Synthesis and Influence of 3-Amino Benzoxaboroles Structure on Their Activity Against Candida Albicans

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Article Synthesis and Inﬂuence of 3-Amino Benzoxaboroles Structure on Their Activity against Candida albicans

Dorota Wieczorek 1, Ewa Kaczorowska 2, Marta Wi´sniewska 2, Izabela D. Madura 2 , Magdalena Le´sniak 2, Jacek Lipok 1 and Agnieszka Adamczyk-Wo´zniak 2,* 1 Faculty of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland; [email protected] (D.W.); [email protected] (J.L.) 2 Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland; [email protected] (E.K.); [email protected] (M.W.); [email protected] (I.D.M.); [email protected] (M.L.) * Correspondence: [email protected]

Academic Editor: Laurent Chabaud Received: 27 October 2020; Accepted: 15 December 2020; Published: 18 December 2020

Abstract: Benzoxaboroles emerged recently as molecules of high medicinal potential with Kerydi® (Tavaborole) and Eucrisa® (Crisaborole) currently in clinical practice as antifungal and anti- inflammatory drugs, respectively. Over a dozen of 3-amino benzoxaboroles, including Tavaborole’s derivatives, have been synthetized and characterized in terms of their activity against Candida albicans as a model pathogenic fungus. The studied compounds broaden considerably the structural diversity of reported benzoxaboroles, enabling determination of the influence of the introduction of a heterocyclic amine, a fluorine substituent as well as the formyl group on antifungal activity of those compounds. The determined zones of the growth inhibition of examined microorganism indicate high diffusion of majority of the studied compounds within the applied media as well as their reasonable activity. The Minimum Inhibitory Concentration (MIC) values show that the introduction of an amine substituent in position “3” of the benzoxaborole heterocyclic ring results in a considerable drop in activity in comparison with Tavaborole (AN2690) as well as unsubstituted benzoxaborole (AN2679). In all studied cases the presence of a fluorine substituent at position para to the boron atom results in lower MIC values (higher activity). Interestingly, introduction of a fluorine substituent in the more distant piperazine phenyl ring does not influence MIC values. As determined by X-ray studies, introduction of a formyl group in proximity of the boron atom results in a considerable change of the boronic group geometry. The presence of a formyl group next to the benzoxaborole unit is also detrimental for activity against Candida albicans.

Keywords: benzoxaboroles; Kerydin; antifungal; piperazine; formyl; Candida albicans

1. Introduction One of the applicable features of benzoxaboroles is their antifungal activity [1]. Over the last few decades, the frequency of infections caused by fungi has increased significantly. Most patients are diagnosed with superficial (mucosal or cutaneous) mycosis. However, invasive opportunistic mycoses associated with the infection of various internal organs such as liver, lungs, spleen, or brain are also often diagnosed. The most exposed group of people for such a type of infections are immune-compromised individuals including solid organ and blood or bone marrow transplant recipients as well as HIV positive or cancer patients [2,3]. The most common causes of mycoses are Candida spp. There are at least over a dozen Candida species that cause human infection but more than 90% of invasive diseases is caused by the five most common pathogens: C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei. Among those infection C. albicans remains the most common one [4]. Despite Candida

Molecules 2020, 25, 5999; doi:10.3390/molecules25245999 www.mdpi.com/journal/molecules MoleculesMolecules 20 202020, ,2 255, ,x x FOR FOR PEER PEER REVIEW REVIEW 22 of of 13 13

Molecules 2020, 25, 5999 2 of 13 thanthan 90%90% ofof invasiveinvasive diseasesdiseases isis causedcaused byby thethe fivefive mostmost commoncommon patpathogens:hogens: C.C. albicansalbicans,, C.C. glabrataglabrata,, C.C. tropicalistropicalis,, C.C. parapsilosisparapsilosis,, andand C.C. kruseikrusei.. AmongAmong thosethose infectioninfection C.C. albicansalbicans remainsremains thethe mostmost commoncommon oneoneis the [[44]] leading.. DespiteDespite reason CandidaCandida of isis the thethe opportunistic leadinleadingg reasonreason mycoses, ofof thethe opportunisticopportunistic there is a limited mycoses,mycoses, number therethere of isis aantifungala limitedlimited numbernumber agents ofofavailable antifungalantifungal for agentstheagents therapy availableavailable [5]. forfor thethe therapytherapy [[55]].. TavaboroleTavaborole (AN2690, (AN2690, Figure Figure 11)1)) isisis aaa small-moleculesmallsmall--moleculemolecule approvedapproved byby thethethe AmericanAmericanAmerican FoodFood andand DrugDrug AdministrationAdministration (FDA)(FDA)(FDA) in inin 2014 20142014 for forfor the thethe treatment treatmenttreatment of onychomycosis ofof onychomycosisonychomycosis caused causedcaused by dermatophyte byby dermatophytedermatophyte fungi [fungifungi6–12]. [[66––1212]].. HHOO BB OO

Tavaborole,Tavaborole, AN2690AN2690

Figure 1. The structure of Tavaborole (AN2690), active ingredient of Keridin–antifungal drug approved Figure 1. The structure of Tavaborole (AN2690), active ingredient of Keridin–antifungal drug Figureby FDA 1. in The 2014 structurefor the treatment of Tavaborole of onychomycosis. (AN2690), active ingredient of Keridin–antifungal drug

