Importance of the 2,6-Difluorobenzamide Motif for FtsZ Allosteric Inhibition: Insights from Conformational Analysis, Molecular Docking and Structural Modifications

A conformational analysis and molecular docking study comparing 2,6-difluoro-3-methoxybenzamide (DFMBA) with 3-methoxybenzamide (3-MBA) has been undertaken for investigating the known increase of FtsZ inhibition related anti S. aureus activity due to fluorination. For the isolated molecules, the calculations reveal that the presence of the fluorine atoms in DFMBA is responsible for its non-planarity, with a dihedral angle of -27° between the carboxamide and the aromatic ring. When interacting with the protein, the fluorinated ligand can thus more easily adopt the non-planar conformation found in reported co-crystallized complexes with FtsZ, than the non-fluorinated one. Molecular docking studies of the favored non-planar conformation of 2,6-difluoro-3-methoxybenzamide highlights the strong hydrophobic interactions between the difluoroaromatic ring and several key residues of the allosteric pocket, precisely between the 2-fluoro substituent and residues Val203 and Val297 and between the 6-fluoro group and the residues Asn263. The docking simulation in the allosteric binding site also confirms the critical importance of the hydrogen bonds between the carboxamide group with the residues Val207, Leu209 and Asn263. Changing the carboxamide functional group of 3-alkyloxybenzamide and 3-alkyloxy-2,6-difluorobenzamide to a benzohydroxamic acid or benzohydrazide led to inactive compounds, confirming the importance of the carboxamide group.


Introduction
Filamentous temperature-sensitive protein Z (FtsZ) plays a major role in bacterial division like tubulin in eukaryotic cells [1]. The Z ring is required for this process and is formed by the FtsZ recruitment and polymerization [2,3]. Subsequently the contraction of the Z ring is leading to the separation of two cells. As a key protein of the bacterial divisome, the FtsZ protein is extensively studied either for the biological understanding of the cell division process in different species but also to interfere with bacterial growth to develop new antibiotics [4,5]. For this purpose, different strategies are commonly investigated such as the design of FtsZ-ZipA interactions inhibitors, or FtsZ inhibitors targeting the GTP binding site or other allosteric pockets [6][7][8][9]. The latter approach is probably the most studied and the discovery of the benzamide scaffold or more precisely the 2,6-difluorobenzamide nucleus has offered new opportunities in the development of FtsZ allosteric inhibitors [10]. The compounds 2,6-difluoro-3-methoxybenzamide (DFMBA), for which full crystallographic data of its complex with FTSZ are available [11], has been indeed found to be more active as antibacterial agent against S. aureus than the 3-methoxybenzamide (3-MBA) and Importance of fluorination in medicinal chemistry is now well recognized for improving metabolic stability and also to induce several possible effects, either electronic, steric or polar vs non-polar interactions [20][21][22].
After having focused on the azole moiety of tripartite benzamide inhibitors of FtsZ [23], we now report our studies focusing on the fluorobenzamide moiety. The work reported herein aimed at understanding why this difluorobenzamide motif is so important, through conformational and molecular docking studies investigating 3-MBA and DFMBA for which co-crystallographic with FtsZ data are known. In addition, slight structural variations of the carboxamide functional group, such as benzohydroxamic acid or benzohydrazide were investigated.

Results and Discussions
The benzamide scaffold is extensively used to develop antibacterial agents in relation with the ability to inhibit FtsZ, with significant increased activity when fluorinated. This difference of can be explained by hydrophobic interactions of the fluorine atoms of the benzamide nucleus with the allosteric site of the FtsZ proteins. In order to visualize these interactions, the complex between DFMBA and the staphylococcal FtsZ [11] was submitted to the Arpeggio webserver [24] which monitors all interactions between a ligand and a protein ( Figure 2). In addition to three hydrogen bonds between the carboxamide functional group and the residues Val207, Leu209 and Asn263, the difluorobenzamide motif develops key interactions via hydrophobic interactions [25] between the 6-fluoro substituent and the central CH of the isopropyl group of the residue Val203 with (distance F→C of 3.3 Å ) and also with the terminal methyl group of the residue Val297 (distance F→C of 2.9 Å ). The 2-fluoro substituent interacts with the residues Asn263 via C-F/C=ONH2 interactions [20] with a F→C=O distance of 3.2 Å and a F→O=C distance of 3.0 Å . Importance of fluorination in medicinal chemistry is now well recognized for improving metabolic stability and also to induce several possible effects, either electronic, steric or polar vs non-polar interactions [20][21][22].
After having focused on the azole moiety of tripartite benzamide inhibitors of FtsZ [23], we now report our studies focusing on the fluorobenzamide moiety. The work reported herein aimed at understanding why this difluorobenzamide motif is so important, through conformational and molecular docking studies investigating 3-MBA and DFMBA for which co-crystallographic with FtsZ data are known. In addition, slight structural variations of the carboxamide functional group, such as benzohydroxamic acid or benzohydrazide were investigated.

