Benzamide Derivatives Targeting the Cell Division Protein Ftsz: Modifications of the Linker and the Benzodioxane Scaffold and Their Effects on Antimicrobial Activity

Article Benzamide Derivatives Targeting the Cell Division Protein FtsZ: Modifications of the Linker and the Benzodioxane Scaffold and Their Effects on Antimicrobial Activity

Valentina Straniero 1,* , Lorenzo Suigo 1 , Andrea Casiraghi 1 , Victor Sebastián-Pérez 2,3, Martina Hrast 4, Carlo Zanotto 5, Irena Zdovc 6, Carlo De Giuli Morghen 7, Antonia Radaelli 5 and Ermanno Valoti 1,*

1 Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Luigi Mangiagalli, 25, 20133 Milano, Italy; [email protected] (L.S.); [email protected] (A.C.) 2 Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain; [email protected] 3 Exscientia, The Schrödinger Building, Oxford Science Park, Oxford OX4 4GE, UK 4 Pharmacy Faculty, University of Ljubljana, Aškerˇcevacesta, 7, 1000 Ljubljana, Slovenia; martina.hrast@ﬀa.uni-lj.si 5 Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Via Vanvitelli, 32, 20129 Milano, Italy; [email protected] (C.Z.); [email protected] (A.R.) 6 Veterinary Faculty, University of Ljubljana, Gerbiˇceva,60, 1000 Ljubljana, Slovenia; [email protected] 7 Department of Chemical – Pharmaceutical and Biomolecular Technologies, Catholic University “Our Lady of Good Counsel”, Rr. Dritan Hoxha, 1025 Tirana, Albania; [email protected] * Correspondence: [email protected] (V.S.); [email protected] (E.V.); Tel.: +39-0250319361 (V.S.); +39-0250319334 (E.V.) Received: 3 March 2020; Accepted: 1 April 2020; Published: 4 April 2020

Abstract: Filamentous temperature-sensitive Z (FtsZ) is a prokaryotic protein with an essential role in the bacterial cell division process. It is widely conserved and expressed in both Gram-positive and Gram-negative strains. In the last decade, several research groups have pointed out molecules able to target FtsZ in Staphylococcus aureus, Bacillus subtilis and other Gram-positive strains, with sub-micromolar Minimum Inhibitory Concentrations (MICs). Conversely, no promising derivatives active on Gram-negatives have been found up to now. Here, we report our results on a class of benzamide compounds, which showed comparable inhibitory activities on both S. aureus and Escherichia coli FtsZ, even though they proved to be substrates of E. coli efflux pump AcrAB, thus affecting the antimicrobial activity. These surprising results confirmed how a single molecule can target both species while maintaining potent antimicrobial activity. A further computational study helped us decipher the structural features necessary for broad spectrum activity and assess the drug-like profile and the on-target activity of this family of compounds.

Keywords: cell division protein FtsZ; benzamide; 1,4-benzodioxane; 1,4-benzoxathiane; multi-drug resistant Staphylococcus aureus; Escherichia coli N43

1. Introduction Antimicrobial resistance is a major cause of concern for public health worldwide, with the ever-growing diﬀusion of multidrug resistant pathogens and the consequent narrowing down of therapeutic options for previously treatable infections.

Antibiotics 2020, 9, 160; doi:10.3390/antibiotics9040160 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 160 2 of 22

According to a recent report by the Centers for Disease Control and Prevention (CDC), the grim predictions of a “post-antibiotic era” have come true, with an estimated over 3 million infections and nearly 50,000 deaths related to drug resistance per year in the U.S. alone [1]. The rise of drug resistant strains is largely propelled by the constant selective pressure associated with extensive use of antimicrobials in human and veterinary medicine and with their diffusion as environmental pollutants. In parallel, the number of novel antibiotics approved by international drug agencies decreased steadily over the last three decades, with only two new classes (lipopeptides and oxazolidinones) developed and approved in the past 20 years [2]. Notably, both classes have selective efficacy against Gram-positive bacteria, while the most recently approved innovative class for the treatment of Gram-negative infections are quinolones, dating back to 1962. In 2016, the World Health Organization (WHO) assessed the clinical relevance of 20 selected drug-resistant strains and compiled a priority list in order to guide future research and development investments. Critical priority was attributed to Gram-negative carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and third-generation cephalosporin-resistant Enterobacteriaceae (including Escherichia coli and Klebsiella pneumoniae) followed by high priority Gram-positive vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus [3]. Relying on more recent data, the WHO list updates and confirms the previous classification of ESKAPE (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) pathogens as high priority pathogens, based on the incidence of nosocomial drug-resistant infections [4]. There are four main molecular mechanisms of antimicrobial resistance: i) mutation in genes encoding the target site, causing a reduction in drug affinity; ii) expression of specific enzymes, enhancing drug metabolism; iii) mutations of drug transporters and reduced drug influx; and iv) (over)expression of efflux pumps, resulting in increased drug efflux. Multiple mechanisms can coexist in the same bacterial cell, often with additive effects, resulting in the inability of the drug to reach the target’s binding site at an adequate inhibitory concentration [5]. Developing molecules able to target some of these ESKAPE pathogens without developing antimicrobial resistance is one of the great goals of medicinal chemistry today. This aim needs to deal with the efflux pumps, which represent a severe issue. They collectively form a ubiquitous cell detoxification system with multiple types of substrates, including antibiotics [6]. There are five main groups of efflux pumps involved in drug resistance: the resistance-nodulation-division (RND) family, the major facilitator superfamily (MFS), the ATP-binding cassette (ABC) superfamily, the small multidrug resistance (SMR) family and the multidrug and toxic compound extrusion (MATE) family [7]. Only RND pumps are exclusively found in Gram-negative species; the others are widely distributed in both Gram-positive and Gram-negative bacteria [8]. The main RND efflux pump is AcrAB-TolC, which is formed by a transporter protein in the inner membrane (AcrB), an accessory protein in the periplasm (AcrA), and an outer membrane protein channel (TolC) [9]. Aiming to achieve broad-spectrum antibiotics while considering the efflux pump susceptibility, structurally different compounds were recently developed. These molecules (zantrins Z2 and Z3 [10], and benzamide derivatives TXY436 (which is a prodrug of the known PC190723 [11]) and benzodioxane-benzamides I and II [12]) are summarized in Table1, together with some relevant details. They all target the essential bacterial cell division protein FtsZ, a protein widely distributed among Gram-positive and Gram-negative strains, with which they interact at two different binding sites (the GTP-binding site for Z2 and Z3, and the interdomain site for the others, which are better explained below). Antibiotics 2019, 8, x FOR PEER REVIEW 3 of 21 Antibiotics 2019, 8, x FOR PEER REVIEW 3 of 21 Antibiotics 2019, 8, x FOR PEER REVIEW 3 of 21 Antibiotics 2019, 8, x FOR PEER REVIEW 3 of 21 Antibiotics 2019, 8, x FOR PEER REVIEW 3 of 21 physiologicalphysiological activity activity of of FtsZ, FtsZ, the the globular globular core core is is highly highly conserved conserved among among bacteria bacteria (both (both Gram Gram physiologicalpositive and Gram activity negative) of FtsZ, and the mycobacteria. globular core is highly conserved among bacteria (both Gram positivephysiologicalphysiological and Gram activityactivity negative) ofof FtsZ, FtsZ,and mycobacteria. thethe globularglobular corecore isis highlyhighly conservedconserved amongamong bacteriabacteria (both(both GramGram positiveThe and second Gram binding negative) site and of FtsZ mycobacteria. inhibitors is the interdomain site, a specific cavity located in a positivepositiveThe second andand GramGram binding negative)negative) site of andFtsZand mycobacteria. mycobacteria.inhibitors is th e interdomain site, a specific cavity located in a long Thecleft second between binding the C-terminal site of FtsZ domain inhibitors and theis th H7e interdomain helix of FtsZ. site, Since a specific this portion cavity of located the protein in a long cleftTheThe between secondsecond the bindingbinding C-terminal sitesite ofof domain FtsZFtsZ inhibitorsinhibitors and the isH7is thth helixee interdomaininterdomain of FtsZ. Since site,site, this aa specificspecific portion cavitycavity of the locatedlocated protein inin aa longis not cleft involved between in any the fundamentalC-terminal domain physiological and the fu H7nction, helix its of degreeFtsZ. 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It is indeed a crucial proteinFtsZ of is theone divisome,of the main a actorscomplex of theformed bacterial by cea llwide division variety process of protein[16]. It iscomponents. indeed a crucial The protein FtsZFtsZof the isis oneonedivisome, ofof thethe mainmaina complex actorsactors ofofformed thethe bacterialbacterial by a cewidecellll divisiondivision variety process processof protein [16].[16]. Itcomponents.It isis indeedindeed aa crucialThecrucial proteinpolymerization of the ofdivisome, FtsZ forms a thecomplex Z-ring, formed which splitsby a the wide mother variety bacterial of proteincell into twocomponents. daughter cellsThe polymerizationproteinprotein ofof thethe of FtsZ divisome,divisome, forms the aa complexZ-ring,complex which formedformed splits byby the aa mother widewide bacterialvarietyvariety of ofcell proteinprotein into two components.components. daughter cells TheThe polymerizationthrough its constriction of FtsZ forms [17]. FtsZ the Z-ring, inhibition which provokes splits the the mother entire blockagebacterial cellof the into division two daughter system cellsand, throughpolymerizationpolymerization its constriction of of FtsZ FtsZ [17]. forms forms FtsZ the the inhibition Z-ring, Z-ring, which whichprovokes splits splits the the the entire mother mother blockage bacterial bacterial of thecell cell division into into two two system daughter daughter and, cells cells throughconsequently, its constriction it results [17]. in a FtsZ bacteriostatic inhibition orprovokes a bactericidal the entire effect. blockage FtsZ ofessentiality the division in systemthe division and, consequently,throughthrough itsits constrictionitconstriction results in [17].[17].a bacteriostatic FtsZFtsZ inhibitioninhibition or a provokesprovokes bactericidal thethe entireentireeffect. blockage blockageFtsZ essentiality ofof thethe divisiondivision in the systemsystemdivision and,and, consequently,system has made it results this protein in a bacteriostatic a fascinating targetor a bactericidal in antibiotic effect. research. FtsZ essentiality in the division systemconsequently,consequently, has made itthisit resultsresults protein inin aaa fascinat bacteriostaticbacteriostaticing target oror aina bactericidal bactericidalantibiotic research. effect.effect. FtsZ FtsZ essentialityessentiality inin thethe divisiondivision systemWhile has madeworking this on protein FtsZ inhibitors, a fascinat ingthe targetpreviously in antibiotic mentioned research. researchers considered the insights systemsystemWhile has hasworking mademade on thisthis FtsZ proteinprotein inhibitors, aa fascinatfascinat theing ingpreviously targettarget inin mentioned antibioticantibiotic research.research.researchers considered the insights comingWhile from working previous on FtsZstudies inhibitors, [18–20], thein whichpreviously it was mentioned shown how researchers the genetic considered inactivation the insightsof RND coming WhileWhilefrom previous workingworking onstudieson FtsZFtsZ [18–20], inhibitors,inhibitors, in thewhichthe previouslypreviously it was shown mentionedmentioned how researcherstheresearchers genetic inactivation consideredconsidered the theof RND insightsinsights comingpumps resultedfrom previous in increased studies Gram-negative [18–20], in which antimicrob it was shownial activity how for the a genetic variety inactivation of other drugs. of RND This pumpscomingcoming resulted fromfrom previousinprevious increased studiesstudies Gram-negative [18–20],[18–20], inin whichantimicrobwhich itit waswasial shownshownactivity howhow for a thethe variety geneticgenetic of inactivationotherinactivation drugs. of ofThis RNDRND pumpsfinding resultedstrongly insupports increased the Gram-negative role of these efflux antimicrob systemsial in activity both intrinsic for a variety and acquired of other resistance drugs. This for Antibioticsfindingpumpspumps2020 strongly resultedresulted, 9, 160 supports inin increasedincreased the role Gram-negativeGram-negative of these efflux antimicrob antimicrobsystems inial ialboth activityactivity intrinsic forfor and aa varietyvariety acquired ofof otherother resistance drugs.drugs. for ThisThis 3 of 22 findingthesefinding microorganisms. strongly strongly supports supports the the role role of theseof these efflux efflux systems systems in bothin both intrinsic intrinsic and and acquired acquired resistance resistance for for thesethesefinding microorganisms. microorganisms. strongly supports the role of these efflux systems in both intrinsic and acquired resistance for thesethese microorganisms.microorganisms. Table 1. Literature FtsZ inhibitors with promising activities on Gram-negative strains. Table 1. Literature FtsZ inhibitors with promising activities on Gram-negative strains. Table 1. Literature FtsZ inhibitors with promising activities on Gram-negative strains. TableTable 1. 1.Literature Literature FtsZ FtsZ inhibitors inhibitors with promis with promisinging activities on activities Gram-negative on Gram-negative strains. strains. Table 1. Literature FtsZ inhibitors with promising activities on Gram-negativeFtsZ binding strains. Compound Structure MIC (μg/mL) FtsZ binding Reference Compound Structure MIC (μg/mL) FtsZ bindingsite Reference CompoundCompound StructureStructure MIC MIC ( (μµg/mL)G/ML) FtsZsite binding FtsZReference Binding Site Reference Compound Structure MIC (μg/mL) FtsZsite binding Reference Compound Structure Methicillin MIC E. ( coliμg/mL) site Reference Methicillin SensitiveMethicillin E. coli E. coli site MethicillinSensitiveE. coliE.Wild WildTypecoli E. coli E. coli AcrAB – S. aureus SensitiveMethicillin Wild E. coli AcrABE. coli – SensitiveS.Methicillin aureus WildTypeE. coli AcrABE.– coli S. aureusSensitive TypeWild AcrABE. coli– S. aureusSensitive TypeWild AcrAB– S. aureus Type AcrAB– S. aureus Type GTP-binding Z2Z2 0.60.6 20 20 2.5 GTP-binding 2.5 GTP-binding[10] site [10] Z2 0.6 20 2.5 GTP-bindingsite [10] Z2 0.6 20 2.5 GTP-bindingsite [10] Z2 0.6 20 2.5 GTP-bindingsite [10] Z2 0.6 20 2.5 site [10] site

N N N N HN N HN GTP-binding Z3 HN 2.1 >40 4 GTP-binding [10] Z3Z3 HNN 2.1 2.1 >40>40 4 GTP-binding 4site GTP-binding[10] site [10] Z3 HNN 2.1 >40 4 GTP-bindingsite [10] Z3 N 2.1 >40 4 GTP-bindingsite [10] Z3 N N 2.1 >40 4 site [10] N N site N N Cl N Cl Cl Cl Cl TXY436 1.0 >40 8 Interdomain site [11] TXY436 1.0 >40 8 Interdomain site [11] TXY436 1.0 >40 8 Interdomain site [11] TXY436TXY436 1.0 1.0 >40>40 8 Interdomain 8 site Interdomain [11] site [11] TXY436 1.0 >40 8 Interdomain site [11]

