Structural Variations in the Central Heterocyclic Scaffold of Tripartite 2,6-Difluorobenzamides: Influence on Their Antibacterial Activity against MDR Staphylococcus aureus

Five series of heterocyclic tripartite 2,6-difluorobenzamides, namely 1,2,3-triazoles, 1,2,4- and 1,3,4-oxadiazoles, analogs of reported model anti-staphylococcal compounds, were prepared. The purpose was to investigate the influence of the nature of the heterocyclic central scaffold on the biological activity against three strains of S. aureus, including two drug-resistant ones. Among the 15 compounds of the new collection, a 3-(4-tert-butylphenyl)-1,2,4-oxadiazole linked via a methylene group with a 2,6-difluorobenzamide moiety (II.c) exhibited a minimal inhibitory concentration between 0.5 and 1 µg/mL according to the strain. Subsequent studies on II.c demonstrated no human cytotoxicity, while targeting the bacterial divisome.


Introduction
Antimicrobial resistance is a worldwide health problem concerning many pathogens including Staphylococcus aureus [1,2]. Compounds with a 2,6-difluorobenzamide moiety are described as interesting antibacterial agents targeting the protein FtsZ of the divisome (Figure 1). Among this class of compounds, 3-methoxybenzamide was first discovered [3] and was the starting point for a great number of derivatives, such as the pyridinothiazole PC190723 [4] and the prodrug TXA709 [5]. Several 2,6-difluorobenzamide derivatives bearing an oxazole [6], an isoxazole [7,8], an oxadiazol-2-one [9], and an oxadiazole ring [10] were also described as interesting FtsZ inhibitors (Figure 1). For the latter, only activity ranges were described, rendering difficult accurate SAR studies.
The general structure of the oxazole or isoxazole derivatives can be divided in three zone: the 2,6-difluorobenzamide moiety, a central five-member ring linked to a methylene group (substituted or not), and the substituted phenyl group (Figure 2). Among reported compounds, there has not been any systematic study of the nature of the central heterocyclic scaffold, with or without oxygen atom, or with the oxygen atom located at all possible positions of the ring. To this aim, we prepared a collection of analogs including 1,2,3-triazole derivatives, two series of 1,2,4-oxadiazoles, and one series of 1,3,4-oxadiazoles ( Figure 2) which were evaluated against three S. aureus strains.  The general structure of the oxazole or isoxazole derivatives can be divided in three zone: the 2,6-difluorobenzamide moiety, a central five-member ring linked to a methylene group (substituted or not), and the substituted phenyl group (Figure 2). Among reported compounds, there has not been any systematic study of the nature of the central heterocyclic scaffold, with or without oxygen atom, or with the oxygen atom located at all possible positions of the ring. To this aim, we prepared a collection of analogs including 1,2,3triazole derivatives, two series of 1,2,4-oxadiazoles, and one series of 1,3,4-oxadiazoles ( Figure 2) which were evaluated against three S. aureus strains.   The general structure of the oxazole or isoxazole derivatives can be divided in three zone: the 2,6-difluorobenzamide moiety, a central five-member ring linked to a methylene group (substituted or not), and the substituted phenyl group ( Figure 2). Among reported compounds, there has not been any systematic study of the nature of the central heterocyclic scaffold, with or without oxygen atom, or with the oxygen atom located at all possible positions of the ring. To this aim, we prepared a collection of analogs including 1,2,3triazole derivatives, two series of 1,2,4-oxadiazoles, and one series of 1,3,4-oxadiazoles ( Figure 2) which were evaluated against three S. aureus strains.

Chemistry
Based on the literature, Zone 3 was established as a phenyl group, by means of reference, and as a 4-trifluoromethyl-phenyl group or 4-tert-butylphenyl group, considering their beneficial effect on the biological activity against S. aureus for other tripartite compounds reported in previous studies [6,7].
The synthetic route for the series I of triazole derivatives is shown in Scheme 1. Benzamide 1 synthetized as previously described [9] was alkylated with propargyl bromide to afford alkyne 2. Various anilines 3 were converted into the corresponding azides using The synthetic route for the series II of 1,2,4-oxadiazoles is described in Scheme 2. The series II was obtained with various benzonitriles 4 converted to corresponding Nhydroxybenzimidamides 5 [12] and acylated with chloroacetyl chloride and cyclized to give compounds 6 [13]. Alkylation of benzamide 1 afforded the oxadiazoles II.a-c [14].
Based on the literature, Zone 3 was established as a phenyl group, by means of reference, and as a 4-trifluoromethyl-phenyl group or 4-tert-butylphenyl group, considering their beneficial effect on the biological activity against S. aureus for other tripartite compounds reported in previous studies [6,7].
The synthetic route for the series II of 1,2,4-oxadiazoles is described in Scheme 2. The series II was obtained with various benzonitriles 4 converted to corresponding N-hydroxybenzimidamides 5 [12] and acylated with chloroacetyl chloride and cyclized to give compounds 6 [13]. Alkylation of benzamide 1 afforded the oxadiazoles II.a-c [14]. The other 1,2,4-oxadiazoles from series III were obtained as described in Scheme 3. Chloroacetonitrile was converted in 2-chloro-N-hydroxyacetimidamide 8 which was then acylated by various benzoyl chlorides and cyclized to afford compounds 9. Subsequent alkylation of benzamide 1 afforded the oxadiazoles III.a-c.