Results and Discussion
The benzamide scaffold is extensively used to develop antibacterial agents in relation with the ability to inhibit FtsZ, with significant increased activity when fluorinated. This difference of can be explained by hydrophobic interactions of the fluorine atoms of the benzamide nucleus with the allosteric site of the FtsZ proteins. In order to visualize these interactions, the complex between DFMBA and the staphylococcal FtsZ [11] was submitted to the Arpeggio webserver [24] which monitors all interactions between a ligand and a protein ( Figure 2). In addition to three hydrogen bonds between the carboxamide functional group and the residues Val207, Leu209 and Asn263, the difluorobenzamide motif develops key interactions via hydrophobic interactions [25] between the 6-fluoro substituent and the central CH of the isopropyl group of the residue Val203 with (distance F→C of 3.3 Å) and also with the terminal methyl group of the residue Val297 (distance F→C of 2.9 Å). The 2-fluoro substituent interacts with the residues Asn263 via C-F/C=ONH 2 interactions [20] with a F→C=O distance of 3.2 Å and a F→O=C distance of 3.0 Å.
To further investigate the difluorobenzamide nucleus, conformational analysis of the 3-methoxybenzamide 3-MBA and the 2,6-difluoro-3-methoxybenzamide DFMBA was then performed to determine their preferential conformations. A first important observation is that the co-crystallized structure of PC190723 in FtsZ, [26,27] like other difluorobenzamide derivatives [11,28] within the allosteric pocket of the protein shows that the difluorobenzamide nucleus is not planar, though conjugated ( Figure 3A). This is also the case for other 2,6-difluorobenzamide-based ligands co-crystallized with FtsZ. For this purpose, a systematic conformational search [29] with an increment of 10 • was applied using VEGA ZZ [30,31] on the dihedral angle defined by the rotation around the bond between the amide functional group and the aromatic ring. As depicted in Figure 3B and as expected the preferential conformation of the conjugated benzamide of 3-MBA is a planar conformation with angle values of 0 • or 180 • allowing the conjugation. For the 2,6-difluorobenzamide scaffold of DFMBA, the curve resulting from the conformational analysis reveals that conformations with the lowest energy are not planar, but with a torsion angle value of −27 • .
Molecules 2023, 28, x FOR PEER REVIEW 3 of 15 Figure 2. Visualization of the interactions revealed by the Arpeggio server [24] indicated in red dashes of the fluorine atoms of the benzamide nucleus with residues of the FtsZ protein and 2D representation of DFMBA in the allosteric pocket (PDB: 6YD1 [11]).
To further investigate the difluorobenzamide nucleus, conformational analysis of the 3-methoxybenzamide 3-MBA and the 2,6-difluoro-3-methoxybenzamide DFMBA was then performed to determine their preferential conformations. A first important observation is that the co-crystallized structure of PC190723 in FtsZ, [26,27] like other difluorobenzamide derivatives [11,28] within the allosteric pocket of the protein shows that the difluorobenzamide nucleus is not planar, though conjugated ( Figure 3A). This is also the case for other 2,6-difluorobenzamide-based ligands co-crystallized with FtsZ. For this purpose, a systematic conformational search [29] with an increment of 10° was applied using VEGA ZZ [30,31] on the dihedral angle defined by the rotation around the bond between the amide functional group and the aromatic ring. As depicted in Figure 3B and These results contrast with the conjugated benzamide scaffold, for which the most stable conformations are planar. Therefore the torsion angle value of −27 • due to the presence of the fluorine atoms is closer to the value of the same torsion measured in cocrystallized ligand (−58 • , Figure 3A). Moreover, the presence of the fluorine atoms, which did not allow conjugation of the π system, reduces the energetic differences of the bound versus unbound ligand. Indeed, the energetic gap to reach a dihedral angle of −58 • from the most stable conformation is 3.71 kcal/mol for benzamide (360 • → 302 • ) while only 1.98 kcal/mol for 2,6-difluorobenzamide (333 • → 302 • ). As a consequence, fluorinated benzamide derivatives, which are now the common pattern of FtsZ allosteric inhibitors may also be considered as conformationally restrained inhibitors [32] showing more potency than their non-fluorinated counterparts. Consistent angles were measured for the hexyl derivatives 3-HBA and DFHBA (data shown in Supplementary Materials see Figure S1).
The preferential conformation of DFMBA with a torsion angle value of −27 • was docked as rigid ligand within the allosteric site of SaFtsZ in order to compare the binding mode with the co-crystallized ligand ( Figure 4). Both conformations, the one with a torsion angle value of −27 • and the co-crystallized ligand (with a torsion angle value of −58 • ) are close.
A short investigation of structural analogs in which the benzamide group is changed to isosteric groups [33] benzohydroxamic acids and benzohydrazides was then conducted, with either non-fluorinated or fluorinated phenyl groups, and comparison to the know FtsZ inhibitors 3-hexyloxybenzamide (3-HBA) and 3-hexyloxy-2,6-difluorobenzamide (DFHBA) used here as model compounds exerting cell division inhibition of B. subtilis [14] with higher activity compared to the methyloxybenzamide derivative ( Figure 5) [15]. DFHBA is also know to induce morphological modifications of B. subtilis and S. aureus bacteria observed by morphometric analysis [14].
With respect to their conformation, 9, 10, 13, 14 show comparable behavior to that of 3-HBA and DFHBA, with the fluorine atoms inducing nonplanar stable conformations in contrast to those adopted by non-fluorinated compounds ( Figure 6). A torsion angle value of −30 • was obtained for the most stable conformations for fluorinated benzohydroxamic acid and benzohydrazides, which is comparable to DFMBA and DFHBA.