approvedapproved byby FDAFDA inin 20142014 forfor thethe treatmenttreatment ofof onychomycosis.onychomycosis. A relatively low molecular weight of Tavaborole results in high penetration into the nailplate [13]. A relatively low molecular weight of Tavaborole results in high penetration into the nailplate ThisA may relatively compensate low molecular its considerably weight of lower Tavaborole antifungal results activity in high determined penetration in intoin vitrothe nailplatestudies [13]. This may compensate its considerably lower antifungal activity determined in in vitro studies [in13 comparison]. This may compensate with three diitsff considerablyerent drugs suchlower as: antifungal Raconazole, activity terbinafine, determined and in fluconazole in vitro studies [14]. in comparison with three different drugs such as: Raconazole, terbinafine, and fluconazole [14]. Tavaborolein comparison acts by with blocking three differentthe fungus’s drugs ability such to produce as: Raconazole, proteins terbinafine,in a highly specific and fluconazole way. It disrupts [14]. Tavaborole acts by blocking the fungus’s ability to produce proteins in a highly specific way. It Tavaborolethe catalytic acts action by of blocking yeast cytoplasmic the fungus enzyme’s ability called to produce leucyl-tRNA proteins synthetase in a highly involved specific in translation way. It disrupts the catalytic action of yeast cytoplasmic enzyme called leucyl-tRNA synthetase involved in processdisrupts [ 15the]. catalytic This mechanism action of named yeast ascytoplasmic the oxaborole enzyme tRNA called trapping leucyl mechanism-tRNA synthetase (OBORT mechanism)involved in translation process [15]. This mechanism named as the oxaborole tRNA trapping mechanism translationis exclusive forprocess benzoxaborole [15]. This compounds, mechanism while named other as popular the oxaborole antifungal tRNA drugs trapping such as fluconazole,mechanism (OBORT mechanism) is exclusive for benzoxaborole compounds, while other popular antifungal (OBORTitraconazole, mechanism) terbinafine, is efinaconazole exclusive for and benz amorolfineoxaborole act compounds, by inhibiting while fungal other cell popularwall synthesis antifungal or by drugs such as fluconazole, itraconazole, terbinafine, efinaconazole and amorolfine act by inhibiting weakeningdrugs such microbialas fluconazole, metabolism itraconazole, [16]. The terbinafine, synthesis ofefinaconazole analogs of chemical and amorolfine compounds act by with inhibiting proven fungal cell wall synthesis or by weakening microbial metabolism [16]. The synthesis of analogs of fungaltherapeutic cell wall effect synthesis is a commonly or by weakening used method microbial in the designmetabolism of new [16 drugs.]. The Itsynthesis allows to of increase analogs the of chemical compounds with proven therapeutic effect is a commonly used method in the design of chemicalefficiency compounds of analogs in with relation proven to the therapeutic lead as well effect as to reduceis a commonly the costs ofused the method entire drug in the development design of new drugs. It allows to increase the efficiency of analogs in relation to the lead as well as to reduce processnew drugs. [17, 18It ].allows to increase the efficiency of analogs in relation to the lead as well as to reduce the costs of the entire drug development process [17,18]. the costsThe of current the entire study drug evaluates development analogs process of Tavaborole [17,18]. (1–5, Figure2) as well as unsubstituted The current study evaluates analogs of Tavaborole (1–5, Figure 2) as well as unsubstituted benzoxaboroleThe current (6 study(AN2679), evaluates Figure analogs3) and itsof Tavaboroleanalogs ( 7 – (151–,5 Figure, Figure3) as2) aspotential well as agents unsubstituted against benzoxaborole (6 (AN2679), Figure 3) and its analogs (7–15, Figure 3) as potential agents against Candidabenzoxaborole albicans (.6 Although(AN2679), severalFigure 3) molecules and its analogs combining (7–15 benzoxaborole, Figure 3) as potentialand heterocyclic agents against amine Candida albicans. Although several molecules combining benzoxaborole and heterocyclic amine Candidastructural albicans motives. Although have displayed several promising molecules antifungal combining activity benzoxaborole [19–21], a nonend heterocyclic of them has amine been structural motives have displayed promising antifungal activity [19–21], none of them has been previouslystructural motives studied have as agents displayed against promisingCandida albicans antifungal. At theactivity same [19 time,–21] some, none of of the them very has recently been previously studied as agents against Candida albicans. At the same time, some of the very recently previouslydescribed piperazine studied as bis(benzoxaboroles) agents against Candida displayed albicans reasonable. At the activitysame time, against some that of microorganism the very recently [22]. described piperazine bis(benzoxaboroles) displayed reasonable activity against that microorganism Thedescribed studied piperazine compounds bis(benzoxaboroles) broaden considerably displayed the structural reasonable diversity activity ofagainst reported that benzoxaboroles,microorganism [22]. The studied compounds broaden considerably the structural diversity of reported [enabling22]. The determination studied compounds of the influence broaden of the considerably presence of a heterocyclic the structural amine, diversity a fluorine of substituent reported benzoxaboroles, enabling determination of the influence of the presence of a heterocyclic amine, a benzoxaboroles,as well as the formyl enabling group determination on antifungal of activity the influence of those of compounds. the presence of a heterocyclic amine, a fluorinefluorine substituentsubstituent asas wellwell asas thethe formylformyl groupgroup onon antifungalantifungal activityactivity ofof thosethose compounds.compounds. HHOO HHOO BB OO BB OO

NN XX NN NN RR

FF FF 4 (R = Me), 5 (R = Ph) 11 (X(X == O),O), 22 (X(X == S),S), 33 (X(X == CHCH2)) 4 (R = Me), 5 (R = Ph) 2 Figure 2. Structures of studied Tavaborole’s derivatives (1–5). FigureFigure 2.2. StructuresStructures ofof studiedstudied TavabTavaborole’sorole’s derivativesderivatives ((11––55).).

Molecules 2020, 25, x FOR PEER REVIEW 3 of 13

Molecules 2020, 25, 5999 3 of 13 MoleculesHO 2020, 25, x FOR PEERHO REVIEW HO 3 of 13 B O B O B O

HO HO HO B O B O N X B O N N R

N X N N R 6 7 (X = O), 8 (X = S), 9 (X = CH ) 10 (R = Me), 11 (R = Ph), 2 12 (R = 2-F-Ph), 13 (R = 4-F-Ph)

HO 6 7 (X = O), 8 (X = S), 9 (X = CH ) HO 10 (R = Me), 11 (R = Ph), O B O 2 O 1B2 (R = O2-F-Ph), 13 (R = 4-F-Ph) HO HO O B O N N O B O N N Me

N 14 N 15 N N Me

Figure 3. Structures14 of unsubstituted benzoxaborole (6) and its studied15 derivatives (7–15).

Figure 3. Structures of unsubstituted benzoxaborole (6) and its studied derivatives (7–15). 2. Results andFigure Discussion 3. Structures of unsubstituted benzoxaborole (6) and its studied derivatives (7–15). 2. Results and Discussion 2.1. Synthesis and Structural Studies 2.1.2. Results Synthesis and and Discussion Structural Studies Most of the studied 3-amino-benzoxaboroles are novel compounds (2–5, 10, 11, 14 and 15) and Most of the studied 3-amino-benzoxaboroles are novel compounds (2–5, 10, 11, 14 and 15) and have have2.1. Synthesis been obtained and Structural according Studies to a very convenient procedure by mixing the starting been obtained according to a very convenient procedure by mixing the starting (2-formylphenyl)boronic (2-formylphenyl)boronicMost of the studied acid3-amino with-benzoxaboroles the corresponding are novelamine compounds at room temperature (2–5, 10, 11 in, 14 diethyl and 15 ether) and acid with the corresponding amine at room temperature in diethyl ether (Scheme1). (Schemehave been 1). obtained according to a very convenient procedure by mixing the starting (2H-formylphenyl)boronicO OH acid with the corresponding amine at roomHO temperature in diethyl ether (SchemeB 1). B O