Compound I 0.6 >40 16 Interdomain site [12] Compound I 0.6 >40 16 Interdomain site [12] Compound I 0.6 >40 16 Interdomain site [12] CompoundCompound I I 0.6 >40 16 Interdomain site [12] Compound I 0.6 0.6 >>4040 16 Interdomain 16 site Interdomain [12] site [12]

Compound II 0.6 >40 16 Interdomain site [12] Compound II 0.6 >40 16 Interdomain site [12] Compound II 0.6 >40 16 Interdomain site [12] Compound II 0.6 >40 16 Interdomain site [12] CompoundCompound II II 0.6 0.6 >>4040 16 Interdomain 16 site Interdomain [12] site [12]

Consequently,Consequently, they they employed employed a a similar similar strategy strategy to to FtsZ FtsZ inhibitors inhibitors [10–12], [10–12], in in order order to to evaluate evaluate a possibleConsequently, restoring they of the employed Gram-negative a similar antimicrobial strategy to FtsZpotency. inhibitors Indeed, [10–12], FtsZ inhibitors in order toare evaluate usually a possibleConsequently,Consequently, restoring of theythethey Gram-negative employedemployed aa similarsimilar antimicrobial strategystrategy potency. toto FtsZFtsZ inhibitorsinhibitors Indeed, FtsZ [10–12],[10–12], inhibitors inin orderorder are to tousually evaluateevaluate adescribed possible restoringas limited of to the Gram-positive Gram-negative species, antimicrobial although potency. this strict Indeed, classification FtsZ inhibitors may be misleading,are usually describedaa possiblepossible as limited restoringrestoring to ofofGram-positive thethe Gram-negativeGram-negative species, antimicrobialantimicrobial although this potency.potency. strict classification Indeed,Indeed, FtsZFtsZ may inhibitorsinhibitors be misleading, areare usuallyusually describedasFtsZ highlighted is as a GTP-bindinglimited in a recent to Gram-positive review protein, [15], species,since the several bacterial althou playersgh homologuethis seemstrict classificationto contribute of eukaryotic mayto the be antimicrobial misleading, tubulin [13 ], and it is highly as describedhighlighteddescribed asas in limitedlimited a recent toto Gram-positivereviewGram-positive [15], since species,species, several althoualthou playersghgh this thisseem strictstrict to contribute classificationclassification to the maymay antimicrobial bebe misleading,misleading, asactivity highlighted on different in a recent Gram review strains. [15], The since most several significant players are hereseem reported: to contribute (i) the to FtsZthe antimicrobial binding sites conservedactivityasas highlightedhighlighted on in different most inin a prokaryoticGrama recentrecent strains. reviewreview The organisms[15],[15], most sincesince signif severalseveral [icant14]. players playersare Its here structure seemseem reported: toto contributecontribute can (i) the conventionally FtsZ toto thethe binding antimicrobialantimicrobial sites be divided into five activityacross different on different species Gram seem strains. to be slightlyThe most to signifstronglyicant different, are here independen reported: (i)tly the of theFtsZ Gram binding staining; sites specificacrossactivityactivity portions,different onon differentdifferent species each seemGramGram characterized to strains.strains. be slightly TheThe mosttomost by stro itssignifsignifngly own icantdifferent,icant degree areare here hereindependen of reported:reported: conservation:tly (i) (i)of the the FtsZGramFtsZ the bindingbinding staining;N-terminal sitessites domain, the across(ii)across there different differentis an evident species species imbalance seem seem to beto in beslightly the slightly number to stroto stroannglydngly in different, the different, variety independen ofindependen Gram-positivetlytly of theof strains the Gram Gram vs. staining; Gram-staining; globular(ii)(ii)across there therecore, is differentis an an evident evident the speciesC imbalance-terminal imbalance seem to in in be linker,the the slightly number number the to an stroanCdd -terminalnglyin in the the different, variety variety tail of ofindependen Gram-positive Gram-positive and, lastly,tly of strains strainsthe the GramC vs. vs.-terminal Gram- staining;Gram- variable region. (ii)(ii) therethere isis anan evidentevident imbalanceimbalance inin thethe numbernumber anandd inin thethe varietyvariety ofof Gram-positiveGram-positive strainsstrains vs.vs. Gram-Gram- Among them, the globular core contains the GTP-binding site, which is one of the two main binding sites of all known FtsZ inhibitors [15]. Considering the cruciality of the GTP-binding site for the physiological activity of FtsZ, the globular core is highly conserved among bacteria (both Gram positive and Gram negative) and mycobacteria. The second binding site of FtsZ inhibitors is the interdomain site, a specific cavity located in a long cleft between the C-terminal domain and the H7 helix of FtsZ. Since this portion of the protein is not involved in any fundamental physiological function, its degree of conservation among bacteria is lower than the GTP-binding site. FtsZ is one of the main actors of the bacterial cell division process [16]. It is indeed a crucial protein of the divisome, a complex formed by a wide variety of protein components. The polymerization of FtsZ forms the Z-ring, which splits the mother bacterial cell into two daughter cells through its constriction [17]. FtsZ inhibition provokes the entire blockage of the division system and, consequently, it results in a bacteriostatic or a bactericidal effect. FtsZ essentiality in the division system has made this protein a fascinating target in antibiotic research. While working on FtsZ inhibitors, the previously mentioned researchers considered the insights coming from previous studies [18–20], in which it was shown how the genetic inactivation of RND pumps resulted in increased Gram-negative antimicrobial activity for a variety of other drugs. This finding strongly supports the role of these efflux systems in both intrinsic and acquired resistance for these microorganisms. Consequently, they employed a similar strategy to FtsZ inhibitors [10–12], in order to evaluate a possible restoring of the Gram-negative antimicrobial potency. Indeed, FtsZ inhibitors are usually described as limited to Gram-positive species, although this strict classification may be misleading, as highlighted in a recent review [15], since several players seem to contribute to the antimicrobial activity Antibiotics 2019, 8, x FOR PEER REVIEW 4 of 21

negative strains tested, with a clear abundance of the former, specifically Staphylococci; (iii) the variety of Gram-negative strains tested was usually limited to E. coli, K. pneumoniae (both expressing AcrAB) and P. aeruginosa, which have an extensive array of chromosomally encoded RND efflux pumps, very limited membrane permeability, and a known intrinsic non-susceptibility to several antimicrobials. AntibioticsAmong2020, 9 the, 160 derivatives in Table 1, Compounds I and II resulted from our recent studies,4 of 22in which we modified the substituent in position 7 of the 1,4-benzodioxane moiety. I and II showed MICs vs. Methicillin Resistant S. aureus (MRSA) and clinical isolates of multi-drug resistant S. aureus on different Gram strains. The most significant are here reported: (i) the FtsZ binding sites across Antibiotics(Sa) between 2019, 8 , 0x. FOR6 and PEER 2.5 REVIEW µg/mL . Furthermore, morphometric analysis by transmission electron4 of 21 dimicroscopyfferent species (TEM seem) on to beSa slightlycells incubated to strongly with diff erent, them independently showed significant of the Gramelongation staining; in (ii) cellular there isnegativedimension, an evident strains bacterial imbalance tested, swelling with in the a clearand number cell abundance desegregation, and in the of the variety former, which of Gram-positive arespecifically the typical Staphylococci strainsalterations vs.; Gram-negative (ofiii )cell the division variety strainsofinhibition. Gram tested,-negative with strains a clear tested abundance was usually of the limited former, to E. specifically coli, K. pneumoniaeStaphylococci (both; expressing (iii) the variety AcrAB) of E. coli K. pneumoniae Gram-negativeand P.I aeruginosaand II were strains, which developed tested have an wasafter exte usually ansive precedent array limited ofdeep chromosomally to Structure, Activity encoded Relation(both RND expressingeffluxship pumps,(SAR AcrAB)) study very andlimited[21,22]P. aeruginosa membraneand after, which the permeability, confirmation have an extensiveand of a FtsZknown array as intrinsic the of chromosomallyreal non target-susceptibility of our encoded derivatives to RND several eusingffl antimicrobials.ux pumps, two in vitrovery limitedbiochemicalAmong membrane assays: the derivatives permeability, a GTPase in activity Table and a assay1, known Compounds and intrinsic a poly Imerization non-susceptibility and II resulted activity from toassay several our [23] recent antimicrobials.. Specifically, studies, wein whichdeterminedAmong we modified how the derivatives our the derivatives substituent in Table affect1 in, Compounds positionSa FtsZ GTPase 7 ofI andthe activity 1,4II resulted-benzodioxane by kinetic from our measurement moiety. recent studies,I and of II inorganic inshowed which weMICphosphate modifieds vs. M release.ethicillin the substituent An Resistant increase in S .in positionaureus polymerization (MRSA) 7 of the and 1,4-benzodioxanewas clinical detected isolates by SDS of moiety. multi-PAGE-Idrug,and after resistantII theshowed progressive S. aureus MICs vs.(additionSa)Methicillin between of our0 Resistant.6 compoundsand 2.5S. µ aureusg/mL, and.(MRSA) Furthermore, was quantified and clinical morphometric by isolates the percentage of anal multi-drugysis FtsZ by resistanttransmission in the pelletsS. aureus electron using(Sa) µ betweenmicroscopydensitometric 0.6 and (TEM analysis 2.5) ong/ mL. [23]Sa . Furthermore,cells incubated morphometric with them analysis showed by significant transmission elongation electron microscopyin cellular (TEM)dimension,In on thisSa bacterialwork,cells incubated with swelling the aim with and of them cell developing desegregation, showed significantbroad which-spectrum elongation are tantimicrobialshe typical in cellular alterations and dimension, starting of cell bacterialdivisionfrom in- swellinginhibition.house lead and compound cell desegregation, III (Figure which1), we aredeveloped the typical novel alterations derivatives, of cell modifying division the inhibition. nature of the 1,4- benzodioxaneI andand IIIIwere were ring developed d eveloped or lengthening after after a precedenta precedent the distance deep deep Structure between Structure Activity the Activity 1,4 Relationship-benzodioxane Relation (SAR)ship and study(SAR the)[ 21study , 2,622]- and[21,22]difluorobenzamide after and the after confirmation the moieties confirmation of ( FtsZFigure as of 2) the FtsZ. real as target the real of our target derivatives of our derivatives using two in using vitro twobiochemical in vitro assays:biochemical a GTPase assays: activity a GTPase assay activity and a polymerizationassay and a poly activitymerization assay activity [23]. Specifically, assay [23]. Specifically, we determined we howdetermined our derivatives how our aderivativesffect Sa FtsZ affect GTPase Sa FtsZ activity GTPase by kineticactivity measurement by kinetic measurement of inorganic of phosphate inorganic release.phosphate An release. increase An in increase polymerization in polymerization was detected was bydetected SDS-PAGE, by SDS after-PAGE the, progressiveafter the progressive addition ofaddition our compounds, of our compounds and was,quantified and was by quantified the percentage by the FtsZ percentage in the pellets FtsZ in using the densitometric pellets using analysisdensitometric [23]. analysis [23]. In this this work, work, with with the the aim aim of of developing developing broad broad-spectrum-spectrum antimicrobials antimicrobials and and starting starting from from inin-househouse leadlead compound compound III (FigureIII (Figure 1), we1), developed we developed novel derivatives, novel derivatives, modifying modifying the nature the of naturethe 1,4- ofbenzodioxane the 1,4-benzodioxane ring or lengthening ring or lengthening the distance the distance between between the 1,4 the-benzodioxane 1,4-benzodioxane and the and 2,6 the- 2,6-difluorobenzamidedifluorobenzamide moieties moieties (Figure (Figure 2). 2 ).

Figure 1. Reference in-house FtsZ inhibitors.

Specifically, we substituted one or both oxygen atoms with sulfur, differently oxidized (1–5), as a conclusion of our study on the importance of the two oxygens of the 1,4-benzodioxane ring. We previously prepared and tested the chromanes IV and V, the tetrahydronapthalene VI [22], the benzoxazines VII and VIII and the tetrahydroquinoxaline IX [23]. None of these modifications resulted in a positive outcome for the antimicrobial activity. Nevertheless, our latest computational studies of our derivatives in the FtsZ binding site defined the cavity as characterized by narrowness and hydrophobicity [12]. As a result, we decided to include the sulfur as bioisostere of the oxygen atom, due to their similar chemicalFigure behavior 1. Reference and in in-house its-house stronger FtsZ inhibitorsinhibitors.lipophilicity..

Specifically, we substituted one or both oxygen atoms with sulfur, differently oxidized (1–5), as a conclusion of our study on the importance of the two oxygens of the 1,4-benzodioxane ring. We previously prepared and tested the chromanes IV and V, the tetrahydronapthalene VI [22], the benzoxazines VII and VIII and the tetrahydroquinoxaline IX [23]. None of these modifications resulted in a positive outcome for the antimicrobial activity. Nevertheless, our latest computational studies of our derivatives in the FtsZ binding site defined the cavity as characterized by narrowness and hydrophobicity [12]. As a result, we decided to include the sulfur as bioisostere of the oxygen atom, due to their similar chemical behavior and its stronger lipophilicity. FigureFigure 2.2. Compounds 1–81–8,, objectobject ofof thethe presentpresent work.work.

Specifically, we substituted one or both oxygen atoms with sulfur, differently oxidized (1–5), as a conclusion of our study on the importance of the two oxygens of the 1,4-benzodioxane ring. We previously prepared and tested the chromanes IV and V, the tetrahydronapthalene VI [22], the benzoxazines VII and VIII and the tetrahydroquinoxaline IX [23]. None of these modifications resulted

Figure 2. Compounds 1–8, object of the present work. Antibiotics 2020, 9, 160 5 of 22 in a positive outcome for the antimicrobial activity. Nevertheless, our latest computational studies of our derivatives in the FtsZ binding site defined the cavity as characterized by narrowness and hydrophobicity [12]. As a result, we decided to include the sulfur as bioisostere of the oxygen atom, due to their similar chemical behavior and its stronger lipophilicity. Moreover, in order to evaluate how deep is the binding site, we varied the length of the linker, moving from the methylenoxy chain of III to an ethylenoxy (6 and 7) or a propylenoxy one (8). As done before, we tested all the compounds against several Gram-positive Sa strains: methicillin sensitive, methicillin resistant and multi-drug resistant. Furthermore, we evaluate their activity not only vs. E. coli ATCC 25922 and extended-spectrum beta-lactamase-positive E. coli, but also on efflux-defective E. coli N43. Docking studies and predictions of a large number of physicochemical properties allowed us to better understand the antimicrobial profile of some compounds.