3-(Hexyloxy) benzohydroxamic acids 9 and 10 were prepared from the corresponding methyl esters 3 and 8 and hydroxylamine in basic conditions. The ester 3 was synthesized starting from 3-hydroxybenzoic acid 1 which was converted to its methyl ester counterpart with thionyl chloride in dry methanol followed by the alkylation of the phenol functional group with hexylbromide using potassium carbonate. The ester 8 was obtained starting from the 2,4-difluorophenol which was alkylated with hexylbromide followed by an ortholithiation and subsequent carboxylation in the presence of dry ice to give the acid 7. The latter was converted to the methyl ester with thionyl chloride in dry methanol. The benzohydrazides 13 and 14 were prepared from the non-fluorinated and fluorinated acids 4 and 7 through a standard coupling reaction with tert-butyl carbazate and DCC and subsequent deprotection in acidic conditions.
The antimicrobial activity was assessed by determining the corresponding MIC values against three S. aureus strains: a reference strain (ATCC29213), a methicillin-resistant strain (SF8300) [34] and a clinically isolated daptomycin-resistant strain (ST20171643) [35] (Table 1). 3-HBA and DFHBA, used as references, exert MIC values of 256 µg/mL and 8 µg/mL consistent with the literature [14][15][16]. For the benzohydroxamic acid or benzohydrazides, either fluorinated of non-fluorinated, the activity is at least decreased to 64 µg/mL. difluorobenzamide scaffold of DFMBA, the curve resulting from the conformational analysis reveals that conformations with the lowest energy are not planar, but with a torsion angle value of −27°.  derivatives 3-HBA and DFHBA (data shown in Supplementary Materials see Figure S1).
The preferential conformation of DFMBA with a torsion angle value of −27° was docked as rigid ligand within the allosteric site of SaFtsZ in order to compare the binding mode with the co-crystallized ligand (Figure 4). Both conformations, the one with a torsion angle value of −27° and the co-crystallized ligand (with a torsion angle value of −58°) are close. A short investigation of structural analogs in which the benzamide group is changed to isosteric groups [33] benzohydroxamic acids and benzohydrazides was then conducted, with either non-fluorinated or fluorinated phenyl groups, and comparison to the know FtsZ inhibitors 3-hexyloxybenzamide (3-HBA) and 3-hexyloxy-2,6-difluorobenzamide (DFHBA) used here as model compounds exerting cell division inhibition of B. subtilis [14] with higher activity compared to the methyloxybenzamide derivative ( Figure  5) [15]. DFHBA is also know to induce morphological modifications of B. subtilis and S. aureus bacteria observed by morphometric analysis [14]. With respect to their conformation, 9, 10, 13, 14 show comparable behavior to that of 3-HBA and DFHBA, with the fluorine atoms inducing nonplanar stable conformations in contrast to those adopted by non-fluorinated compounds ( Figure 6). A torsion angle value of −30° was obtained for the most stable conformations for fluorinated benzohydroxamic acid and benzohydrazides, which is comparable to DFMBA and DFHBA. The modification of the benzamide motif is therefore highly detrimental to the activity, consistently with previous other attempts such as with a substitution to a sulfonamide group [36]. As demonstrated in this study, the 2,6-difluorobenzamide motif could be considered as both, a conformational restrained scaffold [32] with fluorine acting as conformational control element and an optimized structure to develop hydrophobic and C-F/C=O interactions as well as several hydrogen bonds. With respect to the biological activity, the four analogs 9, 10, 13, 14 were evaluated as antimicrobial agents against three S. aureus strains, and compared with compounds 3-HBA and DFHBA used as references. The non-fluorinated and fluorinated 3-hexyloxy-2,6-difluorobenzamide analogues were synthesized as depicted in Scheme 1. 3-HBA and DFHBA were synthesized as previously reported from the compounds 3-hydroxyben zamide and 3-hydroxy-2,6-difluorobenzamide which were alkylated with 1-bromohex ane. [14,15] Scheme 3-(Hexyloxy) benzohydroxamic acids 9 and 10 were prepared from the correspond ing methyl esters 3 and 8 and hydroxylamine in basic conditions. The ester 3 was synthe sized starting from 3-hydroxybenzoic acid 1 which was converted to its methyl ester coun terpart with thionyl chloride in dry methanol followed by the alkylation of the pheno functional group with hexylbromide using potassium carbonate. The ester 8 was obtained starting from the 2,4-difluorophenol which was alkylated with hexylbromide followed by an ortho-lithiation and subsequent carboxylation in the presence of dry ice to give the acid  The antimicrobial activity was assessed by determining the corresponding M ues against three S. aureus strains: a reference strain (ATCC29213), a methicillinstrain (SF8300) [34] and a clinically isolated daptomycin-resistant strain (ST20171 (Table 1). 3-HBA and DFHBA, used as references, exert MIC values of 256 µ g/m µ g/mL consistent with the literature [14][15][16]. For the benzohydroxamic acid or b drazides, either fluorinated of non-fluorinated, the activity is at least decreas µ g/mL. The modification of the benzamide motif is therefore highly detrimental to t ity, consistently with previous other attempts such as with a substitution to a sulfo group [36]. As demonstrated in this study, the 2,6-difluorobenzamide motif could sidered as both, a conformational restrained scaffold [32] with fluorine acting as mational control element and an optimized structure to develop hydrophobic F/C=O interactions as well as several hydrogen bonds.