Biological Evaluation
The series I, II, III, and IV were tested for their antimicrobial activity on three strains of S. aureus, including a reference (ATCC 29213) and two clinically isolated strains respectively resistant to methicillin (SF8300) [16] and daptomycin (ST20171643) [17], with PC190723 as control. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the triazole and oxadiazole series. The results are presented in Table 1. The synthetic route for series IV is described in Scheme 4. Various benzoic acids 10 and tert-butyl carbazate was coupled using DCC as coupling agent. The Boc protecting group was cleaved using TFA, the free hydrazine was acylated by chloroacetyl chloride, and the compound was cyclized in presence of POCl3 [15] to give compounds 12. Alkylation of benzamide 1 afforded the oxadiazoles IV.a-c.

Biological Evaluation
The series I, II, III, and IV were tested for their antimicrobial activity on three strains of S. aureus, including a reference (ATCC 29213) and two clinically isolated strains respectively resistant to methicillin (SF8300) [16] and daptomycin (ST20171643) [17], with PC190723 as control. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the triazole and oxadiazole series. The results are presented in Table 1. The synthetic route for series IV is described in Scheme 4. Various benzoic acids 10 and tert-butyl carbazate was coupled using DCC as coupling agent. The Boc protecting group was cleaved using TFA, the free hydrazine was acylated by chloroacetyl chloride, and the compound was cyclized in presence of POCl3 [15] to give compounds 12. Alkylation of benzamide 1 afforded the oxadiazoles IV.a-c.

Biological Evaluation
The series I, II, III, and IV were tested for their antimicrobial activity on three strains of S. aureus, including a reference (ATCC 29213) and two clinically isolated strains respectively resistant to methicillin (SF8300) [16] and daptomycin (ST20171643) [17], with PC190723 as control. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the triazole and oxadiazole series. The results are presented in Table 1. and tert-butyl carbazate was coupled using DCC as coupling agent. The Boc protecting group was cleaved using TFA, the free hydrazine was acylated by chloroacetyl chloride, and the compound was cyclized in presence of POCl3 [15] to give compounds 12. Alkylation of benzamide 1 afforded the oxadiazoles IV.a-c.

Biological Evaluation
The series I, II, III, and IV were tested for their antimicrobial activity on three strains of S. aureus, including a reference (ATCC 29213) and two clinically isolated strains respectively resistant to methicillin (SF8300) [16] and daptomycin (ST20171643) [17], with PC190723 as control. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the triazole and oxadiazole series. The results are presented in Table 1. The triazole and the 1,3,4-oxadiazole compounds were found deprived of any antimicrobial activity against S. aureus at a concentration up to 256 µg/mL. Compounds from series II, with a 1,2,4-oxadiazole ring, had a better anti-staphylococcal activity than the oxadiazoles regioisomers of series III considering the same substitution of the phenyl ring. The 4-tert-butylphenyl substituent led to an improved activity for all series compared with other substituents, leading to the identification of compound II.c which had an MIC value between 0.5 and 1 µg/mL according to the staphylococcal strain. Interestingly, the activity in each series I-IV was correlated with the calculated LogP with the substituents t-Bu > CF3 > H. Additionally, between the series I-IV with the same phenyl substitution, an increased LogP correlated with an improved activity. The 1,2,4-oxadiazole derivative II.c was thus found to be particularly active with a lower MIC value compared with the corresponding isoxazole derivative [7].