2. Results and Discussion

2.1. Design and Physico-chemical Proﬁle Calculations Compounds 1–8 were designed starting from the chemical structure of III, modifying the heteroatoms of the 1,4-benzodioxane ring for 1–6, or lengthening the distance between the benzodioxane and the benzamide moieties for 7 and 8 (Figure2). Considering the above-mentioned features of the FtsZ binding site and prior to the synthesis, we decided to calculate some important physico-chemical and drug-like properties in silico, as summarized in Table2.

Table 2. Key drug-like properties for the compounds 1–8, object of the present work.

Dipole QlogPo/w PSA QPPCaco QPPMDCK III 7.215 2.920 75.393 1.036.486 1.249.904 1 6.647 3.417 66.311 1.032.637 2.032.874 2 8.801 1.756 105.241 105.625 103.165 3 7.504 3.301 66.536 1.033.096 1.677.064 4 7.720 1.674 104.281 316.001 306.684 5 6.968 3.739 58.274 1.032.481 2.579.522 6 6.036 3.735 64.832 1.033.074 1.953.979 7 6.615 3.294 74.783 1.032.977 1.213.317 8 7.188 3.660 74.847 1.032.656 1.212.923

The octanol/water partition coefficient (QlogPo/w) was chosen as a well-known parameter for the evaluation of their lipophilicity. Molecule dipole moment (Dipole) allowed us to assess the partial charge of the molecule and the impact of two potential charges, separated by a specific distance, which may affect molecular properties. Polar surface area (PSA) is a measure of the hydrogen-bonding capacity of the molecule and it summarizes the fractional contributions of all nitrogen and oxygen atoms to the surface area. Moreover, apparent Caco-2 cell permeability (QPPCaco) and apparent MDCK cell permeability (QPPMDCK) let us understand the permeability profiles, and thus the capability of reaching the target. As highlighted in Table2, dipole and logP calculations were excellent for almost all the derivatives, being in the suitable range for drug-like molecules. Concerning permeability profiles, most of the molecules displayed a great permeability prediction; only sulfones 2 and 4 showed poor predicted values with high PSA, which may translate in poor cellular permeation. Overall, these four characteristics confirmed the high potential of our derivatives as drug candidates and moved us to the definition of peculiar synthetic pathways and to their consequent synthesis.

2.2. Chemistry Scheme1 shows the synthetic pathway of benzoxathiane derivatives 1 and 3, together with the derived sulfones 2 and 4. The benzoxathiane ring was achieved by treating the 1,2-mercaptophenol in Antibiotics 2019, 8, x FOR PEER REVIEW 6 of 21

Moreover, in order to maximize the general overall yield of the synthetic pathway, we decided to keep together 9 and 10 at this stage and to quantitatively separate the regioisomers only at the final step. Therefore, after their obtainment, the mixture of 9 and 10 sequentially underwent (1) reduction with LiAlH4 (11 and 12), (2) mesylation (13 and 14) and (3) O-alkylation of 2,6-difluoro-3- hydroxybenzamideAntibiotics 2020, 9, 160 with the mesylates 13 and 14, with the final isolation and purification by 6flash of 22 chromatography, accomplishing pure 1 and 3. As well as their precursors, 1 and 3 1H-NMR spectra were significantly different and enabled the easy definition of the two chemical structures. a mixture of acetonitrile (ACN) and water (1/1) [24], in the presence of ethyl 2,3-dibromopropionate, 2 and 4 were directly prepared through oxidation of 1 and 3, using large equivalents of 3- prepared as reported in literature [25], with triethylamine (TEA) as a base. chloroperoxybenzoic acid (m-CPBA) and operating at room temperature.

Antibiotics 2019, 8, x FOR PEER REVIEW 6 of 21

Moreover, in order to maximize the general overall yield of the synthetic pathway, we decided to keep together 9 and 10 at this stage and to quantitatively separate the regioisomers only at the final

Schemestep.Scheme Therefore, 1: Reagent 1. afters Reagents and their solvents obtainment and: (a) solvents Ethyl, : 2,3the- (a)dibromopropionate, mixture Ethyl of 2,3-dibromopropionate, 9 and 10 TEA sequentially, ACN/H2O TEA,underwent 1/1, RT ACN, 72/ H(1)h2; O (b)reduction 1 LiAH/1, 4, Twithetrahydrofuran RT, LiAlH 72 h;4 ( 11(T (b) HFand LiAH), Room 124,), TetrahydrofuranTemperature (2) mesylation (RT) (THF),, (3013 minand Room; (c) 14 Mesyl) Temperatureand chloride, (3) O -TEAalkylation (RT),, Dichloro 30 min; ofmethane 2,6 (c)-difluoro Mesyl (DCM-)3, - RThydroxybenzamide, 3 chloride,h; (d) 2,6-Difluoro TEA, Dichloromethane with-3-hydroxybenzamide, the mesylates (DCM), 13 K and2 RT,CO3 , 314 N h;, Nwith- (d)dimethylformamide2,6-Difluoro-3-hydroxybenzamide, the final isolation (DMF and), 60 purification °C , 16 h; K(e)2CO mby-CPBA3 ,flash 1 (4chromatography, eq),N DCM,,N-dimethylformamide RT, 2 h accomplishing. (DMF), pure 60 ◦ C,1 and 16 h; 3 (e). Asm-CPBA well as (4 their eq), DCM, precu RT,rsors, 2 h. 1 and 3 H-NMR spectra were significantly different and enabled the easy definition of the two chemical structures. TheOperating2 and synthesis 4 were with of directly 5 these (Scheme reactionprepared 2) started conditions,through with oxidthe the treatmentation relative of 1 of percentagesand the 3commercial, using of large 2- benzene and equivalents 3-substituted-1,2-dithiol of 3- withbenzoxathianeschloroperoxybenzoic ethyl 2,3-dibromopropionate were acid 60% (m (9-)CPBA) and in 40% aDMFnd (operating10 and), asTEA expected. at as room a base, temperature The affording ratio of.15 regioisomersas quantitative was product. easily 1 Thedefined consecutive through reduction the comparison with LiAlH of H-NMR4 (16) and spectra, mesylation since theyielded chemical 17 that shifts was of condensed the benzoxathiane with the 2,6protons-difluoro were-3- stronglyhydroxybenzamide dependent on(5). the substitution position. Moreover, in order to maximize the general overall yield of the synthetic pathway, we decided to keep together 9 and 10 at this stage and to quantitatively separate the regioisomers only at the final step. Therefore, after their obtainment, the mixture of 9 and 10 sequentially underwent (1) reduction with LiAlH4 (11 and 12), (2) mesylation (13 and 14) and (3) O-alkylation of 2,6-difluoro-3-hydroxybenzamide with the mesylates 13 and 14, with the final isolation and purification by flash chromatography, Scheme 2: Reagents and solvents: (a) Ethyl 2,3-dibromopropionate, TEA, DMF,1 RT, 2 h; (b) LiAH4, THF, RT, 1 h; accomplishingScheme 1: Reagent pures and1 and solvents3. As: (a) well Ethyl as 2,3 their-dibromopropionate, precursors, 1 and TEA3 ,H-NMR ACN/H2 spectraO 1/1, RT were, 72 h significantly; (b) LiAH4, (c) Mesyl chloride, TEA, DCM, RT, 3 h; (d) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 80 °C , 16 h. diTetrahydrofuranfferent and enabled (THF), theRoom easy Temperature definition of(RT the), 30 two min chemical; (c) Mesyl structures. chloride, TEA, Dichloromethane (DCM), RT, 3The 2h; and(d) synthesis 2,64-Difluorowere of directly -63 -hydroxybenzamide,is reported prepared in Scheme through K2CO 33 ,and oxidationN,N -requireddimethylformamide of the1 and initial3, ( usingtreatmentDMF), 60 large °C of, 16 equivalentscommercial h; (e) m-CPBA 3 of- butenoic3-chloroperoxybenzoic(4 eq), DCM, acid RT with, 2 h. bromine acid ((m18-CPBA)) and the and consequent operating atesterif roomication temperature. of the carboxylic group (19). 1,2- mercaptophenolThe synthesis was of then5 (Scheme treated2) startedwith 19 withand TEA, the treatment in a mixture of the of commercial ACN/H2O ( benzene-1,2-dithiol1/1), accomplishing thewith 2-The ethylsubstituted synthesis 2,3-dibromopropionate-benzo of 5 xathiane(Scheme 202) instartedas DMFthe uniquewith and TEAthe re treatmentgioisomer as a base, .of The a fftheording benzo commercial15xathiineas quantitative benzene 20, similarly-1,2 product.-dithiol to 9, with ethyl 2,3-dibromopropionate in DMF and TEA as a base, affording 15 as quantitative product. 10The and consecutive 15, underwent reduction reduction with LiAlHwith LiAlH4 (16)4 and (21) mesylation and the tandem yielded mesylation17 that was (22 condensed) and condensation with the with2,6-difluoro-3-hydroxybenzamideThe consecutivethe 2,6-difluoro reduction-3-hydroxy with- LiAlHbenzamide, (5). 4 (16) andobtaining mesylation 6. yielded 17 that was condensed with the 2,6-difluoro-3-hydroxybenzamide (5).

Scheme 2. Reagents and solvents: (a) Ethyl 2,3-dibromopropionate, TEA, DMF, RT, 2 h; (b) LiAH4, THF, Scheme 2: Reagents and solvents: (a) Ethyl 2,3-dibromopropionate, TEA, DMF, RT, 2 h; (b) LiAH4, THF, RT, 1 h; RT, 1 h; (c) Mesyl chloride, TEA, DCM, RT, 3 h; (d) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 80 (c) Mesyl chloride, TEA, DCM, RT, 3 h; (d) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 80 °C , 16 h. ◦C, 16 h. The synthesis of 6 is reported in Scheme 3 and required the initial treatment of commercial 3- butenoicThe synthesisacid with ofbromine6 is reported (18) and in the Scheme consequent3 and esterif requiredication the initialof the carboxylic treatment ofgroup commercial (19). 1,2- 3-butenoicmercaptophenol acid withwas then bromine treated (18 )with and 19 the and consequent TEA, in a esterificationmixture of ACN/H of the2O carboxylic (1/1), accomplishing group (19). 1,2-mercaptophenolthe 2-substituted--benzo wasxathiane then treated 20 as with the19 uniqueand TEA, regioisomer in a mixture. The of benzo ACNxathiine/H2O (1 /201), , accomplishingsimilarly to 9, the10 and 2-substituted-benzoxathiane 15, underwent reduction20 withas theLiAlH unique4 (21) regioisomer. and the tandem The benzoxathiinemesylation (2220) and, similarly condensation to 9, 10 Scheme 3: Reagents and solvents: (a) Br2, DCM, RT, 16 h; (b) trimethyl orthoformate, H2SO4 conc, MeOH, reflux, andwith15 the, underwent 2,6-difluoro reduction-3-hydroxy with-benzamide, LiAlH4 (21 )obtaining and the tandem 6. mesylation (22) and condensation with 16the h; 2,6-difluoro-3-hydroxy-benzamide, (c) 1,2-mercaptophenol, TEA, ACN/H2 obtainingO, RT, 16 h6; .(d) LiAlH4, THF, 0–5 °C , 1 h; (e) Mesyl chloride, TEA, DCM, RT, 2 h; (f) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 60 °C , 16 h.

Scheme 3: Reagents and solvents: (a) Br2, DCM, RT, 16 h; (b) trimethyl orthoformate, H2SO4 conc, MeOH, reflux,

16 h; (c) 1,2-mercaptophenol, TEA, ACN/H2O, RT, 16 h; (d) LiAlH4, THF, 0–5 °C , 1 h; (e) Mesyl chloride, TEA, DCM, RT, 2 h; (f) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 60 °C , 16 h. Antibiotics 2019, 8, x FOR PEER REVIEW 6 of 21

Moreover, in order to maximize the general overall yield of the synthetic pathway, we decided to keep together 9 and 10 at this stage and to quantitatively separate the regioisomers only at the final step. Therefore, after their obtainment, the mixture of 9 and 10 sequentially underwent (1) reduction with LiAlH4 (11 and 12), (2) mesylation (13 and 14) and (3) O-alkylation of 2,6-difluoro-3- hydroxybenzamide with the mesylates 13 and 14, with the final isolation and purification by flash chromatography, accomplishing pure 1 and 3. As well as their precursors, 1 and 3 1H-NMR spectra were significantly different and enabled the easy definition of the two chemical structures. 2 and 4 were directly prepared through oxidation of 1 and 3, using large equivalents of 3- chloroperoxybenzoic acid (m-CPBA) and operating at room temperature.

Scheme 1: Reagents and solvents: (a) Ethyl 2,3-dibromopropionate, TEA, ACN/H2O 1/1, RT, 72 h; (b) LiAH4, Tetrahydrofuran (THF), Room Temperature (RT), 30 min; (c) Mesyl chloride, TEA, Dichloromethane (DCM), RT, 3 h; (d) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, N,N-dimethylformamide (DMF), 60 °C , 16 h; (e) m-CPBA (4 eq), DCM, RT, 2 h.

The synthesis of 5 (Scheme 2) started with the treatment of the commercial benzene-1,2-dithiol with ethyl 2,3-dibromopropionate in DMF and TEA as a base, affording 15 as quantitative product. The consecutive reduction with LiAlH4 (16) and mesylation yielded 17 that was condensed with the 2,6-difluoro-3-hydroxybenzamide (5).

Scheme 2: Reagents and solvents: (a) Ethyl 2,3-dibromopropionate, TEA, DMF, RT, 2 h; (b) LiAH4, THF, RT, 1 h;

(c) Mesyl chloride, TEA, DCM, RT, 3 h; (d) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 80 °C , 16 h. The synthesis of 6 is reported in Scheme 3 and required the initial treatment of commercial 3- butenoic acid with bromine (18) and the consequent esterification of the carboxylic group (19). 1,2- mercaptophenol was then treated with 19 and TEA, in a mixture of ACN/H2O (1/1), accomplishing the 2-substituted-benzoxathiane 20 as the unique regioisomer. The benzoxathiine 20, similarly to 9, 10Antibiotics and 152020, underwent, 9, 160 reduction with LiAlH4 (21) and the tandem mesylation (22) and condensation7 of 22 with the 2,6-difluoro-3-hydroxy-benzamide, obtaining 6.