R1 HO OH R1 HO B O Et2O, r.t. B O N X + HN X R1 R1 O Et2O, r.t. N X + HN X R R 1 5, 7 15 (36 98% yields) R R R = H or F, R1 = H or CHO; X = CH2 or O or S or NAr or NMe 1 5, 7 15 (36 98% yields) Scheme 1. Synthetic procedure for the studied 3-aminobenzoxaboroles. R = H or F, R = H or CHO; X = CH or O or S or NAr or NMe 1 2 Low solubility of several 3-aminobenzoxaboroles (4, 5, 10, 11, 14 and 15) in diethyl ether caused Scheme 1. Synthetic procedure for the studied 3-aminobenzoxaboroles. their precipitation from a reaction mixture, driving the reaction to completion as well as enabling straightforward isolation and puriﬁcation of the products. In some cases, subsequent crystallization was usedLow tosolubility purifyScheme theof several products 1. Synthetic 3-aminobenzoxaboroles (see procedure experimental for the section). studied (4, 5, 10 3,- Contrary aminobenzoxaboroles.11, 14 and to 15 some) in diethyl of the ether previously caused reportedtheir precipitation synthetic procedures from a reaction for 3-aminobenzoxaboroles mixture, driving the reaction [20,23,24 to] and completion similarly a ass well in the as synthesis enabling ofstraightforward bis(benzoxaboroles)Low solubility isolation of [several25 and] no purification 3 drying-aminobenzoxaboroles agent of the was products. used. (4 Most, 5 In, 10 some, of 11 the, 14cases, compoundsand subsequent 15) in diethyl have crystallization ether been caused fully characterizedwastheir used precipitation to bypurify NMR from the spectroscopy productsa reaction (see mixture, (1 H, experimental13C, driving11B NMR section).the spectra).reaction Contrary to If completion applicable, to some a19 ofsF well theNMR previouslyas spectraenabling havereportedstraightforward also synthetic been provided. isolation procedures Theand unsubstitutedforpurification 3-aminobenzoxaboroles of benzoxaborolethe products. [20, In (6 23some), was24] andcases, obtained similarly subsequent by as reduction in crystallizationthe synthesis of the correspondingofwas bis(benzoxaboroles) used to (2-formylphenyl)boronic purify the [25 products] no drying (see acid age experimental asnt it was was previously used. section). Most reported Contraryof the [compounds26 , to27 ]. some Compounds of have the been previously7–9 [ fully24], 1 13 11 19 12characterizedreportedand 13 [ 20synthetic] wereby NMR obtainedprocedures spectroscopy as for reported 3- amin( H, previously.obenzoxaborolesC, B NMR Most spectra). of[20, the23 If, samples24 applicable,] and similarly used F for NMR as microbiological in spectra the synthesis have studiesalsoof bis(benzoxaboroles) been gave provided. satisfactory The results[25 unsubstituted] no of drying elemental age benzoxaborole analysis,nt was used. which Most (6 conﬁrms) was of the obtained their compounds homogeneity by reduction have and been purity. of fully the However,correspondingcharacterized in case by(2- offNMRormylphenyl)boronic compounds spectroscopy2, 3, 7(–1H,9 acidresults 13C, as 11 Bit of NMRwas elemental previously spectra). analysis If reported applicable, as well [26 ,2719 asF] .1NMR HComp NMR spectraounds spectra 7have–9, showed[24also] 12 been contamination and provided. 13 [20] ofwereThe the products unsubstituted obtained with as diethyl reported benzoxaborole ether previously. and / (or6) the was Most starting obtained of amine. the by samples Those reduction impurities used of for the couldmicrobiologicalcorresponding not be removed (2 studies-formylphenyl)boronic neither gave under satisfactory high acid vacuum results as it was nor of previously elementalby washing reported analysis, with hexane, [26 whi,27ch]. which Comp confirmsounds suggests their 7–9 , co-crystallizationhomogeneity[24] 12 and and13 of purity. those[20] were compounds However, obtained in with case as amine of reported compounds or diethyl previously. ether.2, 3, 7 It– 9 isMost results worth of notingof the elemental samples that the analysis presence used as for 1 ofwellmicrobiological diethyl as H ether NMR was spectra studies previously showed gave detected contamination satisfactory in crystals results of the of 13 ofproducts by elemental X-ray with [20 ]. analysis,diethyl All the ether studied whi and/orch molecules confirms the starting are their homogeneity and purity. However, in case of compounds 2, 3, 7–9 results of elemental analysis as well as 1H NMR spectra showed contamination of the products with diethyl ether and/or the starting

Molecules 2020, 25, x FOR PEER REVIEW 4 of 13 Molecules 2020, 25, 5999 4 of 13 amine. Those impurities could not be removed neither under high vacuum nor by washing with hexane, which suggests co-crystallization of those compounds with amine or diethyl ether. It is stableworth in noting the solid that state the atpresence room temperature. of diethyl ether On was heating previously however, detected most ofin themcrystals decompose of 13 by X prior-ray or instead[20]. All of the melting studied (see molecules experimental are stable section in the for solid decomposition state at room temperatures). temperature. On heating however, mostMost of them of the decompose so far reported prior benzoxaboroles, or instead of meltin includingg (see AN2690experimental [26], section6 [27], 7for[28 decomposition] and 8 [23] form dimerstemperatures). in the solid state. This does not account for the recently reported phenylpiperazine derivatives 12 andMost13, both of the displaying so far reported pretty benzoxaboroles, unusual structural including patterns AN2690 for benzoxaboroles[26], 6 [27], 7 [28] [and20]. 8 In [23 search] form for a structure-activitydimers in the solid relationship, state. This molecular does not structures account for of novel the recently phenylpiperazine reported phenylpiperazine derivatives 11 and 14derivativeswere determined 12 and 13 in, crystallineboth displaying state. pretty Similarly unusual to 12structuraland 13, patterns compounds for benzoxaboroles11 and 14 crystallize [20]. In as ansearch equimolar for a mixture structure of-activity anantiomers relationship, (racemate). molecular Molecular structures structures of of novel11 and phenylpiperazine14 are shown in Figurederivatives4A,B. Both 11 and crystals 14 were exhibit determined symmetry in crystalline of P21/n centrosymmetric state. Similarly to space 12 and group 13, ofcomp theounds monoclinic 11 systemand 14 and crystallize in each caseas an one equimolar molecule mixture is present of anantiomers in the asymmetric (racemate). unit (seeMolecular Table S1,structures Supplementary of 11 Materials).and 14 are While shown compounds in Figure 4A11,Band. Both12 crystalsare isostructural, exhibit symmetry the introduction of P21/n centrosymmetric of the formyl groupspace in 14groupchanges of the the monoclinic geometry system of the boron and in vicinity each case considerably. one molecule In is 11presentand 14 in moleculesthe asymmetric the boron unit (see atom Table S1, Supplementary Materials). While compounds 11 and 12 are isostructural, the introduction shows a trigonal coordination and the B–O and B–C bond lengths are comparable for both molecules of the formyl group in 14 changes the geometry of the boron vicinity considerably. In 11 and 14 (see Table S2, Supplementary Materials). However, in the case of 14 the insertion of the formyl molecules the boron atom shows a trigonal coordination and the B–O and B–C bond lengths are substituent in the ortho position relative to the boronic moiety causes the significant O1–B1–O2 angle comparable for both molecules (see Table S2, Supplementary Materials). However, in the case of 14 narrowing (from 123.1(1) in 11 to 118.4(2) in 14) with simultaneous O1–B1–C1 angle broadening the insertion of the formyl substituent in the◦ ortho position relative to the boronic moiety causes the (from 128.3(1) in 11 to 133.5(2) in 14), whereas the five-membered ring inner O2–B1–C1 angle is significant O1–B1–O2 angle narrowing◦ (from 123.1(1) in 11 to 118.4(2)° in 14) with simultaneous retained.O1–B1–C1 This angle is following broadening the (from findings 128.3(1) by Czerwińskaet in 11 to 133.5(2) al.° in [29 14]), associating whereas the the five strains-membered in the ring boron coordinationinner O2–B1 sphere–C1 angle with is the retained position. This of the is hydrogen following atom the findings in syn (11 by) or Czerwińskaanti position et (14 al.). [ In29] the latterassociating such a the conformation strains in the may boron be imposedcoordination by thesphere presence with the of position a relatively of the strong hydrogen intramolecular atom in O1–Hsyn (...11) O3or antihydrogen position bond (14).(H In ...the Olatter and such O ... a Oconformation distances equal may 2.15(2)be imposed and 2.867(2) by the presence Å, respectively; of a O–Hrelat...ivelyOangle strong equals intramolecular 147(2)◦), leading O1–H…O3 to a formation hydrogen of bond a six-membered (H…O and H-bonded O…O distances ring, essentially equal flat2.15(2) and coplanar and 2.867(2) with Åthe , respectively; benzoxaborole O– unitH…O (deviation angle equals from 147(2) a plane°), defined leading by to all a formation atoms except of athe phenylpiperazinesix-membered H substituent-bonded ring, is essentially only 0.019(2) flat Å). and coplanar with the benzoxaborole unit (deviation from a plane defined by all atoms except the phenylpiperazine substituent is only 0.019(2) Å ).

