**3. Discussions**

The 1,3-oxazoles are five-membered heteromonocyclic scaffolds, containing two heteroatoms, O and N at positions 1 and 3, respectively, of particular importance in synthetic

and medicinal chemistry, since representatives of this class have great structural diversity and are of significant biological interest.

Over time, researchers have paid particular attention to the preparation of new derivatives incorporating the 1,3-oxazole ring [52–55], as well as to the evaluation, among other pharmacological properties, of their antimicrobial and antioxidant activities, a large number of articles being published on this topic. Currently, various bioactive 1,3-oxazole-embedded heterocycles have been reported, such as 1,3-oxazole clubbed pyridyl-pyrazolines having good to excellent antimicrobial action [56], cytisine-containing 1,3-oxazoles as potential inhibitors of *Candida* spp. glutathione reductase [57], thioxo-1,3-oxazole analogs that prevent bacterial growth and with antioxidant ability [58], 1,3-oxazole-quinoxaline amine hybrids showing antibacterial activity [59], steroidal 1,3-oxazole derivatives displaying effective antimicrobial and antibiofilm properties [60], binaphthyl-based, functionalized 1,3 oxazole peptidomimetics with moderate to excellent antimicrobial activity [61], substituted 1,3-oxazole-benzamides as antibacterial inhibitors of the essential bacterial cell division protein FtsZ [62], *N*-(oxazolylmethyl)thiazolidinediones as selective inhibitors of *Candida albicans* biofilm formation [63], and some antimicrobial 2-amino-1,3-oxazoles [64]. Very recently, we investigated the in silico and in vitro antimicrobial and antibiofilm effects of a series of 1,3-oxazole-based compounds and their isosteric analogs derived from alanine and phenylalanine, some of them presenting a promising profile [65]. Quantitative studies also indicated that of all 2,5-diaryl-1,3-oxazoles tested, only some of those with a bromine atom in their molecules showed antimicrobial and/or antibiofilm action. In contrast, all other derivatives from the same class, unsubstituted or with a chlorine atom in their structures, proved to be inactive at the concentrations tested. Other researchers have also observed that the incorporation of the chlorine or bromine atom, usually grafted onto an aromatic nucleus, led to an improvement in the antimicrobial activity of compounds of different classes, as well as that brominated derivatives have shown greater efficacy than chlorinated analogs [66–69]. The antimicrobial effect due to the presence of halogen atoms appears to be related to the compounds' relative hydrophobicity. It was found that hydrophobicity of the compounds enhanced with the addition of a halogen atom in their molecules and that chlorine derivatives displayed a less pronounced effect, while the incorporation of a bromine atom led to analogs with a higher hydrophobic profile [66]. In view of these findings, it is of interest to obtain new bromine-bearing 1,3-oxazoles-type molecules and to explore their application as biologically active agents.

In the present work, we synthesized novel organic compounds, carrying a bromine atom in their molecules, based on the 1,3-oxazole and diphenyl sulfone pharmacophores. The molecular structures of the newly obtained bromine-containing compounds (which are *N*-acyl-*L*-valine, 4-isopropyl-4*H*-1,3-oxazol-5-one, 2-acylamino ketone, and 4-isopropyl-1,3-oxazole derivatives) were elucidated based on spectroscopic investigations. Moreover, the synthesized analogs were assessed for antimicrobial properties, namely qualitative (zone of inhibition) and quantitative (MIC) activities, antibiofilm action (MBIC), and for antioxidant and toxic effects. Furthermore, in silico investigations on the toxicity and potential antimicrobial action were performed.

The structure-based similarity analysis of the new *L*-valine derivatives **5**–**8** highlighted the originality of the proposed structures and indicated that the presence of the bromine atom in the proposed structures of the new compounds can enhance the potential antimicrobial effect. The PASS prediction showed the probability that all new compounds to exhibit anti-infective, antimycobacterial, and antituberculosis activities and, in addition, that 4*H*-1,3-oxazol-5-one **6** to have a glycopeptide-like antibiotic effect.

The antimicrobial activity assays revealed a promising antimicrobial potential against Gram-positive cocci strains, particularly in the case of *E. faecium* biofilm, when the MBIC values were as from 8 to 256 times lower than the corresponding MIC values.

Concerning the results of quantitative tests for the evaluation of antimicrobial and antibiofilm activity, it was also found that the MIC and MBIC values of the standard broadspectrum antibacterial drug ciprofloxacin and antifungal agent fluconazole, which served as

positive controls, were lower than those determined for the compounds tested. This can be explained by the fact that the novel *L*-valine derivatives, which belong to four classes, may have distinct mechanisms of action from those of the used control drugs and, in addition, unlike them, are not standardized active ingredients in optimized drug formulations.