Scheme 3. Reagents and solvents: (a) Br , DCM, RT, 16 h; (b) trimethyl orthoformate, H SO conc, Scheme 3: Reagents and solvents: (a) Br2, DCM,2 RT, 16 h; (b) trimethyl orthoformate, H2SO4 conc, 2MeOH,4 reflux, MeOH, reflux, 16 h; (c) 1,2-mercaptophenol, TEA, ACN/H O, RT, 16 h; (d) LiAlH THF, 0–5 C, 1 h; (e) 16 h; (c) 1,2-mercaptophenol, TEA, ACN/H2O, RT, 16 h; (d) LiAlH2 4, THF, 0–5 °C , 1 h;4, (e) Mesyl chloride,◦ TEA, Mesyl chloride, TEA, DCM, RT, 2 h; (f) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 60 ◦C, 16 h. DCM,Antibiotics RT ,2019 2 h;,, 8(f),, xx 2,6 FORFOR-Difluoro PEERPEER REVIEW-3-hydroxybenzamide, K2CO3, DMF, 60 °C , 16 h. 7 of 21 Intermediate 19 was also used to obtain 7 (see Scheme4). The pathway was very similar Intermediate 19 was also used to obtain 7 (see Scheme 4).. TheThe pathwaypathway waswas very similar to the to the one described for 6, but we used catechol instead of 1,2-mercaptophenol, accomplishing one described for 6,, but but we we used used catechol insteadinstead of of 1,2 1,2-mercaptophenol, accomplishing benzodioxane 23, which underwent reduction (24), mesylation (25) and condensation with benzodioxane 23,, which which underwent underwent reduction reduction ( (24), mesylation (25) and condensation with 2,6- 2,6-difluoro-3-hydroxy-benzamide to afford 7. difluoro-3-hydroxy-benzamide to afford 7..

SchemeScheme 4:: Reagents 4. Reagents and solvents and solvents:: (a)(a) catechol: (a) catechol,,, KK22CO K323,,CO acetone,3, acetone, RT, 16 RT, h 16;; (b)(b) h; (b)LiAlHLiAlH LiAlH4,4, THF,4, THF, 0–50–5 °C,◦ 1C, h 1;; (c) h;(c) (c) MesylMesyl chloride,Mesyl TEA, chloride, DCM, TEA,RT, 2 DCM,h;; (d)(d) 2,62,6 RT,--Difluoro 2 h; (d) 2,6-Diﬂuoro-3-hydroxybenzamide,--3--hydroxybenzamide, K22CO33,, DMF,DMF, K2 6060CO °C,3, DMF, 16 h.. 60 ◦C, 16 h.

Our initial idea to obtain 8 waswas the the application application of the the synthetic synthetic pathway pathway of of 66,, whilewhile while startingstarting starting fromfrom from commercial 4 4-pentenoic-pentenoic acid and catechol catechol... Unfortunately, Unfortunately, after th thee initial quantitativequantitative bromination and esterification, esteriﬁcation, during the condensation with catec catecholhol we obtained,obtained,, isolated and characterized methyl 4-bromopent-4-enoate4-bromopent-4-enoateas as main main reaction reaction by-product by-product (Figure (Figure3). Literature3).. Literature data data [ 26 ][26] were were in line in 1 linewithline with with our ourH-NMR our 11H-NMR spectra. spectra. Diﬀerent Different reaction reaction variables variables were evaluated were evaluated and changed, and changed but the,, reactionbut the reactionoutcome outcome was always was the always same. the same.

Figure 3. ReactionReaction by by-product--product methyl methyl 4 4-bromopent-4-enoate.--bromopent--4--enoate..

We therefore therefore moved moved to to a a diff dierentfferent synthetic synthetic pathway pathway,,, in in in which which which we we we first first first include include include the the 2,6 2,6 the- difluorobenzamide2,6-difluorobenzamide moiety moiety and and we we then then suitably suitably functionalize functionalizedd the the double double bond. bond. As As reported reported in Scheme5 , 5, we we started started from from the mesylation the mesylation of the of commercial the commercial 4-penten-1-ol 4-penten ( 26-)1 and-ol we( 26) soon and condensed we soon condens26 with theed 26 2,6-difluoro-3-hydroxy-benzamide with the 2,6-difluoro-3-hydroxy- (benzamide27). The alkene (27). wasThe thenalkene converted was then into converted the epoxide into the28, whichepoxide was 28,, consequentlywhichwhich waswas consequentlyconsequently reacted with reactedreacted the catechate, withwith thethe accomplishing cateccatechate,, accomplishingaccomplishing29 without 2 any9 without additional any additionalside-products. side- Theproducts final Mitsunobu.. TheThe finafinall MitsunobuMitsunobu reaction let reactionreaction the obtainment letlet tthe ofobtainment the 1,4-benzodioxane of the 1,4-benzodioxane ring and the ringfinal and product the final8. product 8..

Scheme 5:: Reagents and solvents:: (a)(a) TEA,TEA, MsCl,MsCl, DCMDCM,, 33 h;; (b)(b) 2,6--Difluoro--3--hydroxybenzamide,, K₂CO₃,K₂CO₃, DDMF, 80°C,, 16 h;; (c)(c) m--CPBA, DCM,, RT,RT, 18 h;; (d)(d) catechol,, NaH,NaH, DMF,DMF, 100100 °C ,, 72 h;; (e)(e) Triphenylphosphine,, DEAD,DEAD, THF, 60 °C ,, 24 h..

2.3. Antimicrobial Activity Antimicrobial activity of 1–8 was evaluated on both Gram-positive (Table 3) and Gram-negative (Table 4) species.. AsAs aa continuationcontinuation ofof ourour previousprevious studies,studies, consideringconsidering thethe GramGram-positive S. aureus,, we chose a methicillin-sensitive S. aureus (MSSA, ATCC 29213), a methicillin-resistant S. aureus Antibiotics 2019, 8, x FOR PEER REVIEW 7 of 21

Intermediate 19 was also used to obtain 7 (see Scheme 4). The pathway was very similar to the one described for 6, but we used catechol instead of 1,2-mercaptophenol, accomplishing benzodioxane 23, which underwent reduction (24), mesylation (25) and condensation with 2,6- difluoro-3-hydroxy-benzamide to afford 7.

Scheme 4: Reagents and solvents: (a) catechol, K2CO3, acetone, RT, 16 h; (b) LiAlH4, THF, 0–5 °C, 1 h; (c) Mesyl chloride, TEA, DCM, RT, 2 h; (d) 2,6-Difluoro-3-hydroxybenzamide, K2CO3, DMF, 60 °C, 16 h.

Our initial idea to obtain 8 was the application of the synthetic pathway of 6, while starting from commercial 4-pentenoic acid and catechol. Unfortunately, after the initial quantitative bromination and esterification, during the condensation with catechol we obtained, isolated and characterized methyl 4-bromopent-4-enoate as main reaction by-product (Figure 3). Literature data [26] were in line with our 1H-NMR spectra. Different reaction variables were evaluated and changed, but the reaction outcome was always the same.

Figure 3. Reaction by-product methyl 4-bromopent-4-enoate.

We therefore moved to a different synthetic pathway, in which we first include the 2,6- difluorobenzamide moiety and we then suitably functionalized the double bond. As reported in Scheme 5, we started from the mesylation of the commercial 4-penten-1-ol (26) and we soon condensed 26 with the 2,6-difluoro-3-hydroxy-benzamide (27). The alkene was then converted into the epoxide 28, which was consequently reacted with the catechate, accomplishing 29 without any Antibioticsadditional2020 side, 9, 160-products. The final Mitsunobu reaction let the obtainment of the 1,4-benzodioxane8 of 22 ring and the final product 8.

SchemeScheme 5: Reagents 5. Reagents and solvents and solvents: (a) TEA,: (a) MsCl, TEA, DCM MsCl,, 3 DCM,h; (b) 2,6 3 h;-Difluoro (b) 2,6-Diﬂuoro-3-hydroxybenzamide,-3-hydroxybenzamide, K₂CO₃, DMF, 80°C,K 162CO h; 3(c), DMF, m-CPBA, 80 ◦ C,DCM 16,h; RT, (c) 18m h-CPBA,; (d) catechol DCM,, NaH, RT, 18 DMF, h; (d) 100 catechol, °C , 72 h NaH,; (e) T DMF,riphenylphosphine 100 ◦C, 72 h;, (e)DEAD, THF,Triphenylphosphine, 60 °C , 24 h. DEAD, THF, 60 ◦C, 24 h. 2.3. Antimicrobial Activity 2.3. Antimicrobial Activity Antimicrobial activity of 1–8 was evaluated on both Gram-positive (Table3) and Gram-negative Antimicrobial activity of 1–8 was evaluated on both Gram-positive (Table 3) and Gram-negative (Table4) species. As a continuation of our previous studies, considering the Gram-positive S. aureus, (Table 4) species. As a continuation of our previous studies, considering the Gram-positive S. aureus, we chose a methicillin-sensitive S. aureus (MSSA, ATCC 29213), a methicillin-resistant S. aureus (MRSA, we chose a methicillin-sensitive S. aureus (MSSA, ATCC 29213), a methicillin-resistant S. aureus ATCC 43300) and two clinical isolates of S. aureus showing multi-drug resistance (MDRSA). MDRSA 12.1 shows resistance towards gentamicin, kanamycin, rifampicin, streptomycin, sulfamethoxazole and tetracycline, while MDRSA 11.7 is resistant to ciproﬂoxacin, clindamycin, erythromycin, quinupristin and dalfopristin in association, tetracycline, tiamulin and trimethoprim.

Table 3. Inhibitory activity of compounds III and 1–8 against MSSA, MRSA, MDRSA 12.1, MDRSA 11.7 and MRC-5.

MSSA ATCC 29213 MRSA ATCC 43300 MDRSA 12.1 MDRSA 11.7 MRC-5 Cpd MIC MBC MIC MBC MIC MIC TD90 MBC TI90 MBC TI90 (µg/mL) (µg/mL) MIC (µg/mL) (µg/mL) MIC (µg/mL) (µg/mL) (µg/mL) III 5 80 16 - 5 80 16 - 8 16 - 1 1 1 1 >800 1 1 1 >800 2 2 >800 2 >100 >100 - - >100 >100 - - - - - 3 20 20 1 10 20 20 1 10 32 32 200 4 >100 >100 - - >100 >100 - - - - - 5 5 10 2 20 5 10 2 20 8 8 200 6 2.5 5 2 - 2.5 5 2 - 4 8 - 7 5 5 1 76 5 5 1 76 4 4 380 8 10 100 10 - 10 100 10 - 16 32 -

Table 4. Inhibitory activity of compounds I–III and 1–8 against diﬀerent E. coli strains.

E. coli ATCC 25922 ESBL E. coli E. coli N43 Cpd MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) I >100 >100 16 II >100 >100 16 III >100 >100 >128 1 >100 >100 64 2 >100 >100 - 3 >100 >100 128 4 >100 >100 - 5 >100 >100 >128 6 >100 >100 >128 7 >100 >100 8 8 >100 >100 >128 Antibiotics 2020, 9, 160 9 of 22

The inhibitory ability of 1–8 was evaluated by determining the MIC, i.e., the lowest compound dose (µg/mL) at which growth is inhibited, as well as the minimal bactericidal concentration (MBC), i.e., the minimal dose (µg/mL) at which cell growth is irreversibly blocked after compound removal. The compounds showing the most promising activities vs. MRSA were also assessed for their cytotoxicity on human MRC-5 cells. TD90, the concentration (µg/mL) able to reduce by 90% the viability of these cells, was determined together with the therapeutic index (TI), expressed as the ratio between TD90 and MBC values. Considering all the results presented in Table3, di fferent conclusions can be made for compounds 1–5, presenting a modified 1,4-benzodioxane scaffold and 6–8, having an elongated linker between the heteroaromatic moiety and the 2,6-difluorobenzamide. Starting from the former (1–5), the outstanding antimicrobial potency of 1 emerges. This surprising behavior confirm two important features: i) the maintenance of the oxygen in position 1 is essential for potent antibacterial activity on S. aureus; ii) the lipophilicity of this scaffold should be sufficiently high to properly interact with FtsZ. Indeed, the increased lipophilicity of 1, having a sulfur atom in position 4 instead of the oxygen of III, results in five-fold higher MICs on MSSA and MRSA. The inhibitory activity of 1 on MDRSA has a similar outcome; despite its slightly weakened potency, it is four- and eight-fold stronger than III on MDRSA 12.1 and 11.7, respectively. The antimicrobial profile of 3 emphasizes the importance of maintaining the oxygen atom in position 1. The retention of a comparable lipophilicity, while inverting the position of the two heteroatoms, results in a reduced interaction with the target and a consequent mild antimicrobial activity. The MICs ratio value between 1 and 3 is 20, which is entirely in line with what was previously observed for chromanes IV (MIC vs. MSSA: 5 µg/mL) and V (MIC vs. MSSA: 100 µg/mL) [22]. This trend corroborates the essentiality of oxygen (1) for a strong interaction with FtsZ. A similar trend could be detected comparing the MICs of four derivatives with progressively more lipophilic heteroatoms in position 4: VII, IV, III and 1. Indeed, moving from the benzoxazine VII to the chromane IV, to the benzodioxane III and lastly to the benzoxathiane 1, we observe a growing antibacterial potency. Moreover, the position of the sulfur atom affects also the human cytotoxicity, as shown by the results in Table3. Indeed, derivative 1 shows an impressive and promising TI, strongly superior to 3 and 5. Compound 5 shows an antimicrobial profile that is similar to III. Such a result could derive from the balance between the higher lipophilicity due to the presence of two sulfur atoms, and the absence of oxygen (1). Therefore, these two main determinants seem to equally contribute to the interaction with FtsZ and the consequent antibacterial activity. Lastly, in sulfones 2 and 4 the oxidation of the sulfur atoms brought to a crucial loss in lipophilicity and consequently in antimicrobial activity, further corroborating our hypothesis of a hydrophobic binding pocket. Compounds 6–8 were designed starting from our previous computational studies [12], in order to assess the depth of the binding cavity. As highlighted from the MICs in Table3, 6 and 7, bringing the ethylenoxy chain, retain good ability to interact with FtsZ. Conversely, the propylenoxy bridge of 8 seems to be not well tolerated and does not allow a proper fitting of the compound. Finally, the MBC/MIC ratios for the whole series of compounds is quite surprising. The initial value of 16 for III significantly decreases to 1 or 2 for the most promising derivatives (1, 3, 5–7), enhancing their bactericidal effect. Furthermore, we considered our recent and promising results of I and II [12], which highlights their capability to penetrate the E. coli membrane and to have an antibacterial activity on this bacterium. We previously shown how the genetic inactivation of RND-type efflux pump AcrAB (in E. coli N43) imparted a strong potency of I and II on this pathogen, confirming they are substrates of this efflux pump. We thus tested 1–8 and III initially on the reference sensitive E. coli ATCC 25922 and on the extended-spectrum of beta-lactamase-positive (ESBL) E. coli, as Gram-negative bacteria. Moreover, in Antibiotics 2020, 9, 160 10 of 22 order to evaluate the susceptibility of this series of compounds to the efflux pump AcrAB, we employed also the above-mentioned E. coli N43, which is the knockout of the AcrAB cell membrane pump. The values in Table4 show encouraging antimicrobial activities, especially for compound 7. Its MIC is half the ones of the in-house reference compounds I and II. Moreover, surprisingly 7 has MICs vs. S. aureus and E. coli N43 of the same order of magnitude, confirming FtsZ as a valid and real target for both Gram-positive and Gram-negative strains.