FigureFigure 4. 4.(A ,(BA),B Ortep) Ortep drawings drawings [30 ][ of3011] ofand 11 14andmolecules 14 molecules with atoms with numbering atoms numbering scheme, scheme, (C) Molecules (C) overlayMolecules showing overlay the showing diﬀerence the indifference phenyl ringin phenyl orientation; ring orientation; (D) 1-D supramolecular (D) 1-D supramolecular chain formed chain by moleculesformed by of molecules11 joined byof O-H11 joined... N by hydrogen O-H…N bondshydrogen (in orange);bonds (in ( Eorange);) Hydrogen-bonded (E) Hydrogen dimer-bonded of 14 (intra- and intermolecular H-bonds are shown as blue and red lines, respectively. In (D,E) hydrogen atoms not involved in hydrogen bonds are omitted for clarity. MoleculesMolecules2020 2020, 25, 25,, 5999 x FOR PEER REVIEW 5 of 135 of 13

dimer of 14 (intra- and intermolecular H-bonds are shown as blue and red lines, respectively. In Interestingly,(D,E) hydrogen the atoms OH not donor involved in in14 hydrogenis also engagedbonds are inomitted the formationfor clarity. of a weak intermolecular hydrogen bonding with O3 formyl oxygen atom of the molecule related by the inversion center (H ...Interestingly,O and O ... theO OH distances donor in equal 14 is 2.38(2)also engaged and 3.033(2) in the formation Å, respectively; of a weak O–H intermolecular... O angle is hydrogen bonding with O3 formyl oxygen atom of the molecule related by the inversion center 137(2)◦). As a result, a new type of an H-bonded dimer is created (Figure4E), not previously observed(H…O and in structurallyO…O distances characterized equal 2.38(2) benzoxaboroles. and 3.033(2) Å, Whereas respectively; in 11 Oa– relativelyH…O angle strong is 137(2) O1-H°). ...As N2 a result, a new type of an H-bonded dimer is created (Figure 4E), not previously observed in intermolecular hydrogen bond is observed (H ... N and O ... N distances equal 2.13(2) and 2.962(1) Å, structurally characterized benzoxaboroles. Whereas in 11 a relatively strong O1-H…N2 respectively; O–H ... N angle is 171(1)◦), leading to the formation of an infinite chain along 2 screw intermolecular hydrogen bond is observed (H…N and O…N distances equal 2.13(2) and 2.962(1)1 Å, axis (Figure4D). It is noteworthy that the same motif was previously observed in the isostructural respectively; O–H…N angle is 171(1)°), leading to the formation of an infinite chain along 21 screw 3-[4-(2-fluorophenyl)piperazin-1-yl]-2,1-benzoxaborol-1(3H)-ol (12)[20]. The other difference between axis (Figure 4D). It is noteworthy that the same motif was previously observed in the isostructural 11 14 3-and[4-(2-fluorophenyl)piperazinmolecules is a twist-1 of-yl] the-2,1 phenyl-benzoxaborol ring with-1(3H) respect-ol (12 to) the[20] piperazine. The other ring di (Figurefference4 C), arisingbetween probably 11 and 14 from molecules packing is factorsa twist asof inthe both phenyl cases ring these with rings respect are to not the involved piperazine in ring any ( significantFigure intermolecular4C), arising probably interactions. from packing factors as in both cases these rings are not involved in any significant intermolecular interactions. 2.2. Studies of Activity against Candida albicans 2.2. TheStudies antifungal of Activity activity against Candida of all the Albicans studied benzoxaboroles (1–15) against C. albicans has been evaluatedThe byantifungal the diff usion activity agar of methodall the studied as well benzoxaboroles as by determination (1–15) of against the MIC C. values.albicans Resultshas been were comparedevaluated to by that the ofdiffusion Tavaborole agar (AN2690,method as Keridin)well as by reported determination previously of the and MIC carried values. out Results under were strictly thecompared same protocol to that [31 of ]. Tavaborole Additionally, (AN2690, amphotericin Keridin) B reported was used previously as a positive and standardcarried out and under DMSO asstrictly a negative the same probe. protocol Antifungal [31]. Additionally, activity was evaluatedamphotericin by B measuring was used theas a diameter positive ofstandard the clear and zone surroundingDMSO as a negative the holes. probe. The Antifungal results are activity shown was in Tableevaluated1. Figure by measuring5 shows picturesthe diameter of agar of the di clearffusion methodzone surrounding for chosen compounds the holes. The (5, 6 results, 11 and are14 shown) in three in repetitions. Table 1. Figure 5 shows pictures of agar diffusion method for chosen compounds (5, 6, 11 and 14) in three repetitions.

FigureFigure 5. 5.Three Three repetitionsrepetitions of the agar agar diffusion diﬀusion method method tests tests for for compounds: compounds: 6 (A6),(A 5), (B5),( B11), (11C) (andC) and 1414(D (D).).