Optimization of Series II
The promising MIC values obtained for II.c led to a structural optimization of this compound in order to improve its anti-staphylococcal activity. As described in the literature [6], the addition of a hydrophobic methyl group on the methylene group between the phenol and the five members heteroaromatic ring could improve the biological activity. As a consequence, the synthetic route for series V was investigated and is shown in Scheme 5. The N-hydroxybenzimidamides 5 were acylated with 2-chloropropionyl chlo- The triazole and the 1,3,4-oxadiazole compounds were found deprived of any antimicrobial activity against S. aureus at a concentration up to 256 µg/mL. Compounds from series II, with a 1,2,4-oxadiazole ring, had a better anti-staphylococcal activity than the oxadiazoles regioisomers of series III considering the same substitution of the phenyl ring. The 4-tert-butylphenyl substituent led to an improved activity for all series compared with other substituents, leading to the identification of compound II.c which had an MIC value between 0.5 and 1 µg/mL according to the staphylococcal strain. Interestingly, the activity in each series I-IV was correlated with the calculated LogP with the substituents t-Bu > CF 3 > H. Additionally, between the series I-IV with the same phenyl substitution, an increased LogP correlated with an improved activity. The 1,2,4-oxadiazole derivative II.c was thus found to be particularly active with a lower MIC value compared with the corresponding isoxazole derivative [7].

Optimization of Series II
The promising MIC values obtained for II.c led to a structural optimization of this compound in order to improve its anti-staphylococcal activity. As described in the litera-ture [6], the addition of a hydrophobic methyl group on the methylene group between the phenol and the five members heteroaromatic ring could improve the biological activity. As a consequence, the synthetic route for series V was investigated and is shown in Scheme 5. The N-hydroxybenzimidamides 5 were acylated with 2-chloropropionyl chloride and cyclized to give compounds 13. Alkylation of benzamide 1 afforded the oxadiazoles V.a-c. Enantiopure synthesis was first attempted, but partial racemization occurred. The study of racemic compounds was, therefore, preferred, by using racemic chloropropionyl chloride during their synthesis.
an increased LogP correlated with an improved activity. The 1,2,4-oxadiazole derivative II.c was thus found to be particularly active with a lower MIC value compared with the corresponding isoxazole derivative [7].

Optimization of Series II
The promising MIC values obtained for II.c led to a structural optimization of this compound in order to improve its anti-staphylococcal activity. As described in the literature [6], the addition of a hydrophobic methyl group on the methylene group between the phenol and the five members heteroaromatic ring could improve the biological activity. As a consequence, the synthetic route for series V was investigated and is shown in Scheme 5. The N-hydroxybenzimidamides 5 were acylated with 2-chloropropionyl chloride and cyclized to give compounds 13. Alkylation of benzamide 1 afforded the oxadiazoles V.a-c. Enantiopure synthesis was first attempted, but partial racemization occurred. The study of racemic compounds was, therefore, preferred, by using racemic chloropropionyl chloride during their synthesis. The MIC values for series V are shown in Table 2. Interestingly, the additional hydrophobic methyl group significantly increases the antibacterial ability for the less active compounds (phenyl and trifluoromethylphenyl derivatives), reaching 1 µg/mL for V.b, comparable to the MIC values of the most active II.c. The MIC values for series V are shown in Table 2. Interestingly, the additional hydrophobic methyl group significantly increases the antibacterial ability for the less active compounds (phenyl and trifluoromethylphenyl derivatives), reaching 1 µg/mL for V.b, comparable to the MIC values of the most active II.c.

Mechanism of Action
Filamentous temperature-sensitive mutant Z (FtsZ) is an essential protein for bacterial cytokinesis, a homologue to mammalian tubulin. During division, GTP-bound FtsZ polymerizes into protofilaments to form the Z-ring, a mid-cell scaffold, allowing for the recruitment and assembly of other division proteins [18,19]. Therefore, FtsZ inhibition is detrimental for the bacteria and is leading to cell death, and this protein is considered to be a valuable pharmacological target [20][21][22][23][24][25]. Since 2,6-difluorobenzamide derivatives are regularly described as FtsZ inhibitors and exert an alteration of the morphology of the bacteria by inducing aberrant localization of division septum, the action of II.c on the bacterial divisome was studied by cell wall staining. Bacteria (strain ATCC 29213) were incubated with compound II.c at the minimal inhibitory concentration for 4 h before fluorescent staining of the wall with a vancomycin-bodipy conjugate [26]. PC190723 was used as a positive control. As presented in Figure 3, dividing control bacteria are small (1 µm of diameter) with a centered division septum. On the contrary, bacteria incubated with II.c show the same morphology as bacteria incubated with PC190723, including larger overall size (approximately 1.5 µm of diameter) and wrongly localized division septum, indicating bacterial divisome targeting. Identical results were obtained with SF8300 (MRSA) S. aureus (see Figure S1, supplementary information). These results, added to structural similarities between II.c and described compounds, strongly suggest FtsZ inhibition.