2.4. Microscopy Evaluation After having assessed the antimicrobial profile of this class of derivatives, we chose 1 to be further analyzed by transmission electron microscopy (TEM), in order to evaluate any morphological changes in septum formation, as done before [12,22,23] as a simple FtsZ target validation assay. We cultured S. aureus ATCC 43300 alone or in the presence of 1 and 2,6-difluorononyloxybenzamide (DFNB)[27] as a reference FtsZ inhibitor (positive control). No morphological changes were noticed in untreated S. aureus, as reported in Figure4. On the contrary, when treated with 1 or DFNB, S. aureus cells showed strong alterations in the septum formation and bacterial desegregation, with consequent loss of the cytoplasmic content. These are the typical effects of the inhibition of FtsZ assembly and consequently ofAntibiotics the block 2019 of, 8,bacterial x FOR PEER cell REVIEW division. 10 of 21

Figure 4. Ultrastructural analysis by TEM of S. aureus (MRSA) cultured overnight in the presence of Figure 4. Ultrastructural analysis by TEM of S. aureus (MRSA) cultured overnight in the presence of 1 at 1 µg/mL. Bacteria with no compound (NT) or in the presence of DFNB (0.25 µg/mL) were used 1 at 1 μg/mL. Bacteria with no compound (NT) or in the presence of DFNB (0.25 μg/mL) were used as negative and positive control, respectively. Arrows indicate the septum. Alterations in septum as negative and positive control, respectively. Arrows indicate the septum. Alterations in septum formation are clearly visible in the samples treated with DFNB and 1. Scale bar: 1 µm. formation are clearly visible in the samples treated with DFNB and 1. Scale bar: 1 μm. 2.5. Computational Studies 2.5. Computational Studies Physical-chemical properties predictions allowed us to design and propose compounds with a promisingPhysical- drug-likechemical proper profile.ties Moreover,predictions these allowed properties us to design gave and preliminary propose compounds insights into with the a structure–activitypromising drug-like relationship profile. Moreover, of some compounds these properties of this gave family. preliminary Indeed, theyinsights might into explain the structure the lack– ofactivity activity relationship of both compounds of some compounds2 and 4 in of the this MRSA family. and Indeed MSSA, they biological might explain assays, the because lack of ofactivity their poorof both permeability compounds profiles. 2 and On 4 thein the contrary, MRSA the and rest MSSA of the compounds biological assay displayeds, because excellent of their predicted poor permeationpermeability profiles, profile beings. On able the tocontrary, exert their the action rest of on the FtsZ compounds inside the bacteria. displayed excellent predicted permeationWe decided profile to furthers, being evaluate able to exert derivatives their action1–8 from on aFtsZ computational inside the bacteria. perspective; docking analyses wereWe performed decided to to study further the evaluate binding mode derivatives of these 1– molecules8 from a computational in the interdomain perspective; active site docking of the protein.analyses According were performed to our previous to study dockingthe binding model, mode three of hydrogenthese molecules bonds in drive the theinterdomain interaction active between site theof the 2,6-difluorobenzamide protein. According to moiety our previo andus the dockingS. aureus modelFtsZ, three protein hydrogen [12]. Here, bonds we drive confirmed the interaction that the amidebetween group the of2,6 the-difluorobenzamide 2,6-difluorobenzamide moiety sca andffold the can S. interact aureusvia FtsZ two protein hydrogen [12] bond. Here donors, we confirm betweened that the amide group of the 2,6-difluorobenzamide scaffold can interact via two hydrogen bond the NH2 and Val207 and Asn263 and via a hydrogen bond acceptor between the carbonyl and Leu209. Figuredonors5 depictsbetween the the docking NH2 and results Val207 for theand most Asn263 active and compound via a hydrogen of this series,bond acceptor1. between the carbonyl and Leu209. Figure 5 depicts the docking results for the most active compound of this series, 1.

Figure 5. Docking results for compound 1 (depicted in green) in the FtsZ protein (cyan), full protein view and detailed view of the binding site and main ligand-protein interactions.

On the other hand, our previous studies highlighted the specific FtsZ region involved in binding the 1,4-benzodioxane. Indeed, this scaffold extends its conformation towards the inner part of the pocket and this hydrophobic subpocket is surrounded by hydrophobic residues such as Met98, Antibiotics 2019, 8, x FOR PEER REVIEW 10 of 21

Figure 4. Ultrastructural analysis by TEM of S. aureus (MRSA) cultured overnight in the presence of 1 at 1 μg/mL. Bacteria with no compound (NT) or in the presence of DFNB (0.25 μg/mL) were used as negative and positive control, respectively. Arrows indicate the septum. Alterations in septum formation are clearly visible in the samples treated with DFNB and 1. Scale bar: 1 μm.

2.5. Computational Studies Physical-chemical properties predictions allowed us to design and propose compounds with a promising drug-like profile. Moreover, these properties gave preliminary insights into the structure– activity relationship of some compounds of this family. Indeed, they might explain the lack of activity of both compounds 2 and 4 in the MRSA and MSSA biological assays, because of their poor permeability profiles. On the contrary, the rest of the compounds displayed excellent predicted permeation profiles, being able to exert their action on FtsZ inside the bacteria. We decided to further evaluate derivatives 1–8 from a computational perspective; docking analyses were performed to study the binding mode of these molecules in the interdomain active site of the protein. According to our previous docking model, three hydrogen bonds drive the interaction between the 2,6-difluorobenzamide moiety and the S. aureus FtsZ protein [12]. Here, we confirmed that the amide group of the 2,6-difluorobenzamide scaffold can interact via two hydrogen bond donors between the NH2 and Val207 and Asn263 and via a hydrogen bond acceptor between the Antibiotics 2020, 9, 160 11 of 22 carbonyl and Leu209. Figure 5 depicts the docking results for the most active compound of this series, 1.

FigureFigure 5. DockingDocking results results for for compound compound 11 (depicted(depicted in in green) green) in in the the FtsZ FtsZ protein protein (cyan), (cyan), full full protein protein view and detailed view of the binding site and main ligand ligand-protein-protein interactions.

OnOn the the other other hand, hand, our our previous previous studies studies highlighted highlighted the the specific specific FtsZ FtsZ region region involved involved in in binding binding thethe 1,4 1,4-benzodioxane.-benzodioxane. Indeed, Indeed, this this scaffold scaffold extends extends its its conformation conformation towards towards the the inner inner part part of of the the pocket and and this this hydrophobic hydrophobic subpocket subpocket is surrounded is surrounded by hydrophobic by hydrophobic residues residues such as such Met98, as Phe100,Met98, Val129, Ile162, Gly193, Ile197, Val214, Met218, Met226, Leu261 and Ile311 [12]. The high hydrophobicity of this subpocket suggests that apolar tails are preferred, while polar moieties are not well-tolerated. This finding, in combination with the poor permeation predictions, justifies the lack of activity of sulfones 2 and 4, products of sulfur atom oxidation in the benzoxathiane scaffold. In addition, docking results of 2 and 4 clearly displayed significant clashes with FtsZ, as a result of the steric hindrance by the sulfonyl group. Moreover, the second main aim of our computational study was to decipher the influence of the linker length in FtsZ interaction and thus in the antimicrobial activity. The biological data in Table3 suggest that the presence of an ethylenoxy linker (compounds 6 and 7) let the maintenance of a biological activity that is comparable to the one of their homologues 1 and III, respectively. We thus in silico analyzed 6 and 7 and we compared their binding poses. The docking results were almost the same, as exemplified in Figure6 presenting derivative 7. Finally, we considered the propylenoxy chain of 8. Docking studies showed that several binding modes were possible, due to the flexibility of the linker. As a result, a rearrangement of the 2,6-difluorobenzamide moiety may be required for properly fitting the subpocket binding site. This might translate to the decrease in the biological activity of 8, when compared to its homologues III and 7, presenting the methylenoxy and ethylenoxy linkers, respectively. In conclusion, all the results presented in this paper entirely support our hypothesis that the deep cavity of the FtsZ interdomain binding site, where the 1,4-benzodioxane scaffold fits, is characterized by narrowness and hydrophobicity. As a result, the transformation of the 1,4-benzodioxane moiety into a more lipophilic one, as the 2-substituted 1,4-benzoxathiane or the 2-substituted 1,4-benzodithiane, let a proper accommodation into this pocket. The sulfur atom, among all the heteroatoms evaluated in these years, shows to be beneficial for the substitution of 1,4-benzodioxane oxygen(4), obtaining potent antimicrobials with a bactericidal effect and a strong effect on bacteria morphological changes. Antibiotics 2019, 8, x FOR PEER REVIEW 11 of 21

Phe100, Val129, Ile162, Gly193, Ile197, Val214, Met218, Met226, Leu261 and Ile311 [12]. The high hydrophobicity of this subpocket suggests that apolar tails are preferred, while polar moieties are not well-tolerated. This finding, in combination with the poor permeation predictions, justifies the lack of activity of sulfones 2 and 4, products of sulfur atom oxidation in the benzoxathiane scaffold. In addition, docking results of 2 and 4 clearly displayed significant clashes with FtsZ, as a result of the steric hindrance by the sulfonyl group. Moreover, the second main aim of our computational study was to decipher the influence of the linker length in FtsZ interaction and thus in the antimicrobial activity. The biological data in Table 3 suggest that the presence of an ethylenoxy linker (compounds 6 and 7) let the maintenance of a biological activity that is comparable to the one of their homologues 1 and III, respectively. We thus in silico analyzed 6 and 7 and we compared their binding poses. The docking results were almost the same, as exemplified in Figure 6 presenting derivative 7. Finally, we considered the propylenoxy chain of 8. Docking studies showed that several binding modes were possible, due to the flexibility of the linker. As a result, a rearrangement of the 2,6- difluorobenzamide moiety may be required for properly fitting the subpocket binding site. This mightAntibiotics translate2020, 9, 160to the decrease in the biological activity of 8, when compared to its homologues12 ofIII 22 and 7, presenting the methylenoxy and ethylenoxy linkers, respectively.

FigureFigure 6. 6. DockingDocking results results for for compound compound 7 7(depicted(depicted in in pink) pink) in in the the FtsZ FtsZ protein protein (cyan), (cyan), full full protein protein viewview and and detailed detailed view view of of the the binding binding site site and and main main ligand ligand-protein-protein interactions. interactions.

InMoreover, conclusion, the promisingall the results antimicrobial presented activities in this paper on E. colientirelyN43 suggestsupport that our this hypothesis lipophilic that cavity the is deepalmost cavity conserved of the among FtsZ Gram-negative interdomain binding and Gram-positive site, where strains. the 1,4 Unfortunately,-benzodioxane toscaffold our knowledge, fits, is characterizedthere are no byE. narrowness coli FtsZ crystallographic and hydrophobicity structures. reported in literature thus far, including the interdomainAs a result binding, the transformation site, where our of class the 1,4 of- derivativesbenzodioxane binds. moiety As sooninto a as more the X-ray lipophilic structure one, willas the be 2available,-substituted we 1,4 could-benzoxathiane move to a better or the design 2-substituted of novel 1,4 broad-benzodit spectrumhiane FtsZ, let a inhibitors, proper accommodation aided by sharp intocomputational this pocket. studies. The sulfur atom, among all the heteroatoms evaluated in these years, shows to be beneficialLastly, for wethe confirmedsubstitution the of susceptibility 1,4-benzodioxane of our oxygen(4), compounds obtaining to the potentE. coli AcrABantimicrobials cell membrane with a bactericidalpump, corroborating effect and oura strong preliminary effect on results bacteria for morphological benzodioxane-based changes inhibitors. [12]. Similar findings wereMoreover, reported the in apromising previous antimicrobial study by Kaul activit et al.ies [11 on], E. in coli which N43 thesuggest antibacterial that this elipophilicffect of TXA436 cavity is(Table almost1) was conserved restored inamong selected Gram Gram-negative-negative and strains Gram after-positive the genetic strains. inactivation Unfortunately, of AcrAB to or our its knowledge,inhibition by there the polyselective are no E. coli RND FtsZ e ffl crystallographicux pump inhibitor structures phenylalanine reported arginyl in literatureβ-naphthylamide thus far, includ(PaβN),ing reachingthe interdomain MIC of 8bindingµg/mL. site Similarly,, where synergyour class studies of derivatives with specific binds. e Asfflux soon pump as the inhibitors X-ray structure(EPIs) will will be be needed available, in order we could to better move characterize to a better thedesign efflux of pumpnovel broad susceptibility spectrum and FtsZ the inhibitors, activity of abenzodioxane-basedided by sharp computational FtsZ inhibitors studies. in a larger number of Gram-negative strains, as well as to evaluate the potentialLastly, we of confirm EPI-FtsZed inhibitors the susceptibility associations of our as broadcompounds spectrum to the therapeutic E. coli AcrAB options cell for membrane multidrug pump,resistant corroborating bacterial infections. our preliminary results for benzodioxane-based inhibitors [12]. Similar findings

3. Materials and Methods

3.1. Chemistry

All reagents (1,2-mercaptophenol, benzene-1,2-dithiol, catechol, LiAH4, mesyl chloride, K2CO3, TEA, m-CPBA, trimethyl orthoformate, H2SO4 conc) were purchased from Sigma Aldrich and were used without further purification. The solvents, such as ACN, THF, DCM, DMF, methanol and acetone, were purchased from Sigma Aldrich. Silica gel matrix, with fluorescent indicator 254 nm, was used in analytical thin-layer chromatography (TLC on aluminium foils), and silica gel (particle size 40-63 µm, Merck) was used in flash chromatography on Biotage IsoleraTM One or Sepachrom Puriflash XS 420; visualizations were accomplished with UV light (λ 254 nm). 1H-NMR spectra were measured by Varian Mercury 300 NMR spectrometer/Oxford Narrow Bore superconducting magnet operating at 300 MHz. 13C-NMR spectra were acquired operating at 75 MHz. Chemical shifts (δ) are reported in ppm relative to residual solvent as internal standard. Signal Antibiotics 2020, 9, 160 13 of 22 multiplicity is used according to the following abbreviations: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quadruplet, m = multiplet, bs = broad singlet. The final products, 1–8, were analyzed by reverse-phase HPLC using a Waters XBridge C-18 column (5 µm, 4.6 mm 150 mm) on × an Elite LaChrom HPLC system with a diode array detector. Mobile phase: A, H2O with 0.10% TFA; B, acetonitrile with 0.10% TFA; gradient, 90% A to 10% A in 25 min with 35 min run time and a flow rate of 1 mL/min. Their purity was quantified at peculiar λ max, depending on each sample and resulted to be >95%. The relative retention times are reported in each experimental section. Melting points were determined by DSC analysis over a TA Instruments DSC 1020 apparatus.