It was also found that all compounds analyzed show antioxidant activity through all spectroscopic tests used (DPPH, ABTS, and ferric reducing power methods), but lower than the bioactive standards (AA, BHA, and BHT). The tested compounds thus demonstrated weak electron-donating properties.

The in vitro evaluation regarding antimicrobial, antioxidant, and toxicity features highlighted that acyclic intermediate **5** is promising due to its very good antibiofilm activity, low antioxidant effect, and moderate toxicity. It is worth noting that by cyclodehydration of compound **5** to the corresponding 4*H*-1,3-oxazol-5-one **6**, an increase in the antibiofilm effect and antioxidant activity was observed, but also an increase in its toxicity. Moreover, it appears that the conversion of 2-oxazolin-5-one **6** to α-acylamino ketone **7b** and the cyclization of **7b** to 1,3-oxazole **8b** leads to a decrease in antimicrobial activity, and compound **7a** and corresponding 1,3-oxazole **8a** were found to be inactive at the tested concentrations. No regular variation in the antioxidant effect of compounds **7a**, **7b**, **8a**, and **8b** was noticed, but of all the new compounds tested, **7a** and **8b** stood out, which showed the best activity by all three methods used. It was also shown that by converting saturated azlactone **6** to α-acylamino ketones **7a**,**b**, the toxicity increases, and by cyclization of open-chain intermediates **7a**,**b** to 1,3-oxazoles **8a**,**b**, the toxicity decreases significantly, **8b** being non-toxic. However, the *Daphnia magna* assay showed medium to high toxicity for compounds **5**, **6**, and **7b**, and significant-high toxicity for compounds **3** and **7a**. Our findings indicate a high potential for biological targets, whereas the 1,3-oxazole-containing compound **8b** showed no toxicity at concentrations lower than 50 μg/mL. The toxicity study also showed that, with the exception of the LC50 value obtained for key intermediate **3**, the experimental values of LC50 for all other compounds were significantly higher than those predicted by the GUSAR analysis.

Recently, we reported the synthesis and results of the antimicrobial and antibiofilm assessment of a series of similar *N*-{4-[(4-X-phenyl)sulfonyl]benzoyl}-*L*-valine derivatives (X = H, Cl) [70,71]. The non-substituted analog of **7b**, *N*-[3-methyl-1-oxo-1-(*p*-tolyl)butan-2 yl]-4-(phenylsulfonyl)benzamide presented a better antimicrobial effect compared with **7b** or its chloro-derivative. The increase in the lipophilic character was perhaps detrimental because of the associated lower water solubility. The introduction into the molecule of the bromine atom proved slightly beneficial in the case of the 4*H*-1,3-oxazol-5-one **6**.

Subsequent structural and biological investigations into these kinds of privileged scaffolds can lead to the discovery of new more potent derivatives that can act as optimized candidates for the development of new effective preventive and therapeutic agents. In this respect, we consider three critical positions on the molecular templates that may have an effect on biological action. A first possibility for the future improvement of the antimicrobial property of these analogs is the replacement of the bromine atom in the arylsulfonylphenyl fragment with different substituents, such as fluorine, iodine, trifluoromethyl, or nitro. For this purpose, other benzene analogs can be used as starting molecules in the Friedel–Crafts sulfonylation reaction. As a second optimization choice, in the *N*-acylation reaction instead of *L*-valine, we consider the use of other natural or unnatural α-amino acids (such as histidine and tryptophan) or the incorporation of several α-amino acid residues in the molecules as it is well-known that some natural polypeptides bearing the 1,3-oxazole ring (e.g., microcin B17) are inhibitors of DNA-gyrase [3]. The use of other aromatic compounds with different substituents (e.g., I, NO2, CF3, OCH3) in the reaction with **6** is the third alternative route of synthesis to enhance the biological activity of these molecular scaffolds. The iodine atom proved more active than other halogens, CF3-functionalized compounds showed higher antibacterial activity than those with a CH3 group, hydrophobic substituents (e.g., benzyloxy, *tert*-butyl) [72], or electron-withdrawing groups (such as nitro) on aromatic cores increased the antibacterial effect [73], small substituents (e.g., hydroxy) at 4-position of

the phenyl nucleus improved the antibacterial potency [62]. In addition, it was noticed that the electron-donating substituents (such as OCH3) on the aromatic moiety enhanced the antioxidant activity compared with the electron-withdrawing groups and that, in general, this pharmacological action increased with the increasing electron-donating effect of the substituent [74–77].