TheThe agar agar di ﬀ diffusionusion tests tests show show that that most most of the of investigated the investigated benzoxaboroles benzoxaboroles display display higher higher diameter ofdiameter the inhibition of the inhibition zone in comparison zone in comparison with a standard with a standard amphotericin amphotericin B (9 mm B at(9 100mmµ atg). 100 μg). TheThe resultsresults indicateindicate high diffusion diﬀusion of of the the investigated investigated benzoxaboroles benzoxaboroles with with the the applied applied solid solid mediummedium as as well well as as their their reasonable antifungal antifungal activity. activity. The The highest highest values values are are observed observed for for the the unsubstitutedunsubstituted benzoxaborolebenzoxaborole ( 6)) with with the the diameter diameter of of 44 44 mm mm at at100 100 μgµ (gFigure (Figure 5A),5A), which which is smaller is smaller yetyet comparable comparable to to that that recently recently reported report fored AN2690 for AN2690 (54 mm (54 at mm 100 atµ g) 100 [31 μg)]. In [ case31]. ofIn the case investigated of the 3-substitutedinvestigated benzoxaboroles 3-substituted benzoxaboroles1–5 and 7–13 1the–5 and diameters 7–13 the of the diamet zonesers of of totally the zones inhibited of totally growth ofinhibitedCandida growth albicans ofare Candida considerably albicans are lower considerably and range lower from and 21 range mm for from11 21to mm 34 mm for 11 for to4 34at mm 100 µg. Twofor of4 at the 100 3-aminobenzoxaboroles μg. Two of the 3-aminobenzoxaboroles studied (14 and 15 studied), both containing (14 and 15), formyl both containing group in boron formyl atom proximity,group in boronreveal atom no inhibition proximity, of thereveal growth no inhibition of Candida of albicans the growthat applied of Candida conditions albicans (Figureat applied5D for conditions (Figure 5D for 14). Similar results have been previously reported for the 14). Similar results have been previously reported for the (2,6-diformylphenyl)boronic acid [32]. (2,6-diformylphenyl)boronic acid [32]. The determined values of the Minimal Inhibition Concentration (MIC) allow to arrange the The determined values of the Minimal Inhibition Concentration (MIC) allow to arrange the investigated compounds 1–15 with respect to their activity against Candida albicans. The most potent investigated compounds 1–15 with respect to their activity against Candida albicans. The most potent one is the unsubstituted benzoxaborole (6) with MIC value of 8 µg/mL, which is however four times one is the unsubstituted benzoxaborole (6) with MIC value of 8 μg/mL, which is however four times higherhigher than than that that ofof TavaboroleTavaborole (AN2690).(AN2690). Introduction Introduction of of the the amine amine substituent substituent at position at position “3”“3” of the of the heterocyclic ring results in a considerable drop of the activity. The trend is similar in case of analogues of Tavaborole (1–5) as well as compound 6 and its derivatives (7–15). Molecules 2020, 25, 5999 6 of 13

Table 1. The average diameter of the zone of totally inhibited growth of Candida albicans at various amounts (mm) and Minimal Inhibitory Concentration (µg/mL).

Compound 10 µg 25 µg 50 µg 100 µg DMSO MIC (µg/mL) amphotericine B 9 1 9 1 8 1 9 1 0 <=1 ± ± ± ± Tavaborole [31] 40 1 44 1 48 2 54 2 0 2 ± ± ± ± 1 20 1 25 2 28 2 31 3 0 62.5 ± ± ± ± 2 20 1 25 1 27 2 29 1 0 62.5 ± ± ± ± 3 20 2 25 1 27 2 31 1 0 62.5 ± ± ± ± 4 12 1 27 1 30 1 34 1 0 31.2 ± ± ± ± 5 19 1 25 2 28 1 32 1 0 31.2 ± ± ± ± 6 30 1 36 1 39 1 44 2 0 8 ± ± ± ± 7 11 1 17 1 23 1 27 1 0 125 ± ± ± ± 8 7 1 13 2 20 2 25 2 0 125 ± ± ± ± 9 10 1 16 2 22 1 26 2 0 125 ± ± ± ± 10 6 * 1 14 1 21 1 28 1 0 62.5 ± ± ± ± 11 5 * 1 10 1 18 1 21 1 0 62.5 ± ± ± ± 12 5 * 1 15 1 22 2 26 1 0 62.5 ± ± ± ± 13 12 1 18 2 22 1 27 1 0 62.5 ± ± ± ± 14 0 0 0 0 0 >250 15 0 0 0 0 0 >500 * only inhibition of the growth of C. albicans was observed in that case.

In both cases the piperazine derivatives–compounds 4, 5 and 10–13 display higher activity in comparison of morpholine, thiomorpholine and piperidine derivatives (1–3 and 7–9 respectively). The MIC values range from about 31 to 62 µg/mL for the 3-amino analogues of Tavaborole (1–5) and from about 62 to 125 µg/mL for analogues of 6, which contain no fluorine substituent at position para to the boron atom. These results show that the presence of a fluorine atom at position para to the boronic group enhances antifungal activity. Interestingly, no such effect was observed for introduction of the fluorine atom in a more distant part of the molecule i.e., in the phenyl piperazine ring in compounds 12 and 13.

3. Conclusions The structure of reported benzoxaboroles influences considerably their activity against Candida albicans. The unsubstituted benzoxaborole (6) displays the highest activity amongst all the investigated compounds. In all of the studied cases, introduction of the amino substituent in position “3” of the heterocyclic ring results in a significant drop in activity. The unfavorable effect is higher for simple heterocyclic amines derivatives such as morpholine, thiomorpholine and piperidine than for the piperazine derivatives that still display a reasonable activity. The other structural factor considerably influencing performance of investigated benzoxaboroles is the presence of a fluorine substituent at position para to the boron atom, which results in lower MIC values (higher activity). This effect is the most profound for unsubstituted benzoxaborole compared with Tavaborole, where the lack of the fluorine atom results in four times higher MIC value (lower activity) for the unsubstituted benzoxaborole in comparison with the known drug. In case of investigated 3-amino-benzoxaboroles the effect is smaller, yet noticeable. All derivatives containing fluorine substituent at position para to the boron atom display twice lower MIC values comparing to derivatives containing no such a substituent. Interestingly, no influence on activity was observed for derivatives containing fluorine atom in the more distant phenylpiperazine substituent. It is worth noticing that the presence of the Molecules 2020, 25, 5999 7 of 13 formyl group in the boron vicinity influences the geometry of the boronic group considerably and is detrimental for the activity against Candida albicans.

4. Experimental Section

4.1. Materials and Methods All reactions were performed under air, using undried glassware and undistilled solvents. Reagents and solvents were obtained from commercial sources (Sigma-Aldrich, St. Louis, MO, USA, POCH, ChemPur) and used without further purification. Deuterated solvents were obtained from Armar Chemicals and used as received. The reported yields correspond to isolated products. NMR spectra were recorded on a Bruker Avance 300 MHz or Varian NMR System 500 MHz spectrometers in CDCl3 or (CD3)2CO at room temperature. Chemical shifts are reported in parts per million (ppm), relative to the residual undeuterated solvent signal as an internal standard. All signals are reported as follows: chemical shift (multiplicity, number of corresponding nuclei), using the following abberviations: s = singlet, m = multiplet, d = doublet, dd = doublet of doublets, t = triplet, br = broad signal. Elemental analyses were obtained using a Perkin Elmer 2400 apparatus. All the Yeast Peptone Dextrose (YPD) medium components as well as Amphotericin B, were obtained from Sigma– Aldrich. DMSO was purchased from POCh. Candida albicans LOCK 001 was obtained from the collection of microorganisms of Institute of Fermentation Technology and Microbiology (University of Technology, Łód´z,Poland). Studies of the activity against C. albicans by the diffusion agar method: 0.5 mL of inoculum containing 106–107 fungal cells were spread on the surface of the solidified yeast extract peptone dextrose (YPD) medium and allowed to dry. Portions of 100, 50, 25 and 10 µg of the tested compounds dissolved in DMSO were placed in 2 mm diameter holes, which were cut into the media. The duration of incubation of the fungi was established as 48 h at 27 ◦C. Each experiment, including control, was carried out in three repetitions. Determination of MIC values: A 24-h-old culture of C. albicans was diluted with a fresh YPD medium to obtain optical density of 0.02 at 600 nm (OD600). The investigated compounds were dissolved in DMSO and placed in the initial culture of yeast (2 mL), ensuring final concentration 1 ranging from 0.9 to 500 µg mL− ). Cells were incubated for 24 h at 25 ◦C on a rotary shaker (60 rpm). After that time, the OD600 measurements were performed using a spectrophotometer (UV-2601, Rayleigh). The MIC endpoints defined as the lowest concentration at which no visible growth of yeast was observed were read visually following 24 h of incubation. Each experiment (control or compound-containing medium) was repeated three times. The single crystal X-ray experiments were conducted at room temperature on the Gemini A Ultra Diffractometer (Rigaku Oxford Diffraction) using graphite monochromated Mo/Kα radiation (λ = 0.71073 Å). Data collection and reduction were performed in the CrysAlisPro program [33]. The empirical absorption corrections using spherical harmonics, implemented in multi-scan scaling algorithm were applied. OLEX-2 suite [34] with implemented ShelXT [35] structure solution program using Intrinsic Phasing and the ShelXL [36] refinement package (the full-matrix least-squares technique) were used to solve and refine the structures. All non-hydrogen atoms were refined with anisotropic temperature factors. The carbon hydrogen atoms were placed in calculated positions while those bonded with oxygen atoms were refined freely. For all hydrogen atoms fixed isotropic thermal parameters (U (H) = 1.2 [Ueq(C) or Ueq(O)] were used. The final refinement indices for the iso × observed data converged to R1 = 4.39% and wR2 = 9.79% for 11 and R1 = 5.73% and wR2 = 12.34% for 14 (see Table S1 in the Supplementary Materials). Selected geometrical parameters are collected in Table S2 in the Supplementary Materials file. Molecular and packing diagrams were generated using MERCURY [37] and ORTEP-3 for Windows [30] programs. CCDC 1998837-1998838 contain the Molecules 2020, 25, 5999 8 of 13 supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures.