Synthesis Ethyl 1,4-benzoxathian-2-carboxylate (9) and ethyl 1,4-benzoxathian-3-carboxylate (10): TEA (0.50 mL, 3.52 mmol) was added dropwise to a solution of ethyl-2,3-dibromopropionate (0.44 g, 1.76 mmol) into a mixture 1/1 of ACN and water (10 mL). After stirring at room temperature for 30 min, 1,2-mercaptophenol (0.22 g, 1.76 mmol) was slowly added. The reaction mixture was stirred at RT for 72 h, and then, it was diluted with diethyl ether and water. The organic phase was dried over Na2SO4, filtered and concentrated to give a yellow oil as the residue. The crude was purified by flash chromatography on silica gel, using cyclohexane/ethyl acetate 95/5 as the elution solvent; the yielded ratio between 9 and 10 was quantified by 1H NMR and resulted in 60% for 9 and of 40% for 10. Yield: 65% 1 Ethyl 1,4-benzoxathian-2-carboxylate (9). H NMR (300 MHz, CDCl3, δ): 7.04 (m, 2H), 6.96 (d, J = 7.7 Hz, 1H), 6.87 (t, J = 7.4 Hz, 1H), 4.99 (dd, J = 5.3, 3.8 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 3.29 (d, J = 3.8 Hz, 1H), 3.28 (d, J = 5.3 Hz, 1H), 1.28 ppm (t, J = 7.1 Hz, 3H). 1 Ethyl 1,4-benzoxathian-3-carboxylate (10). H NMR (300 MHz, CDCl3, δ): 7.06 (m, 1H), 7.00 (dd, J = 7.6, 2.0 Hz, 1H), 6.88 (t, J = 7.4 Hz, 2H), 4.56 (dd, J = 11.5, 3.0 Hz, 1H), 4.46 (dd, J = 11.5, 6.5 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 4.12 (dd, J = 6.5, 3.0 Hz, 1H), 1.30 ppm (t, J = 7.1 Hz, 3H). 2-hydroxymethyl-1,4-benzoxathiane (11) and 3-hydroxymethyl-1,4-benzoxathiane (12): LiAlH4 (85 mg, 2.23 mmol) was suspended in dry THF (5 mL) at 0 ◦C under nitrogen atmosphere. The mixture of 9 and 10 (0.5 g, 2.23 mmol) was dissolved in THF (5 mL) and was added to the reaction. The mixture was warmed to RT and stirred for 30 minutes; at completion, it was cooled to 0 ◦C and slowly quenched with ethyl Acetate (5 mL). Further ethyl acetate (10 mL) was added, the organic layer was washed with brine (3 10 mL), dried over Na SO and concentrated under vacuum to give 0.38 g (81%) as a × 2 4 mixture of 60% 11 and 40% 12 as a colorless oil. 1 2-hydroxymethyl-1,4-benzoxathiane (11). H NMR (300 MHz, CDCl3, δ): 7.03 (m, 2H), 6.86 (m, 2H), 4.32 (m, 1H), 3.90 (dd, J = 11.7, 4.2 Hz, 1H), 3.84 (dd, J = 11.7, 5.6 Hz, 1H), 3.14 (dd, J = 13.0, 9.0 Hz, 1H), 2.97 ppm (dd, J = 13.0, 2.1 Hz, 1H). 1 3-hydroxymethyl-1,4-benzoxathiane (12). H NMR (300 MHz, CDCl3, δ): 7.02 (m, 2H), 6.86 (m, 2H), 4.50 (dd, J = 11.7, 4.2 Hz, 1H), 4.34 (dd, J = 11.7, 2.1 Hz, 1H), 3.88 (m, 2H), 3.42 ppm (ddd, J = 7.2, 4.2, 2.1 Hz, 1H). 2-mesyloxymethyl-1,4-benzoxathiane (13) and 3-mesyloxymethyl-1,4-benzoxathiane (14): Mesyl chloride (0.55 mL, 7.13 mmol) was added dropwise to a solution of 11 and 12 (1.0 g, 5.49 mmol) and TEA (1.0 mL, 7.13 mmol) in DCM (5 mL) at 0 ◦C. The mixture was stirred at room temperature for 3 h, diluted with DCM (20 mL), washed firstly with 10% aqueous NaHCO3 (10 mL), secondly with 10% aqueous HCl (10 mL) and finally with brine (10 mL), dried over Na2SO4, filtered and concentrated under vacuum to yield 1.58 g (80%) of 13 and 14 as an oil residue. 1 2-mesyloxymethyl-1,4-benzoxathiane (13). H NMR (300 MHz, CDCl3, δ): 7.04 (m, 2H), 6.88 (ddd, J = 14.2, 7.7, 1.2 Hz, 2H), 4.57 (m, 1H), 4.45 (m, 2H), 3.10 (s, 3H), 3.20–2.99 ppm (m, 2H). 1 3-mesyloxymethyl-1,4-benzoxathiane (14). H NMR (300 MHz, CDCl3, δ): 7.03 (m, 2H), 6.89 (m, 2H), 4.63 (ddd, J = 11.8, 3.2, 1.5 Hz, 1H), 4.42 (m, 2H), 4.24 (m, 1H), 3.59 (dddd, J = 9.5, 6.4, 3.2, 1.7 Hz, 1H), 3.07 ppm (s, 3H). Antibiotics 2020, 9, 160 14 of 22

3-(1,4-benzoxathiane-2-yl)-2,6-difluorobenzamide (1) and 3-(1,4-benzoxathiane-3-yl)-2,6- difluorobenzamide (3): Potassium carbonate (0.42 g, 3.01 mmol) was added to a solution of 2,6-difluoro-3-hydroxybenzamide (0.50 g, 2.88 mmol) in dry DMF (2 mL). After stirring at room temperature for 30 min, a solution of 13 and 14 (0.71 g, 2.74 mmol) in DMF (2 mL) was added. The reaction mixture was stirred at 60 ◦C for 16 h, concentrated under vacuum, diluted with Ethyl Acetate (20 mL), washed with brine (3 10 mL), dried over Na SO , filtered and concentrated, to give a × 2 4 residue, which was purified by flash chromatography on silica gel. Elution with 9/1 Cyclohexane/Ethyl Acetate gave 1 (35%) and 3 (20%) as white solids. mp (1): 117 ◦C and mp (3): 109–112 ◦C. 3-(1,4-benzoxathiane-2-yl)-2,6-difluorobenzamide (1). Tr (HPLC): 13.3 min (A% = 98.0% at 1 λ = 288 nm). H NMR (300 MHz, d6-DMSO, δ): 8.14 (bs, 1H), 7.85 (bs, 1H), 7.29 (m, 1H), 7.04 (m, 3H), 6.85 (m, 2H), 4.56 (m, 1H), 4.31 (m, 2H), 3.29 (dd, J = 14.1, 13.1 Hz, 1H), 3.15 ppm (dd, J = 13.1, 8.4 Hz, 13 1H). C NMR (75 MHz, d6-DMSO, δ): 161.7, 152.6 (dd, J = 240.5, 6.9 Hz), 151.2, 148.4 (dd, J = 247.4, 9.1 Hz), 143.2 (dd, J = 10.9, 2.8 Hz), 127.7, 126.2, 122.1, 118.8, 117.5, 117.1 (dd, J = 24.0, 20.6 Hz), 116.5 (d, J = 9.1 Hz), 111.5 (dd, J = 22.9, 3.4 Hz), 73.1, 71.2, 26.1 ppm. 3-(1,4-benzoxathiane-3-yl)-2,6-difluorobenzamide (3): Tr (HPLC): 13.5 min (A% = 95.1% at 1 λ = 288 nm). H NMR (300 MHz, d6-DMSO, δ): 8.13 (bs, 1H), 7.85 (bs, 1H), 7.27 (m, 1H), 7.03 (m, 3H), 6.86 (m, 2H), 4.48 (dd, J = 11.8, 4.3 Hz, 1H), 4.24 (m, 3H), 3.88 ppm (m, 1H). 13C NMR (75 MHz, d6-DMSO, δ): 161.7, 152.6 (dd, J = 240.5, 6.8 Hz), 151.5, 148.3 (dd, J = 247.3, 8.0 Hz), 143.0 (dd, J = 10.9, 2.8 Hz), 127.7, 126.0, 122.5, 118.7, 117.3, 117.1 (dd, J = 24.6, 20.0 Hz), 116.4 (d, J = 9.2 Hz), 111.5 (dd, J = 22.9, 4.6 Hz), 69.3, 65.3, 37.4 ppm. 3-(1,4-benzoxathiane-4,4-dioxide-2-yl)-2,6-difluorobenzamide (2): m-CPBA (0.10 g, 0.59 mmol) was added to a cooled solution of 1 (0.10 g, 0.30 mmol) in acetone (5 mL) at 0 ◦C. The reaction mixture was warmed to RT and stirred for 2 h, then 10 mL of 10% aqueous NaHCO3 was added, followed by 15 mL of ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated to give an oily residue. The further treatment with DCM lets the precipitation of 0.03 g (28%) of 2 as a white solid. 1 mp: 192 ◦C. Tr (HPLC): 10.1 min (A% = 95.9% at λ = 280 nm). H NMR (300 MHz, d6-DMSO, δ): 8.13 (bs, 1H), 7.85 (bs, 1H), 7.76 (dd, J = 8.0, 1.6 Hz, 1H), 7.57 (m, 1H), 7.31 (td, J = 9.3, 5.3 Hz, 1H), 7.20 (m, 1H), 7.14-7.06 (m, 2H), 5.10 (m, 1H), 4.49 (dd, J = 11.3, 3.2 Hz, 1H), 4.42 (dd, J = 11.3, 5.1 Hz, 1H), 13 4.03 (dd, J = 14.2, 1.6 Hz, 1H), 3.82 ppm (dd, J = 14.2, 12.0 Hz, 1H). C-NMR (75 MHz, d6-DMSO, δ): 162.0, 153.1, 152.7 (dd, J = 240.8, 6.7 Hz), 148.5 (dd, J = 247.5, 9.0 Hz), 142.9 (dd, J = 10.9, 3.4 Hz), 135.4, 125.7, 123.9, 122.9, 119.2, 117.1 (dd, J = 7.9, 1.9 Hz), 116.8 (dd, J = 24.7, 20.2 Hz), 111.7 (dd, J = 23.3, 3.7 Hz), 75.1, 71.0, 50.2 ppm. 3-(1,4-benzoxathiane-4,4-dioxide-3-yl)-2,6-difluorobenzamide (4): m-CPBA (0.06 g, 0.36 mmol) was added to a cooled solution of 2 (60 mg, 0.18 mmol) in acetone (5 mL) at 0 ◦C. The reaction mixture was warmed to RT and stirred for 2 h, then 10 mL of 10% aqueous NaHCO3 was added, followed by 15 mL of ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated to give an oily residue. The further treatment with DCM lets the precipitation of 0.03 g (28%) of 4 as a white solid. 1 mp: 184 ◦C. Tr (HPLC): 10.3 min (A% = 97.9% at λ = 281 nm). H NMR (300 MHz, d6-DMSO, δ): 8.11 (bs, 1H), 7.82 (bs, 1H), 7.76 (dd, J = 8.0, 1.6 Hz, 1H), 7.56 (ddd, J = 8.9, 7.3, 1.6 Hz, 1H 1H), 7.31 (td, J = 9.3, 5.3 Hz, 1H), 7.20 (m, 1H), 7.12-6.99 (m, 2H), 4.87 (dd, J = 13.1, 2.0 Hz, 1H), 4.81 (dd, J = 13.1, 13 5.6 Hz, 1H), 4.53 (m, 1H), 4.44-4.27 ppm (m, 2H). C-NMR (75 MHz, d6-DMSO, δ): 161.5, 154.0, 152.8 (dd, J = 242.2, 6.7 Hz), 148.4 (dd, J = 249.8, 8.6 Hz), 142.7 (dd, J = 11.4, 3.4 Hz), 135.0, 126.0, 124.3, 122.8, 119.1, 117.1 (dd, J = 24.9, 19.9 Hz), 116.7 (d, J = 10.4 Hz), 111.5 (dd, J = 23.0, 4.0 Hz), 67.1, 64.3, 57.0 ppm. Ethyl 1,4-benzodithian-2-carboxylate (15): TEA (0.56 mL, 4.03 mmol) was added dropwise to a solution of ethyl-2,3-dibromopropionate (0.52 g, 2.01 mmol) in DMF (5 mL). After stirring at room temperature for 20 min; therefore, 1,2-benzenedithiol (0.26 g, 1.83 mmol) in 2 mL of DMF was slowly added. The reaction mixture was stirred at RT for 2 h, and then it was concentrated under vacuum and then diluted with 15 mL of ethyl acetate. The organic phase was washed with brine (3 10 mL), dried 1 × over Na2SO4, filtered, and concentrated to give 0.38 g (86%) of 15 as a yellow oil. H NMR (300 MHz, Antibiotics 2020, 9, 160 15 of 22

