaerobic pathogens and determined the MIC for the compound 21 and metronidazole, which served as a controlled drug  (Table 3). Compound 21 demonstrated higher antimicrobial activity against Gram-positive pathogens (C. difficile and C. perfringens). The weak activity was also observed against Gram-negative pathogens, suggesting that under anaerobic conditions, compound 21 could confer some weak activity against Gram-negative anaerobic bacteria. Compound 21 did not exhibit greater activity than metronidazole, which was used as a control agent.  Collectively, these results demonstrate that 5-oxopyrrolidine derivative 21 shows promising and selective antimicrobial activity against Gram-positive pathogens with the highest activity against multidrug-resistant S. aureus with genetically defined and emerging resistance profiles. The 5-oxopyrrolidine derivatives could be potentially explored as promising pharmacophores for a further hit to lead development as antimicrobial candidates targeting challenging resistance mechanisms in the high priority pathogens.

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 by TLC using aluminum plates precoated with Silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). Melting points were determined with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and were uncorrected. NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts were reported in (d) ppm relative to tetramethylsilane (TMS) with the residual solvent as internal reference (DMSO-d 6 , d = 2.50 ppm for 1H 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. 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.

Minimal Inhibitory Concentration Determination
The minimal inhibitory concentrations (MICs) of compounds 2 and 4-22, as well as various antibiotics, were determined according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [38]. 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. 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× of assay concentrations (0.5-64 µg/mL) and were then transferred to the assay plates. A standardized inoculum was prepared using 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, CAMBH was replaced with CAMBH supplemented with vitamin K, leaked horse blood and plates were incubated in an anaerobic environment. 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.

The Cytotoxic Activity Characterization
The A549 and HSAEC1-KT cells were obtained from American Type Culture Collection. The cytotoxic activity of compounds 2 and 4-22, as well as cisplatin (Sigma-Aldrich, Saint Louis, Missouri, USA), was determined by using an MTT assay (ThermoFisher Scientific, Waltham, Massachusetts USA). Briefly, cells were plated in 96-well plates at a density of 1 × 10 4 cells/well in DMEM with 10% FBS (for A549) or SAGM BulletKit medium (Lonza CC-3119 and CC-4124) containing supplements (AGM™ SingleQuots™, Lonza CC-4124) (for HSAEC1-KT). After overnight attachment at 37 • C, 5% CO 2 , cells were treated with compounds (100 µM) in triplicate. After 20 h treatment, the MTT reagent was added, and cells were further incubated for 4 h. The formazan was then extracted with anhydrous DMSO. The samples were measured using a microplate reader at a wavelength of 570 nm. The following formula was used to calculate the percentage 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.

Statistical Analysis
The data are expressed as a mean ± SD value from three separate experiments unless stated otherwise. The statistical significance was determined using a test. Data were considered significant when p < 0.05.

Conclusions
In conclusion, the synthesis, chemical transformations, and biological assessment of some 1-(4-acetamidophenyl)-and 1-(4-aminophenyl)-5-oxopyrrolidine derivatives bearing hydrazone and azole fragments are provided herein. All the prepared compounds were characterized using spectroscopic techniques and elemental analysis.
Compound 21 showed promising and selective antimicrobial activity targeting multidrugresistant Staphylococcus aureus harboring emerging multidrug-resistance mechanisms. The activity of compound 21 was comparable to or greater than clinically approved antibiotics against S. aureus with challenging resistance mechanisms, demonstrating the great potency of compound 21 for the further hit to lead optimization. It is worth mentioning that the in vitro antimicrobial activity exhibited by compound 21 is reduced in linezolid/tedizolidresistant S. aureus strains, suggesting that pre-existing resistance mechanisms conferring S. aureus resistance to linezolid/tedizolid perhaps can overcome compound 21 mediated antimicrobial activity.
In addition to that, compounds 18-21 showed promising anticancer activity against A549 human lung adenocarcinoma cells, demonstrating that the 1-(4-aminophenyl)-5oxopyrrolidine scaffold could be further explored as a promising anticancer pharmacophore for the development and optimization of novel antimicrobial and anticancer candidates.