4.2. Synthetic Procedures and Characterization Data 5-Fluoro-3-morpholin-4-yl-2,1-benzoxaborol-1(3H)-ol (1)

(4-ﬂuoro-2-formylphenyl)boronic acid (0.235 g, 1.39 mmol) and diethyl ether (10 mL) were placed in a glass vial and mixed until dissolved. Morpholine (0.122 mL, 1.39 mmol) was added to the solution and mixed on a magnetic stirred for several minutes. The reaction mixture was left in the open vessel for one day to self-evaporate most of the Et2O (longer time in the open vessel did not result in the evaporation of more solvent). On the next day, the glassy body obtained was subjected to evaporation in a vacuum to remove remnant volatiles resulting in 0.322 g of the solid, yield: 97%. 1H NMR (300 MHz, Acetone) δ 8.11 (s, br, 1H, B-OH), 7.73 (dd, J = 8.0, 5.6 Hz, 1H), 7.28–7.00 (m, 2H), 5.80 (s, 1H), 3.62–3.99 (m, 4H), 2.72–2.59 (m, 2H), 2.58–2.43 (m, 2H). 13C NMR (75 MHz, Acetone) 1 3 3 2 δ 166.1 (d, JcF = 247.7 Hz), 156.7 (d, JcF = 8.1 Hz), 133.1 (d, JcF = 9.0 Hz), 116.7 (d, JcF = 22.3 Hz), 2 3 19 110.5 (d, JcF = 22.0 Hz), 96.6 (d, JcF = 3.5 Hz), 67.50, 48.05. F NMR (282 MHz, Acetone) δ 111.12 (m). 11 − B NMR (96 MHz, Acetone) δ 31 (s). EA calculated for C11H13BFNO3: C(55.74%), H(5.53%), N(5.91%), determined: C(56.29%), H(5.87%), N(7.00%). m.p. = 65–105 ◦C (decomposition).

5-Fluoro-3-thiomorpholin-4-yl-2,1-benzoxaborol-1(3H)-ol (2)

(4-ﬂuoro-2-formylphenyl)boronic acid (0.234 g, 1.39 mmol) and diethyl ether (10 mL) were placed in a glass vial and mixed until dissolved. Tiomorpholine (0.139 mL, 1.39 mmol) was added to the solution and mixed on a magnetic stirred for several minutes. The reaction mixture was left in the open vessel for one day to self-evaporate most of the Et2O (longer time in the open vessel did not result in the evaporation of more solvent). On the next day, the glassy body obtained was subjected to evaporation in a vacuum to remove remnant volatiles resulting in 0.342 g of the solid, yield: 97%. 1 H NMR (300 MHz, CDCl3): δ (ppm) = 7.70–7.67 (m, 1H, Ar), 7.14–7.08 (m, 2H, Ar), 5.79 (s, 1H, 19 CH), 3.22–3.19 (m, 2H, CH2N), 2.93–2.83(m, 2H, CH2N), 2.68–2.64 (m, 4H, (CH2)2S). F NMR (282 MHz, CDCl ): δ (ppm) = 108.8 (s, br). 11B NMR (96 MHz, CDCl ): δ (ppm) = 31. EA calculated 3 − 3 for C11H13BFNO2S: C(52.20%), H(5.18%), N(5.53%), determined: C(52.28%), H(5.40%), N(5.42%). m.p. = 70–85 ◦C (decomposition).

5-Fluoro-3-piperidin-1-yl-2,1-benzoxaborol-1(3H)-ol (3)

(4-Fluoro-2-formylphenyl)boronic acid (0.238 g, 1.42 mmol) and diethyl ether (10 mL) were placed in a glass vial and mixed until dissolved. Piperidine (0.140 mL, 1.42 mmol) was added to the solution and mixed on a magnetic stirred for several minutes. The reaction mixture was left in the open vessel for one day to self-evaporate most of the Et2O (longer time in the open vessel did not result in the evaporation of more solvent). On the next day, the glassy body obtained was subjected to evaporation in a vacuum to remove remnant volatiles resulting in 0.313 g of the solid, yield: 94%. 1 H NMR (300 MHz, CDCl3): δ (ppm) = 7.59 (s, br, 1H, Ar), 7.11–7.08 (m, 2H, Ar), 5.77 (s, br, 1H, 19 CH) 2.94, (s, br, 2H, CH2), 2.59 (s, br, 2H), 1.63–1.47), 6H, (CH2)3). F NMR (282 MHz, CDCl3): δ (ppm) = 109.6. 11B NMR (96 MHz, CDCl ): δ (ppm) = 30. EA calculated for C H BFNO : C(61.31%), − 3 12 15 2 H(6.43%), N(5.96%); determined: C(63.44%), H(6.97%), N(7.04%). m.p. = 67–75 ◦C (decomposition).