Mechanism of Action
Filamentous temperature-sensitive mutant Z (FtsZ) is an essential protein for bacterial cytokinesis, a homologue to mammalian tubulin. During division, GTP-bound FtsZ polymerizes into protofilaments to form the Z-ring, a mid-cell scaffold, allowing for the recruitment and assembly of other division proteins [18,19]. Therefore, FtsZ inhibition is detrimental for the bacteria and is leading to cell death, and this protein is considered to be a valuable pharmacological target [20][21][22][23][24][25]. Since 2,6-difluorobenzamide derivatives are regularly described as FtsZ inhibitors and exert an alteration of the morphology of the bacteria by inducing aberrant localization of division septum, the action of II.c on the bacterial divisome was studied by cell wall staining. Bacteria (strain ATCC 29213) were incubated with compound II.c at the minimal inhibitory concentration for 4 h before fluorescent staining of the wall with a vancomycin-bodipy conjugate [26]. PC190723 was used as a positive control. As presented in Figure 3, dividing control bacteria are small (1 µm of diameter) with a centered division septum. On the contrary, bacteria incubated with II.c show the same morphology as bacteria incubated with PC190723, including larger overall size (approximately 1.5 µm of diameter) and wrongly localized division septum, indicating bacterial divisome targeting. Identical results were obtained with SF8300 (MRSA) S. aureus (see Figure S1, Supplementary Information). These results, added to structural similarities between II.c and described compounds, strongly suggest FtsZ inhibition. a positive control. As presented in Figure 3, dividing control bacteria are small (1 µm of diameter) with a centered division septum. On the contrary, bacteria incubated with II.c show the same morphology as bacteria incubated with PC190723, including larger overall size (approximately 1.5 µm of diameter) and wrongly localized division septum, indicating bacterial divisome targeting. Identical results were obtained with SF8300 (MRSA) S. aureus (see Figure S1, supplementary information). These results, added to structural similarities between II.c and described compounds, strongly suggest FtsZ inhibition.

Molecular Docking
A docking study of II.c was performed on the crystal structure of S. aureus FtsZ within the allosteric binding site (PDB code 6KVP [27]). The side chain of 13 amino acids of this allosteric pocket (namely Met98, Val129, Ile197, Leu200, Val203, Asn208, Leu209, Val214, Met218, Ile228, Asn263, Val297, and Thr309) were set as flexible in order to best mimic potential movements of the pocket which was found to be dynamic [28].
The three parts of the molecule show multiple interactions with the protein (Figure 4): the benzamide moiety forms an extensive hydrogen bond network with the protein, the tert-butylphenyl group is deeply inserted in the hydrophobic part of the pocket, generating van der Waals interactions, and the oxadiazole ring is able to form an H-bond with Thr309. Therefore, the position of the oxygen atom plays a central role for the activity of compounds of series II by increasing their lipophilicity and by possibly interacting with Thr309 via H-bond. The importance of the presence of an oxygen atom has been also observed in benzodioxane derivatives [29,30]. Docking simulations of this type of compounds and of compound II.c led to a superimposition of the oxygen atoms (see Figure S2, Supplementary  Information). Docking simulations of both enantiomers of compounds V.b and V.c were also performed and showed good superimpositions between the (R) derivatives and the co-crystallized inhibitor [6] (see Figure S3, Supplementary Information). These results are in agreement with the literature for substituted oxazole derivatives, showing better activity for (R) enantiomers.
served in benzodioxane derivatives [29,30]. Docking simulations of this type of compounds and of compound II.c led to a superimposition of the oxygen atoms (see Figure  S2, supplementary information). Docking simulations of both enantiomers of compounds V.b and V.c were also performed and showed good superimpositions between the (R) derivatives and the co-crystallized inhibitor [6] (see Figure S3, Supplementary Information). These results are in agreement with the literature for substituted oxazole derivatives, showing better activity for (R) enantiomers.

Cytotoxicity Assay
Due to its interesting anti-staphylococcal activity, the potential mammalian cytotoxicity of II.c was examined. Therefore, a 7 h propidium iodide (PI)-based assay was used on adenocarcinomic human alveolar basal epithelial cells (A549), with recombinant S. aureus α-hemolysin as positive control. As depicted in Figure 5, a fluorescent maximum was reached within two hours in the presence of α-hemolysin. A small rise was observed for negative control and II.c after 6 h. The two curves are superimposed, showing an absence of cytotoxicity for II.c after 7 h at a concentration of 64 µg/mL (64 times the MIC).