CDCl3, δ): 7.26 (m, 2H), 7.06 (m, 2H), 4.30 (t, J = 6.5 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 3.32 (d, J = 6.5 Hz, 2H), 1.28 ppm (t, J = 7.2 Hz, 3H). 2-hydroxymethyl-1,4-benzodithiane (16): LiAlH4 (60 mg, 1.58 mmol) was suspended in dry THF (2 mL) at 0 ◦C under nitrogen atmosphere. The solution of 15 (0.38 g, 1.58 mmol) in THF (5 mL) was slowly added to the reaction. The mixture was then warmed to RT and stirred for 1 h; at completion, it was cooled to 0 ◦C and slowly quenched with ethyl acetate (5 mL). Further ethyl acetate (10 mL) was added, the organic layer was washed with brine (3 10 mL), dried over Na2SO4 and concentrated × 1 under vacuum to give 0.26 g (84%) of 16 as a brown oil. H NMR (300 MHz, CDCl3, δ): 7.20 (m, 2H), 7.02 (m, 2H), 3.90 (dd, J = 11.1, 7.3 Hz, 1H), 3.81 (dd, J = 11.1, 6.0 Hz, 1H), 3.70 (m, 1H), 3.27 (dd, J = 13.4, 3.7 Hz, 1H), 3.10 ppm (dd, J = 13.4, 6.4 Hz, 1H). 2-mesyloxymethyl-1,4-benzodithiane (17): Mesyl chloride (0.14 mL, 1.76 mmol) was added dropwise to a solution of 16 (0.25 g, 1.26 mmol) and TEA (0.25 mL, 1.76 mmol) in DCM (4 mL) at 0 ◦C. The mixture was stirred at that temperature for 3 h, diluted with DCM (20 mL), washed firstly with 10% aqueous NaHCO3 (10 mL), secondly with 10% aqueous HCl (10 mL) and finally with brine (10 mL), dried over Na2SO4, filtered and concentrated under vacuum to yield 0.34 g (quantitative) of 1 17 as a pink oil. H NMR (300 MHz, CDCl3, δ): 7.18 (ddd, J = 9.4, 4.8, 2.3 Hz, 2H), 7.04 (m, 2H), 4.57 (t, J = 10.0 Hz, 1H), 4.40 (dd, J = 10.3, 5.1 Hz, 1H), 3.91 (m, 1H), 3.29 (dd, J = 14.0, 3.5 Hz, 1H), 3.19 (dd, J = 14.0, 5.1 Hz, 1H), 3.07 ppm (s, 3H). 3-(1,4-benzodithiane-2-yl)-2,6-difluorobenzamide (5): Potassium carbonate (0.19 g, 1.35 mmol) was added to a solution of 2,6-difluoro-3-hydroxybenzamide (0.22 g, 1.29 mmol) in dry DMF (2 mL). After stirring at room temperature for 30 min, a solution of 17 (0.34 g, 1.23 mmol) in DMF (2 mL) was added. The reaction mixture was stirred at 60 ◦C for 16 h, concentrated under vacuum, diluted with Ethyl Acetate (15 mL), washed with brine (3 10 mL), dried over Na SO , filtered and concentrated, × 2 4 to give a residue, which was purified by flash chromatography on silica gel. Elution with 6/4 Cyclohexane/Ethyl Acetate and further crystallization from chloroform gave 0.22 g (50%) of 5 as a white 1 solid. mp: 134 ◦C. Tr (HPLC): 14.3 min (A% = 98.8% at λ = 268 nm). H NMR (300 MHz, d6-DMSO, δ): 8.14 (bs, 1H), 7.85 (bs, 1H), 7.26 (m, 1H), 7.21 (m, 2H), 7.05 (m, 3H), 4.35 (m, 1H), 4.21 (m, 1H), 4.09 (m, 1H), 3.30 (dd, J = 13.6, 3.0 Hz, 1H), 3.18 ppm (dd, J = 13.6, 6.1, 2.5 Hz, 1H). 13C NMR (75 MHz, d6-DMSO, δ): 161.6, 152.6 (dd, J = 240.4, 6.8 Hz), 148.4 (dd, J = 247.2, 8.3 Hz), 143.0 (dd, J = 10.8, 3.2 Hz), 131.5, 131.0, 129.5, 128.8, 126.4, 125.6, 117.1 (dd, J = 24.9, 20.4 Hz), 116.6 (dd, J = 9.3, 2.1 Hz), 111.5 (dd, J = 22.8, 3.9 Hz), 71.6, 41.6, 29.9 ppm. 3,4-dibromobutyric acid (18): Bromine (2.98 mL, 58.1 mmol) was added dropwise to a solution of 3-butenoic acid (5.0 g, 58.1 mmol) in DCM (50 mL) at 0 ◦C. Once added, the reaction mixture was warmed to RT and kept stirring for 16 h. The reaction mixture was then quenched with aqueous 10% sodium thiosulfate solution, dried over Na2SO4, filtered and concentrated under reduced pressure, 1 affording 13.25 g (93%) of 18 as a yellowish oil. H NMR (300 MHz, CDCl3, δ): 9.58 (bs, 1H); 4.48 (m, 1H), 3.92 (dd, J = 10.4, 4.3 Hz, 1H), 3.72 (t, J = 10.4 Hz, 1H), 3.44 (dd, J = 17.1, 3.8 Hz, 1H), 2.93 ppm (dd, J = 17.1, 9.2 Hz, 1H). Methyl 3,4-dibromobutyrate (19): 0.5 mL of H2SO4 conc. was added dropwise to a solution of 18 (13.25 g, 53.8 mmol) and trimethyl orthoformate (11.35 g, 107.0 mmol) in methanol (150 mL) at 0 ◦C. Once added, the reaction mixture was warmed to RT and then refluxed, keeping stirring, for 16 h. The reaction mixture was then concentrated under reduced pressure, resumed with ethyl acetate (150 mL), washed with aqueous 10% NaHCO (2 100 mL) and brine (100 mL), dried over Na SO , filtered 3 × 2 4 and concentrated under vacuum, affording 10.9 g (78%) of 19 as a yellowish oil. 1H NMR (300 MHz, CDCl3, δ): 4.50 (m, 1H), 3.91 (dd, J = 10.4, 4.3 Hz, 1H), 3.76 (s, 3H), 3.72 (dd, J = 10.2, 2.7 Hz, 1H), 3.34 (dd, J = 17.1, 3.8 Hz, 1H), 2.87 ppm (dd, J = 16.7, 9.2 Hz, 1H). Methyl (1,4-benzoxathian-2-yl)-acetate (20): Methyl 3,4-dibromobutirrate (1.50 g, 5.77 mmol) was added dropwise to a solution of 1,2-mercaptophenol (0.73 g, 5.77 mmol) and TEA (1.16 mL, 11.54 mmol) in acetonitrile/water 1:1 (15 mL) and stirred at room temperature for 18 h. The reaction mixture is then added with ethyl acetate (15 mL), washed firstly with 10% aqueous NaOH (15 mL) and Antibiotics 2020, 9, 160 16 of 22

lastly with brine (15 mL). The organic phase is then dried over NaSO4, filtered and concentrated under vacuum, affording 1.01 g (78%) of 20 as a yellowish oil. 1H NMR (300 MHz, CDCl3, δ): δ 7.15–6.93 (m, 2H), 6.94–6.74 (m, 2H), 4.70 (m, 1H), 3.74 (s, 3H), 3.14 (dd, J = 13.0, 2.3 Hz, 1H), 3.04 (dd, J = 13.0, 7.6 Hz, 1H), 2.87 (dd, J = 15.8, 6.7 Hz, 1H), 2.74 ppm (dd, J = 15.8, 6.5 Hz, 1H). 2-hydroxyethyl-1,4-benzoxathiane (21): LiAlH4 (30 mg, 1.78 mmol) was suspended in dry THF (2 mL) at 0 ◦C under nitrogen atmosphere. The solution of 20 (0.18 g, 0.71 mmol) in THF (5 mL) was slowly added to the reaction. The mixture was then warmed to RT and stirred for 1 h; at completion, it was cooled to 0 ◦C and slowly quenched with ethyl acetate (5 mL). Further ethyl acetate (10 mL) was added, the organic layer was washed with brine (3 10 mL), dried over Na2SO4 and concentrated × 1 under vacuum to give 0.13 g (92%) of 21 as an orange oil. H NMR (300 MHz, CDCl3, δ): δ 7.10–6.91 (m, 2H), 6.91–6.75 (m, 2H), 4.44 (m, 1H), 4.02–3.79 (m, 2H), 3.12–2.95 (m, 2H), 2.15–1.87 (m, 2H), 1.78 ppm (bs, 1H). 2-mesyloxyethyl-1,4-benzoxathiane (22): Mesyl chloride (0.24 mL, 3.12 mmol) was added dropwise to a solution of 21 (0.51 g, 2.59 mmol) and TEA (0.44 mL, 3.12 mmol) in DCM (5 mL) at 0 ◦C. The mixture was stirred at that temperature for 3 h, diluted with DCM (20 mL), washed firstly with 10% aqueous NaHCO3 (10 mL), secondly with 10% aqueous HCl (10 mL) and finally with brine (10 mL), dried over Na2SO4, filtered and concentrated under vacuum to yield 0.71 g (quantitative) of 1 22 as a yellowish oil. H NMR (300 MHz, CDCl3, δ): δ 7.11–6.93 (m, 2H), 6.85 (m, 2H), 4.61–4.32 (m, 3H), 3.04–3.01 (m, 2H), 3.03 (s, 3H), 2.18 ppm (m, 2H). 3-(1,4-benzoxathian-2-yl)ethoxy)-2,6-difluorobenzamide (6): Potassium carbonate (0.40 g, 2.86 mmol) was added to a solution of 2,6-difluoro-3-hydroxybenzamide (0.49 g, 2.73 mmol) in dry DMF (5 mL). After stirring at room temperature for 30 min, a solution of 22 (0.70 g, 2.55 mmol) in DMF (5 mL) was added. The reaction mixture was stirred at 60 ◦C for 16 h, concentrated under vacuum, diluted with ethyl acetate (30 mL), washed with brine (3 20 mL), dried over Na SO , filtered × 2 4 and concentrated, to give a residue, which was purified by flash chromatography on silica gel. Elution with 1/1 cyclohexane/ethyl acetate and further crystallization from IPA gave 0.36 g (39%) of 6 as a white 1 solid. mp: 109.75 ◦C. Tr (HPLC): 14.1 min (A% = 99.4% at λ = 282 nm). H NMR (300 MHz, d6-DMSO, δ): 8.09 (bs, 1H), 7.82 (bs, 1H), 7.26 (dt, J = 9.3, 5.3 Hz, 1H), 7.10–6.94 (m, 3H), 6.89–6.74 (m, 2H), 4.38 (tdd, J = 8.1, 5.2, 2.1 Hz, 1H), 4.29–4.22 (m, 2H), 3.27 (dd, J = 13.2, 2.1 Hz, 1H), 3.07 (dd, J = 13.2, 8.1 Hz, 13 1H), 2.27–2.05 ppm (m, 2H). C NMR (75 MHz, d6-DMSO, δ): 161.8, 152.3 (dd, J = 241.2, 6.8 Hz), 151.3, 148.3 (dd, J = 248.4, 8.4 Hz), 143.3 (dd, J = 10.9, 3.3 Hz), 127.5, 126.0, 121.9, 118.8, 117.7, 117.0 (dd, J = 24.9, 20.5 Hz), 115.9 (dd, J = 9.4, 2.5 Hz), 111.4 (dd, J = 22.9, 4.0 Hz), 71.5, 66.0, 34.1, 29.1 ppm. Methyl 1,4-benzodioxane-2-yl-acetate (23): Potassium carbonate (4.78 g, 34.00 mmol) was added to a solution of catechol (1.2 g, 11.00 mmol) in acetone (20 mL). After stirring at room temperature for 30 min, a solution of 19 (3.00 g, 11.00 mmol) in acetone (20 mL) was added. The reaction mixture was stirred at 60 ◦C for 16 h, concentrated under vacuum, diluted with Ethyl Acetate (50 mL), washed with brine (3 40 mL), dried over Na SO , filtered and concentrated, to give a residue, which was purified × 2 4 by flash chromatography on silica gel. Elution with 8/2 Cyclohexane/Ethyl Acetate gave 1.98 g (87%) of 1 23 as a colourless oil. H NMR (300 MHz, CDCl3, δ): 6.86 (m, 4H); 4.63 (tt, J = 6.7, 3.3 Hz, 1H), 4.32 (dd, J = 11.3, 2.2 Hz, 1H), 4.00 (dd, J = 11.3, 6.8 Hz, 1H), 3.75 (s, 3H), 2.80 (dd, J = 16.1, 6.8 Hz, 1H), 2.65 ppm (dd, J = 16.1, 6.7 Hz, 1H). 2-hydroxyethyl-1,4-benzodioxane (24): LiAlH4 (0.37 g, 10.00 mmol) was suspended in dry THF (2 mL) at 0 ◦C under nitrogen atmosphere. A solution of 23 (1.98 g, 9.5 mmol) in THF (5 mL) was added dropwise to the mixture; once added, the reaction was warmed to RT and stirred for 1 h; at completion, it was cooled to 0 ◦C and slowly quenched with ethyl acetate (10 mL). Further ethyl acetate (10 mL) was added, the organic layer was washed with brine (3 20 mL), dried over Na2SO4 and concentrated × 1 under vacuum to give 1.60 g (93%) of 24 as a colorless oil. H NMR (300 MHz, CDCl3, δ): 6.87 (m, 4H), 4.38 (m, 1H), 4.28 (dd, J = 11.3, 2.2 Hz, 1H), 3.94 (m, 3H), 1.91 ppm (m, 2H). 2-mesiloxyethyl-1,4-benzodioxane (25): Mesyl chloride (1.00 mL, 13.32 mmol) was added dropwise to a solution of 24 (1.60 g, 8.88 mmol) and TEA (1.80 mL, 13.32 mmol) in DCM (20 mL) at Antibiotics 2020, 9, 160 17 of 22