5-Fluoro-3-(4-methylpiperazin-1-yl)-2,1-benzoxaborol-1(3H)-ol (4)

A solution of (4-fluoro-2-formylphenyl)boronic acid (1.5 g; 9 mmol) in diethyl ether (85 mL) was stirred in a 250 mL flask at room temperature. Next, 1-methylpiperazine (0.9 g; 1.0 mL; 9 mmol) was added during 3 min resulting immediately in precipitation of a white solid. It was filtered, washed with diethyl ether (2 3 mL) and dried to give the title compound as white solid. Yield: 1.48 g; 6 mmol; 65%; × Molecules 2020, 25, 5999 9 of 13

1 H NMR (500 MHz, CDCl3) δ 7.66 (dd, J = 8.0, 5.6 Hz, 1H, Ar), 7.16–6.95 (m, 2H, Ar), 5.78 (s, 1H, CH), 13 1 3.20–2.00 (m, br, 8H, CH2N), 2.32 (s, 3H, CH3). C NMR (126 MHz, CDCl3) δ 165.3 (d, JCF = 248.9 Hz), 3 3 2 2 155.6 (d, JCF = 8.3 Hz), 132.0 (d, JCF = 8.9 Hz), 116.0 (d, JCF = 21.9 Hz), 109.8 (d, JCF = 21.7 Hz), 4 11 19 95.8 (d, JCF = 3.1 Hz), 55.2 (s), 45.8 (s). B NMR (75 MHz, CDCl3): δ 30 (s, br). F NMR (282 MHz, CDCl ): δ 110.09–110.18 (m). EA calculated for C H N BO F: C(57.63%), H(6.45%), N(11.20%); 3 − 12 16 2 2 determined: C(57.72%), H(6.51%), N(11.12%). m.p. = 166–169 ◦C (decomposition, browning).

5-Fluoro-3-(4-phenylpiperazin-1-yl)-2,1-benzoxaborol-1(3H)-ol (5)

A solution of (4-fluoro-2-formylphenyl)boronic acid (1.5 g; 9 mmol) in diethyl ether (90 mL) was stirred in a 250 mL flask at room temperature. Next, 1-phenylpiperazine (1.4 g; 1.4 mL; 9 mmol) was added during 3 min resulting in precipitation of a white solid after 24 h. It was filtered, washed with diethyl ether (2 3 mL) and dried to give the title compound as a white solid. Yield: 0.99 g; × 3 mmol; 36%; 1 H NMR (300 MHz, CDCl3) δ 7.69 (dd, J = 8.0, 5.6 Hz, 1H, Ar), 7.31–7.22 (m, 2H, Ar), 7.19–7.06 (m, 2H, Ar), 6.93 (d, J = 8.0 Hz, 2H, Ar), 6.87 (t, J = 7.3 Hz, 1H, Ar), 5.92 (s, 1H), 13 1 3.18 (t, J = 5.0 Hz, 4H), 3.01–2.64 (m, 4H). C NMR (126 MHz, CDCl3) δ 165.5 (d, JCF = 249.4 Hz), 151.2, 3 2 2 132.2 (d, JCF = 9.6 Hz), 129.3, 129.1, 119.9, 116.9, 116.4 (d, JCF = 22.2 Hz), 116.3, 110.1 (d, JCF = 22.0 Hz), 96.7, 49.4, 46.7. 11B NMR (96 MHz, CDCl ): δ 30. 19F NMR (282 MHz, CDCl ): δ 108.4 (m). 3 3 − EA calculated for C17H18N2B02F: C(65.41%), H(5.81%), N(8.97%); determined: C(65.60%), H(5.90%), N(9.11%). m.p. = 160–163 ◦C (decomposition).

2,1-Benzoxaborol-1(3H)-ol (6)

1H NMR (400 MHz, Acetone) δ 8.13 (s, 1H, B(OH)), 7.73 (d, J = 7.3 Hz, 1H, Ar), 7.49–7.43 (m, 1H, Ar), 7.42–7.38 (m, 1H, Ar), 7.33 (td, J = 7.3, 1.0 Hz, 1H, Ar), 5.01 (s, 2H).

3-Morpholin-4-yl-2,1-benzoxaborol-1(3H)-ol (7)

(2-formylphenyl)boronic acid (0.252 g, 1.68 mmol) and diethyl ether (10 mL) were placed in a glass vial and mixed until dissolved. Morpholine (0.147 mL, 1.68 mmol) was added to the solution and mixed on a magnetic stirred for several minutes. The reaction mixture was left in the open vessel for one day to self-evaporate most of the Et2O (longer time in the open vessel did not result in the evaporation of more solvent). On the next day, the glassy body obtained was subjected to evaporation in a vacuum to remove remnant volatiles resulting in 0.350 g of the solid, yield: 95%. 1 H NMR (300 MHz, CDCl3) δ 7.69 (d, J = 6.6 Hz, 1H, Ar), 7.54–7.32 (m, 3H, Ar), 5.85 (s, 1H, 11 CH), 3.71–3.68 (m, 4H), 2.73–2.61 (m, 4H). B NMR (96 MHz, CDCl3): δ (ppm) 31. EA calculated for C11H14BNO3: C(60.32%), H(6.44%), N(6.39%); determined: C(60.27%), H(6.42%), N(6.37%). m.p. = 63–80 ◦C (decomposition).

3-Thiomorpholin-4-yl-2,1-benzoxaborol-1(3H)-ol (8)

(2-formylphenyl)boronic acid (0.202 g, 1.35 mmol) and diethyl ether (10 mL) were placed in a glass vial and mixed until dissolved. Thiomorpholine (0.135 mL, 1.35 mmol) was added to the solution and mixed on a magnetic stirred for several minutes. The reaction mixture was left in the open vessel for one day to self-evaporate most of the Et2O (longer time in the open vessel did not result in the evaporation of more solvent). On the next day, the glassy body obtained was subjected to evaporation in a vacuum to remove remnant volatiles resulting in 0.310 g of the solid, yield: 98%. 1H NMR (300 MHz, CDCl3) δ 7.74–7.64 (m, 1H, Ar), 7.54–7.44 (m, 1H, Ar), 7.45–7.33 (m, 2H, 11 Ar), 5.82 (s, 1H, CH), 3.18–3.05 (m, 4H, CH2), 3.03–2.83 (m, 4H, CH2). B NMR (96 MHz, CDCl3): δ (ppm) = 31. EA calculated for C11H14BNO2S: C(56.19%), H(6.00%), N(5.96%); determined: C(54.31%), H(6.55%), N(8.12%). m.p. = 56–57 ◦C (decomposition). Molecules 2020, 25, 5999 10 of 13

3-Piperidin-1-yl-2,1-benzoxaborol-1(3H)-ol (9)

(2-formylphenyl)boronic acid (0.255 g, 1.70 mmol) and diethyl ether (10 mL) were placed in a glass vial and mixed until dissolved. Piperidine (0.169 mL, 1.70 mmol) was added to the solution and mixed on a magnetic stirred for several minutes. The reaction mixture was left in the open vessel for one day to self-evaporate most of the Et2O (longer time in the open vessel did not result in the evaporation of more solvent). On the next day, the glassy body obtained was subjected to evaporation in a vacuum to remove remnant volatiles resulting in 0.353 g of the solid, yield: 95%. 1 H NMR (300 MHz, CDCl3) δ 7.78–7.61 (m, br, 1H, Ar), 7.58–7.34 (m, 3H, Ar), 5.88 (s, br, 11 1H, CH), 2.72–2.52 (m, br, 4H), 1.67–1.48 (m, br, 6H). B NMR (96 MHz, CDCl3): δ (ppm) = 30. EA calculated for C12H16BNO2: C(66.40%), H(7.43%), N(6.45%); determined: C(67.30%), H(7.93%), N(7.51%). m.p. = 66–70 ◦C (decomposition). 3-(4-Methylpiperazin-1-yl)-2,1-benzoxaborol-1(3H)-ol (10)