Conformational Analysis
The compounds DFMBA, 3-MBA and analogues were drawn using VEGA Z and were minimized using the SP4 force field with Gasteiger charges. Conform analyses were carried out using the conformational search module by varying th angle defined by the bond between the benzamide, the hydroxamic acid or the hy and the aromatic ring using a 10° increment and minimization of each generated c ers. Subsequently to the conformational analyses, energy graphs were created to the conformation landscape of the compounds and to find preferential conforma

Molecular Docking
Subsequently to the conformational analysis, the preferential conformatio DFMBA with a torsion angle value of −27° was retrieved and a rigid ligand dock performed on Staphylococcus aureus SaFtsZ (PDB: 6YD1) with Arguslab [37] with a box of 15 Å × 15 Å × 15 Å centered on the ligand using the genetic algorithm with parameters. The docking result was superimposed to the structure of the co-cry DFMBA (PDB: 6YD1). 3D-Visualization was performed with PyMOL which was create Figure 1 and ues against three S. aureus strains: a reference strain (ATCC29213), a methicillinstrain (SF8300) [34] and a clinically isolated daptomycin-resistant strain (ST20171 (Table 1). 3-HBA and DFHBA, used as references, exert MIC values of 256 µ g/m µ g/mL consistent with the literature [14][15][16]. For the benzohydroxamic acid or b drazides, either fluorinated of non-fluorinated, the activity is at least decreas µ g/mL. The modification of the benzamide motif is therefore highly detrimental to t ity, consistently with previous other attempts such as with a substitution to a sulfo group [36]. As demonstrated in this study, the 2,6-difluorobenzamide motif could sidered as both, a conformational restrained scaffold [32] with fluorine acting as mational control element and an optimized structure to develop hydrophobic F/C=O interactions as well as several hydrogen bonds.