0 ◦C. The mixture was warmed to RT and stirred at that temperature for 2 h, diluted with further DCM (20 mL), washed firstly with 10% aqueous NaHCO3 (30 mL), secondly with 10% aqueous HCl (30 mL) and finally with brine (30 mL), dried over Na2SO4, filtered and concentrated under vacuum to yield 1 2.10 g (91%) of 25 as yellowish oil. H NMR (300 MHz, CDCl3, δ): 6.87 (m, 4H), 4.48 (ddd, J = 10.2, 7.0, 5.1 Hz, 2H), 4.34 (m, 1H), 4.27 (dd, J = 11.4, 2.3 Hz, 1H), 3.96 (dd, J = 11.4, 7.0 Hz, 1H), 3.05 (s, 3H), 2.09 ppm (m, 2H). 3-(1,4-benzodioxane-2-yl)ethoxy)-2,6-difluorobenzamide (7): Potassium carbonate (0.42 g, 3.01 mmol) was added to a solution of 2,6-difluoro-3-hydroxybenzamide (0.50 g, 2.88 mmol) in dry DMF (5 mL). After stirring at room temperature for 30 min, a solution of 25 (0.71 g, 2.75 mmol) in DMF (5 mL) was added. The reaction mixture was stirred at 60 ◦C for 16 h, concentrated under vacuum, diluted with ethyl acetate (30 mL), washed with brine (3 20 mL), dried over Na SO , × 2 4 filtered and concentrated, to give a residue which was crystallized from 3/2 cyclohexane/ethyl Acetate, accomplishing 0.50 g (54%) of 7 as a white solid. mp: 96 ◦C. Tr (HPLC): 13.2 min (A% = 98.0% at 1 λ = 278 nm). H NMR (300 MHz, d6-DMSO, δ): 8.12 (bs, 1H), 7.85 (bs, 1H), 7.27 (td, J = 9.4, 5.3 Hz, 1H), 7.07 (t, J = 8.9 Hz, 1H), 6.84 (m, 4H), 4.37 (dd, J = 16.8, 5.7 Hz, 2H), 4.24 (t, J = 6.1 Hz, 2H), 3.97 13 (dd, J = 11.2, 7.2 Hz, 1H), 2.08 ppm (dd, J = 11.0, 6.2 Hz, 2H). C NMR (75 MHz, d6-DMSO, δ): 161.8, 152.3 (dd, J = 239.9, 6.8 Hz), 148.3 (dd, J = 246.9, 8.4 Hz), 143.4, 143.3 (d, J = 10.8 Hz), 143.2, 121.8, 121.7, 117.6, 117.3, 117.1 (dd, J = 25.6, 21.0 Hz), 115.9 (d, J = 7.1 Hz), 111.4 (dd, J = 22.8, 3.9 Hz), 70.5, 67.6, 66.8, 30.4 ppm. Pent-4-en-1-yl methanesulfonate (26): Mesyl chloride (1.08 mL, 13.93 mmol) was added dropwise to a solution of 4-penten-1-ol (1.00 g, 11.61 mmol) and TEA (1.90 mL, 13.93 mmol) in DCM (10 mL) at 15 C. The mixture was stirred at that temperature for 3 h, diluted with DCM (20 mL), washed firstly − ◦ with 10% aqueous NaHCO3 (10 mL), secondly with 10% aqueous HCl (10 mL) and finally with brine (10 mL), dried over Na2SO4, filtered and concentrated under vacuum to yield 1.52 g (80%) of 26 as a 1 colorless oil. H NMR (300 MHz, CDCl3, δ): 5.78 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.12–4.97 (m, 2H), 4.23 (t, J = 6.5 Hz, 2H), 3.00 (s, 3H), 2.25–2.12 (m, 2H), 1.94–1.79 ppm (m, 2H). 2,6-difluoro-3-(pent-4-en-1-yloxy)benzamide (27): Potassium carbonate (0.48 g, 3.48 mmol) was added to a solution of 2,6-difluoro-3-hydroxybenzamide (0.57 g, 3.32 mmol) in dry DMF (5 mL). After stirring at room temperature for 30 min, a solution of 26 (0.52 g, 3.17 mmol) in DMF (5 mL) was added. The reaction mixture was stirred at 80 ◦C for 16 h, concentrated under vacuum, diluted with ethyl acetate (30 mL), washed firstly with 10% aqueous NaHCO3 (10 mL), secondly with 10% aqueous HCl (10 mL) and finally with brine (10 mL), dried over Na2SO4, filtered and concentrated, to give 0.71 g 1 (93%) of 27 as a brown oil. H NMR (300 MHz, CDCl3, δ): 6.99 (td, J = 9.1, 5.2 Hz, 1H), 6.86 (td, J = 9.1, 1.9 Hz, 1H), 5.98 (bs, 2H), 5.83 (ddt, J = 16.9, 10.2, 7.0 Hz, 1H), 5.12–4.92 (m, 2H), 4.01 (t, J = 6.4 Hz, 2H), 2.24 (dd, J = 14.1, 7.0 Hz, 2H), 1.98–1.81 ppm (m, 2H). 2,6-difluoro-3-(3-(oxiran-2-yl)propoxy)benzamide (28): m-CPBA (0.31 g, 1.78 mmol) was added to a solution of 27 (0.43 g, 1.78 mmol) in DCM (5 mL) at 0 ◦C. The reaction mixture was stirred at room temperature for 18 h, diluted with DCM, washed with 10% aqueous NaHCO3 (2 15 mL) dried 1 × over NaSO4, filtered and concentrated to give 0.42 g (91%) of 28 as a colorless oil H NMR (300 MHz, CDCl3, δ): 7.00 (td, J = 9.1, 5.2 Hz, 1H), 6.86 (td, J = 9.1, 1.9 Hz, 1H), 6.16 (bs, 1H), 6.03 (bs, 1H), 4.19–3.94 (m, 2H), 3.07–2.89 (m, 1H), 2.78 (dd, J = 4.8, 4.1 Hz, 1H), 2.51 (dd, J = 4.8, 2.7 Hz, 1H), 2.03–1.74 (m, 2H), 1.74–1.43 ppm (m, 2H). 2,6-difluoro-3-((4-hydroxy-5-(2-hydroxyphenoxy)pentyl)oxy)benzamide (29): NaH (0.04 g, 1.63 mmol) was suspended in dry DMF (5 mL) at 0 ◦C under nitrogen atmosphere. A solution of catechol (0.18 g, 1.63 mmol) in DMF (5 mL) was added dropwise and, after stirring at room temperature for 30 minutes, a solution of 28 (0.42 g, 1.63 mmol) in DMF (5 mL) was then added dropwise. The reaction mixture is heated at 100 ◦C and stirred for 72 h. The mixture was then cooled to RT and slowly quenched with 1/1 ethyl acetate and 10% aqueous HCl (40 mL). The phases were separated and the organic one was washed with brine (10 mL), dried over NaSO4, filtered and 1 concentrated to yield 0.46 g (77%) of 29 as a reddish oil. H NMR (300 MHz, d6-DMSO, δ): 8.62 Antibiotics 2020, 9, 160 18 of 22

(bs, 1H), 8.08 (bs, 1H), 7.80 (s, 1H), 7.20 (td, J = 9.3, 5.3 Hz, 1H), 7.03 (td, J = 9.3, 1.9 Hz, 1H), 6.89 (d, J = 6.9 Hz, 1H), 6.79–6.60 (m, 3H), 5.00 (s, 1H), 4.11–3.94 (m, 2H), 3.94–3.78 (m, 2H), 3.72 (dd, J = 8.8, 6.3 Hz, 1H), 1.95–1.45 ppm (m, 4H). 3-(1,4-benzodioxane-2-yl)propoxy)-2,6-difluorobenzamide (8): Under nitrogen atmosphere, to a solution of triphenylphosphine (0.48 g, 1.84 mmol) in THF (2 mL), DIAD (0.36 mL, 1.84 mmol) and a solution of 28 (0.45 g, 1.23 mmol) in THF (1 mL) were added dropwise at 0 ◦C. The reaction mixture was stirred at reflux for 24 h, concentrated under vacuum, diluted with ethyl acetate (10 mL), washed firstly with brine (10 mL), secondly with 10% aqueous NaOH (10 mL) and lastly with brine (10 mL), dried over NaSO4, filtered and concentrated, to give a residue which was purified by flash chromatography on silica gel. Elution with 1/1 cyclohexane/ethyl acetate gave 0.05 g (11%) of 8 as a white wax. Tr 1 (HPLC): 13.7 min (A% = 99.2% at λ = 279 nm). H NMR (300 MHz, d6-DMSO, δ): 8.11 (bs, 1H), 7.83 (bs, 1H), 7.20 (td, J = 9.3, 5.3 Hz, 1H), 7.04 (td, J = 9.0, 1.8 Hz, 1H), 6.87–6.70 (m, 4H), 4.31 (dd, J = 11.4, 2.2 Hz, 1H), 4.25–4.14 (m, 1H), 4.08 (t, J = 6.3 Hz, 2H), 3.87 (dd, J = 11.4, 7.5 Hz, 1H), 2.00–1.79 (m, 2H), 13 1.79–1.59 ppm (m, 2H). C NMR (75 MHz, d6-DMSO, δ): 161.81, 152.13 (dd, J = 240.8, 6.8 Hz), 148.29 (dd, J = 248.2, 8.5 Hz), 143.46, 143.50 (dd, J =10.6, 3.2 Hz), 143.45, 121.72, 121.51, 117.51, 117.23, 117.0 (dd, J = 23.0, 18.6 Hz), 115.83 (dd, J = 9.3, 2.5 Hz), 111.33 (dd, J = 22.8, 4.0 Hz), 72.80, 69.59, 67.67, 27.22, 24.68 ppm.

3.2. Cells Normal human lung ﬁbroblasts (MRC-5) were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin in an incubator at 5% CO2 atmosphere and 37 ◦C. The Gram-positive Staphylococcus aureus (methicillin-sensitive, MSSA ATCC 29213, and methicillin-resistant, MRSA ATCC 43300) and the Gram-negative Escherichia coli (ESBL, extended-spectrum beta-lactamase-positive) bacterial cells were grown in Luria-Bertani broth (LB) at 37 ◦C under constant shaking at 300 rpm.

3.3. Antibacterial Activity

3.3.1. MSSA, MRSA and ESBL Protocols The antibacterial activity was tested on both a methicillin-sensitive and a methicillin-resistant S. aureus strain, and an ESBL E. coli clinical isolate. All the compounds were dissolved at the final concentration of 20 mg/mL in dimethyl sulfoxide (DMSO) and serially diluted in LB. Fresh cell cultures were used at 105 cells/mL in a final volume of 2 mL. Each bacterial sample was grown with different compound concentrations that ranged from 100 to 0.1 µg/mL. After incubation at 37 ◦C for 16 h in aerobic culture tubes, the concentration of prokaryotic cells was determined by optical density measurement, at 600 nm (OD600) in a SmartSpecTM 3000 spectrophotometer (Bio-Rad, Oceanside, CA, USA) to determine the MIC. To determine the MBC, the bacteria were then washed three times with LB, centrifuged at 900 g for 10 min at 4 C, and the pellet resuspended in fresh LB. After overnight × ◦ incubation at 37 ◦C, the absence of growth was confirmed by OD measurement. The presence of antibacterial activity, at any concentration tested, was established for values of absorbance < 0.1 OD600. All tests were performed in quadruplicate and for each series of experiments, both positive (no compounds) and negative (no bacteria) controls were included.

3.3.2. MDRSA, E. coli N43, E. coli D22 Protocols Antibacterial activities of compounds were determined by the broth microdilution method against MDRSA 11.7, MDRSA 12.1 and E. coli N43 following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations and Clinical and Laboratory Standards Institute (CLSI) guidelines. Strains MDRSA ST-11.7 and ST-12.1 were obtained through the interlaboratory control organized by the EU Reference Laboratory—Antimicrobial Resistance. Only data on their resistance to antibiotics are known, but other data are not available. Antibiotics 2020, 9, 160 19 of 22

Bacterial suspension of specific bacterial strain equivalent to 0.5 McFarland turbidity standard was diluted with cation-adjusted Mueller Hinton broth with TES (Thermo Fisher Scientific), to obtain a final inoculum of 105 CFU/mL. Compounds dissolved in DMSO and inoculum were mixed together and incubated for 20 h at 37 ◦C. After incubation, the minimal inhibitory concentration (MIC) values were determined by visual inspection as the lowest dilution of compounds showing no turbidity. Tetracycline was used as a positive control on every assay plate.

3.4. Thiazolyl Blue Tetrazolium Bromide (MTT) Cytotoxicity Assay Compounds showing an antibacterial activity at a concentration lower than 10 µg/mL were serially diluted in DMEM and tested on MRC-5 cells by the MTT cytotoxicity assay (Sigma, St Louis, MO, USA). Cells (104 cells/well) were tested in a 96-well plate, in quadruplicate, using serially two-fold-diluted concentrations of the compound in 100 µL DMEM medium. After a 24-h incubation, the compound was removed, and the cells were overlaid with 1 mg/mL MTT in 100 µL serum-free DMEM for 3 h at 37 ◦C. The MTT solution was then replaced with DMSO for 10 min, and the absorbance was measured at 570 nm. The percentage of cytotoxicity was calculated by the formula [100 (sample OD/untreated − cells OD) 100]. The compound concentration reducing cell viability by 90% was deﬁned as the TD90 × cytotoxic dose. The “therapeutic index” (TI) was also determined and deﬁned as the ratio between TD90 and the MBC values.

3.5. Transmission Electron Microscopy S. aureus ATCC 43300 (109 cells/mL) were cultured in the presence of 1 and DFNB (positive control), at the same concentrations used for optical microscopy and, after 16 h incubation at 37 ◦C, the cells were harvested and processed for transmission electron microscopy, as already described [28]. Untreated S. aureus was used as negative control. After centrifugation at 3100 g for 5 min at room × temperature, pelleted bacteria were fixed in 2.5% glutaraldehyde (Polysciences, Warrington, PA) in 0.1 M Na cacodylate buffer, pH 7.4, for 1 h at 4 ◦C, rinsed twice and post-fixed in Na cacodylate-buffered 1% OsO4 for 1 h at 4 ◦C. The samples were dehydrated through a series of graded ethanol solutions and propylene oxide and embedded in Poly/Bed 812 resin mixture. Ultrathin sections were obtained using a Reichert-Jung ultramicrotome equipped with a diamond knife. Samples were then stained with water-saturated uranyl acetate and 0.4% lead citrate in 0.1 M NaOH. The specimens were viewed under a Philips CM10 electron microscope.

3.6. Computational Studies Ligand preparation. The preparation of the compounds synthesized in this work was performed before carrying out further computational studies. The preparation, together with the two-dimensional-to-three-dimensional conversion, was performed using LigPrep [29], included in the Schrödinger software package. In the preparation, we generated different steps such as the addition of hydrogens, the calculation of the ionization state of the molecules at a specific pH or the generation of potential tautomers. Additionally, low-energy ring conformations were generated for every molecule, followed by a final energy minimization step using the OPLS-2005 force [30,31]. With the aim of mimicking cell conditions, physiological pH states were used to prepare the molecules, all of them were desalted and the compounds were minimized as default in the last step. Protein preparation. The FtsZ crystal structure of S. aureus (PDB code: 5XDT) was used as the protein structure for the computational studies. The protein was prepared for docking studies following a protocol described in our previous studies [12]. Ligand Characterization. The compounds previously prepared were analyzed using the Qikprop module in the Schrödinger software package. QikProp allowed us to calculate and predict a total number of 44 pharmaceutically relevant ADME or ADME Tox properties among others. These properties include both simple molecular descriptors and relevant computational predictions for drug discovery. Properties were considered to assess key parameters such as lipophilicity or cellular permeability. Antibiotics 2020, 9, 160 20 of 22

Docking studies. Docking studies were performed by employing the Glide module included in the Schrödinger software package [32]. Once the protocol was validated in our previous studies [12], the FtsZ structure from S. aureus and the compounds synthesized in this work were used as a starting point for the analysis. Docking studies were performed applying the same protocol in terms of conformational search and evaluation parameters for all the molecules, considering the center of the grid the center of the previously crystallized ligand in the catalytic pocket. In the grid generation, a scaling factor of 1.0 in van der Waals radius scaling and a partial charge cut-oﬀ of 0.25 were used. The xtra precision (XP) mode with no constraints was applied during the docking [33]. The ligand sampling was ﬂexible, epik state penalties were added to the docking score and an energy window of 2.5 kcal/mol was utilized for ring sampling. In the energy minimization step, distance dependent dielectric constant was 4.0 with a maximum number of minimization steps of 100,000. In the clustering, poses were discarded as duplicates if both the RMS deviation was less than 0.5 Å and the maximum atomic displacement was less than 1.3 Å.

Author Contributions: Conceptualization, V.S.; Data curation, C.D.G.M.; Formal analysis, V.S.-P.; Funding acquisition, E.V.; Investigation, L.S., A.C., V.S.-P.,M.H. and C.Z.; Methodology, A.R. and E.V.; Project administration, V.S.; Supervision, E.V.; Validation, C.Z. and I.Z.; Visualization, V.S., L.S., A.C. and M.H.; Writing—original draft, V.S., A.C. and V.S.-P. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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