A solution of (2-formylphenyl)boronic acid (1.5 g; 10 mmol) in diethyl ether (70 mL) was stirred in a 100 mL flask at room temperature. Next, 1-methylpiperazine (1.0 g; 1.11 mL; 10 mmol) was added during 4 min resulting immediately in precipitation of a white solid. It was filtered, washed with diethyl ether (2 3 mL) and dried to give the desired product. Due the presence of residual substrate × in the product at a ratio of 0.18: 1, the contaminated product was crystallized in a mixture of CH2Cl2 (30 mL) and hexane (30 mL). It was again filtered, washed with diethyl ether (2 3 mL) and dried to × give the title compound. Yield: 1.85 g; 8 mmol; 79%. 1 H NMR (300 MHz, CDCl3) δ 7.71 (dt, J = 7.1, 1.0 Hz, 1H, Ar), 7.48–7.29 (m, 3H, Ar), 5.84 13 (s, 1H, CH), 3.04–2.39 (m, br, 8H, CH2), 2.33 (s, 3H, CH3). C NMR (75 MHz, CDCl3): δ 152.8; 11 130.7; 130.3; 128.2; 122.7; 96.5; 55.3; 45.9. B NMR (96 MHz, chloroform-d6): δ 31. EA calculated for C12H17N2B02: C(62.10%), H(7.38%), N(12.07%); determined: C(62.08%), H(7.26%), N(12.13%). m.p. = 150–151 ◦C (decomposition). 3-(4-Phenylpiperazin-1-yl)-2,1-benzoxaborol-1(3H)-ol (11)

A solution of (2-formylphenyl)boronic acid (1.5 g; 10 mmol) in diethyl ether (70 mL) was stirred in a 100 mL flask at room temperature. Next, 1-phenylpiperazine (1.6 g; 1.5 mL; 10 mmol) was added during 5 min resulting immediately in precipitation of a white solid. It was filtered, washed with diethyl ether (2 3 mL) and dried to give the desired product. The crude product was crystallized in a × mixture of CH2Cl2 (100 mL) and hexane (100 mL). It was again filtered, washed with diethyl ether (2 3 mL) and dried to give the title compound. Yield: 2.21 g, 7.5 mmol, 75%. × 1 H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 7.0 Hz, 1H, Ar), 7.57–7.37 (m, 3H, Ar), 7.31–7.19 (m, 2H), 6.95–6.89 (m, 2H), 6.85 (t, J = 7.3 Hz, 1H), 5.98 (s; 1H), 3.18 (t, J = 5.0 Hz, 4H, CH2), 2.90–2.27 (m, 4H, 13 CH2). C NMR (75 MHz, CDCl3): δ 152.6, 151.5, 131.4, 130.3, 129.2, 128.6, 123.0, 119.9, 116.4, 97.6, 11 49.6, 46.9. B NMR (96 MHz, CDCl3): δ 31. EA calculated for C17H19N2BO2: C(69.41%), H(6.51%), N(9.52%); determined: C(69.40%), H(6.48%), N(9.51%). m.p. = 149–151 ◦C (decomposition). 1-Hydroxy-3-(4-phenylpiperazin-1-yl)-1,3-dihydro-2,1-benzoxaborole-7-carbaldehyde (14)

A solution of (2,6-diformylphenyl)boronic acid (1.0 g; 5 mmol) in diethyl ether (180 mL) was stirred in a 250 mL ﬂask at room temperature. Next, 1-phenylpiperazine (0.83 g; 0.78 mL; 5 mmol) was added during 3 min resulting in precipitation of a white solid after 2 h. It was ﬁltered, washed with diethyl ether (2 3 mL) and dried to give the title compound. Yield: 0.80 g; 2 mmol; 49%. 1 × H NMR (300 MHz, CDCl3) δ 10.05 (s, 1H), 8.07 (s, 1H), 8.00–7.86 (m, 1H, Ar), 7.82–7.67 (m, 2H, Ar), 7.35–7.15 (m, 2H, Ar), 6.93–6.88 (m, 2H, Ar), 6.88–6.81 (m, 1H, Ar), 6.02 (s, 1H, CH), 3.17 (t, J = 5.1 Hz, 13 4H, CH2), 2.95–2.83 (m, 2H, CH2), 2.80–2.70 (m, 2H, CH2). C NMR (75 MHz, CDCl3): δ 196.3; 154.7; 11 151.4; 138.3; 135.2; 132.0; 129.4; 129.2; 119.8; 116.3; 97.6; 49.4; 47.0. B NMR (96 MHz, CDCl3): δ 31. EAsw calculated for C18H19N2B03: C(67.11%) H(5.94%) B(3.36%) N(8.70%); determined: C(67.03%) H(5.97%) B(3.36%) N(8.79%). m.p. = 171–173 ◦C (decomposition). Molecules 2020, 25, 5999 11 of 13

1-Hydroxy-3-(4-methylpiperazin-1-yl)-1,3-dihydro-2,1-benzoxaborole-7-carbaldehyde (15)

A solution of (2,6-diformylphenyl)boronic acid (1.0 g; 5 mmol) in diethyl ether (180 mL) was stirred in a 250 mL ﬂask at room temperature. Next, 1-methylpiperazine (0.5 g; 0.56 mL; 5 mmol) was added during 2 min resulting in precipitation of a white solid after 2 h. It was ﬁltered, washed with diethyl ether (2 3 mL) and dried to give the title compound. Yield: 1.1 g; 4.2 mmol; 83%. 1 × H NMR (300 MHz, CDCl3) δ 10.11 (s, 1H, CHO), 8.01–7.83 (m, 1H, Ar), 7.75–7.66 (m, 2H, Ar), 13 5.95 (s, 1H, CH), 2.89–2.33 (m, br, 8H, CH2), 2.28 (s, 3H, CH3). C NMR (126 MHz, CDCl3): δ 195.8; 11 154.6; 138.3; 133.9; 131.7; 129.1; 97.3; 55.2; 46.0. B NMR (96 MHz, CDCl3): δ 31. EA calculated for C13H17N2BO3: C(60.03%), H(6.59%), N(10.77%); determined: C(60.02%), H(6.63%), N(10.70%). m.p. = 173–176 ◦C (decomposition, browning).

Supplementary Materials: The following are available online. Structures of investigated compounds (Figures S1 and S2). Crystal data and structure refinement for 11 and 14 (Table S1). Selected geometrical parameters for 11 and 14 (Table S2). NMR spectra of 1–11, 14 and 15. Author Contributions: Conceptualization, D.W. and A.A.-W.; investigation, D.W., E.K., M.W., M.L., I.D.M.; writing—original draft preparation, D.W.; writing—review and editing, A.A.-W., J.L.; supervision, A.A.-W.; project administration, A.A.-W.; funding acquisition, A.A.-W., J.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Polish National Science Centre (NCN, grant No. 2016/23/B/ST5/02847). Conflicts of Interest: The authors declare no conflict of interest.

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