Cytotoxicity Assay
Due to its interesting anti-staphylococcal activity, the potential mammalian cytotoxicity of II.c was examined. Therefore, a 7 h propidium iodide (PI)-based assay was used on adenocarcinomic human alveolar basal epithelial cells (A549), with recombinant S. aureus α-hemolysin as positive control. As depicted in Figure 5, a fluorescent maximum was reached within two hours in the presence of α-hemolysin. A small rise was observed for negative control and II.c after 6 h. The two curves are superimposed, showing an absence of cytotoxicity for II.c after 7 h at a concentration of 64 µg/mL (64 times the MIC).

Materials and Methods
All commercial materials were used as received without further purification. Flash chromatography was carried out using Macherey-Nagel Kieselgel 60 M silica. Analytical thin-layer chromatography was realized using aluminum-backed plates coated with Macherey-Nagel Kieselgel 60 XtraSIL G/UV254 and were visualized under UV light (at 254

Materials and Methods
All commercial materials were used as received without further purification. Flash chromatography was carried out using Macherey-Nagel Kieselgel 60 M silica. Analytical thin-layer chromatography was realized using aluminum-backed plates coated with Macherey-Nagel Kieselgel 60 XtraSIL G/UV254 and were visualized under UV light (at 254 nm or 365 nm) or stained using ninhydrin. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AV300, Bruker AV400, or Bruker AV500 spectrometers, operating at 300 MHz, 400 MHz, and 500 MHz, respectively, for the proton ( 1 H) NMR and at 75 MHz, 100 MHz, and 125 MHz, respectively, for the carbon ( 13 C) NMR. Chemical shifts were reported in parts per million (ppm) on a scale relative to residual solvent signals. Multiplicities are abbreviated as: s, singlet; d, doublet; t, triplet; q, quadruplet; dd, doublet of doublets; dt, doublet of triplets; td, triplet of doublets; ddd, doublet of doublet of doublets; and m, multiplet. Coupling constants were measured in Hertz (Hz). Copies of NMR spectra of final products are available in Supplementary Materials. High-resolution mass spectra (HRMS) and low-resolution mass spectra were obtained by the Centre Commun de Spectrométrie de Masse (CCSM), University of Lyon 1, Lyon, France. LogP calculations were performed using Molinspiration Cheminformatics free web services, https://www.molinspiration.com (accessed on 6 December 2021), Slovensky Grob, Slovakia.

Cytotoxicity Assay
Cytotoxicity was evaluated using a propidium iodide assay. Briefly, human lung adenocarcinomic A549 cells were plated on black flat 96-well plates (Greiner, Pleidelsheim, Germany) at a concentration of 0.5 × 10 6 cells/mL in DMEM GlutaMAX (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum for 24 h in a humidified atmosphere at 37 • C and 5% CO 2 . The medium was then removed, and fresh medium with 2.5 µg/mL of propidium iodide and tested compounds (64 µg/mL of II.c and 0.2 µg/mL for recombinant Hla) was added. Plates were incubated for 7 h at 37 • C, and the cytotoxicity induced was evaluated by measuring propidium iodide (PI) incorporation into cells exposed to toxin or compounds using a fluorescence cell sorter (Spark R, TECAN, Zürich, Switzerland). Each concentration was made as a triplicate, and mean values are displayed with standard deviation.

Conclusions
A collection of 15 new analogs of tripartite benzamide FtsZ inhibitors, varying in the nature of the central heterocyclic motif, was synthesized and evaluated against three S. aureus strains. It was found that the activities were correlated to the nature of the central heterocycle and also to the overall hydrophobicity of the compounds. Compound II.c with a central 1,2,4-oxadiazole ring, was found to be the most active in this study with MIC values of 0.5 µg/mL (for methicillin-sensitive and daptomycin-resistant S. aureus) and 1 µg/mL (for methicillin-resistant S. aureus). This compound is also deprived of any toxicity and induces an abnormal bacterial morphology due to a wrongly localized division septum.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27196619/s1. Figure S1 (fluorescence microscope images with SF8300 bacteria), Figure S2 (docking studies of benzodioxane derivative), Figure  S3 (docking studies of both enantiomers of compounds V.b and V.c) and copies of 1H and 13C NMR spectra of final products are available in supplementary information.