Conformational Analysis
The compounds DFMBA, 3-MBA and analogues were drawn using VEGA Z and were minimized using the SP4 force field with Gasteiger charges. Conform analyses were carried out using the conformational search module by varying th angle defined by the bond between the benzamide, the hydroxamic acid or the hy and the aromatic ring using a 10° increment and minimization of each generated c ers. Subsequently to the conformational analyses, energy graphs were created to the conformation landscape of the compounds and to find preferential conforma

Molecular Docking
Subsequently to the conformational analysis, the preferential conformatio DFMBA with a torsion angle value of −27° was retrieved and a rigid ligand dock performed on Staphylococcus aureus SaFtsZ (PDB: 6YD1) with Arguslab [37] with a box of 15 Å × 15 Å × 15 Å centered on the ligand using the genetic algorithm with parameters. The docking result was superimposed to the structure of the co-cry DFMBA (PDB: 6YD1). 3D-Visualization was performed with PyMOL which was create Figure 1 and

Conformational Analysis
The compounds DFMBA, 3-MBA and analogues were drawn using VEGA ZZ [30,31] and were minimized using the SP4 force field with Gasteiger charges. Conformational analyses were carried out using the conformational search module by varying the torsion angle defined by the bond between the benzamide, the hydroxamic acid or the hydrazide and the aromatic ring using a 10 • increment and minimization of each generated conformers. Subsequently to the conformational analyses, energy graphs were created to analyze the conformation landscape of the compounds and to find preferential conformations.

Molecular Docking
Subsequently to the conformational analysis, the preferential conformation of the DFMBA with a torsion angle value of −27 • was retrieved and a rigid ligand docking was performed on Staphylococcus aureus SaFtsZ (PDB: 6YD1) with Arguslab [37] with a docking box of 15 Å × 15 Å × 15 Å centered on the ligand using the genetic algorithm with default parameters. The docking result was superimposed to the structure of the co-crystallized DFMBA (PDB: 6YD1). 3D-Visualization was performed with PyMOL which was used to create Figures 1 and 4. 2D-representation was created with LigPlot+ [38] for Figure 1.

Chemistry
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, 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, quadruplets; dd, doublet of doublets; dt, doublet of triplets; td, triplet of doublets; ddd, doublet of doublet of doublets; m, multiplet. Coupling constants were measured in Hertz (Hz). 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. 3-HBA and DFHBA were synthesized by alkylation of 3-hydroxybenzamide and 2,6-difluoro3-hydroxybenzamide with 1-bromohexane as previously reported [14,15]

(iv), (v) Procedure for 2,6-difluoro-3-(hexyloxy)benzoic Acid
To a solution of 2,4-difluoro-1-(hexyloxy)benzene (7.4 mmol, 1.0 eq.) in THF (15 mL) under a dried and inert atmosphere (N 2 ) at −78 • C was added dropwise n-BuLi (1.6 M in THF, 8.1 mmol, 1.1 eq.). The solution was stirred at −78 • C for 1 h. The mixture was then added dropwise on crushed dry ice, and the solution was stirred at room temperature for 2 h. To the mixture was then added NaOH solution (2 M, 11 mL) and was extracted with Et 2 O to remove unreacted reagent. The solution was then acidified with an HCl 2 M solution, and the product was extracted with EtOAc, dried over Na 2 SO 4 and concentrated under vacuum to give the desired product.

Conclusions
This study contributes to the knowledge on the important fluorinated benzamide pharmacophore for allosteric inhibition of SaFtsZ. While the benzamide function was confirmed to be essential for reaching FtsZ inhibiting activity, the reason for increased activity due to fluorination has been scrutinized from the conformational viewpoint. The comparison of conformational analyses of 3-MBA and DFMBA unveiled the influence of the fluorine atoms on the conformation of the 2,6-difluorobenzamide by inducing a nonplanar conformation, rending easier for the ligand to adopt the known active conformation. Docking studies of the calculated preferential conformation shows results very close to the co-crystallized structure, indicating van der Walls interactions of the fluorine atoms with the residues Val203, Val297 and Asn263, while the benzamide functional group develops interactions with Val207, Leu209 and Asn263 via hydrogen bonds.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28052055/s1. Figure S1: Conformational analysis of DFHBA and 3-HBA obtained as a result of variation of the torsion angle between the carboxamide and the phenyl group.