molecules Article Antibacterial Activities of Azole Complexes Combined with Silver Nanoparticles NestorJ.Bello-Vieda1 ID,HomeroF.Pastrana2 ID,ManuelF.Garavito3,AlbaG.Ávila2, AdrianaM.Celis3 ID,AlvaroMuñoz-Castro4,SilviaRestrepo3andJohnJ.Hurtado1,* ID 1 DepartmentofChemistry,UniversidaddelosAndes,Carrera1No.18A-12,Bogotá111711,Colombia; [email protected] 2 EléctricayElectrónica,CentrodeMicroelectrónica,UniversidaddeLosAndes,Carrera1No.18A-12, Bogotá111711,Colombia;[email protected](H.F.P.);[email protected](A.G.Á.) 3 LaboratoriodeMicologíayFitopatología,DepartamentodeCienciasBiológicas,UniversidaddeLosAndes, Carrera1No.18A-12,Bogotá111711,Colombia;[email protected](M.F.G.); [email protected](A.M.C.);[email protected](S.R.) 4 GrupodeQuímicaInorgánicayMaterialesMoleculares,UniversidadAutonomadeChile, ElLlanoSubercaseaux2801,Santiago,Chile;[email protected] * Correspondence:[email protected];Tel.:+57-1-339-4949(ext.3468) Received:4January2018;Accepted:6February2018;Published:8February2018 Abstract: Growingantimicrobialresistanceisconsideredapotentialthreatforhumanhealthsecurity by health organizations, such as the WHO, CDC and FDA, pointing to MRSA as an example. New antibacterial drugs and complex derivatives are needed to combat the development of bacterial resistance. Six new copper and cobalt complexes of azole derivatives were synthesized andisolatedasair-stablesolidsandcharacterizedbymeltingpointanalyses, elementalanalyses, thermogravimetricanalyses(TGA),andinfraredandultraviolet/visiblespectroscopy. Theanalyses andspectraldatashowedthatthecomplexeshad1:1(M:L)stoichiometriesandtetrahedralgeometries, thelatterbeingsupportedbyDFTcalculations. Theantibacterialactivitiesofthemetalcomplexesby themselvesandcombinedwithsilvernanoparticles(AgNPs;2µgmL−1)wereassessedinvitroby brothmicrodilutionassaysagainsteightbacterialstrainsofclinicalrelevance.Theresultsshowedthat thecomplexesaloneexhibitedmoderateantibacterialactivities. However,whenthemetalcomplexes were combined with AgNPs, their antibacterial activities increased (up to 10-fold in the case of complex5),whilehumancellviabilitiesweremaintained. Theminimuminhibitoryconcentration (MIC )valueswereintherangeof25–500µgmL−1. Thisstudythuspresentsnovelapproachesfor 50 thedesignofmaterialsforfightingbacterialresistance. Theuseofazolecomplexescombinedwith AgNPsprovidesanewalternativeagainstbacterialinfections,especiallywhencurrenttreatments areassociatedwiththerapiddevelopmentofantibioticresistance. Keywords: azoleligands;copperandcobaltcomplexes;silvernanoparticles;antibacterialresistance; antibacterialactivity;cytotoxicity 1. Introduction Thebioactivityofsomemetalnanoparticlesandtheirusesindiagnosticsensorsareresearchareas ofgrowinginterest[1]. Nanosizedmetalparticlesshownotablephysical, chemicalandbiological propertiescomparedwithbulksolids[2]. Silvernanoparticles(AgNPs)haveattractedtheinterest ofthescientificcommunitybecausesilverhasbeenusedasanantisepticandantimicrobialagainst differentspeciesofbacteria[1,2]. Thegrowingresistanceofpathogenicbacterialandfungalstrainsto traditionaltreatmentsisanotherkeyfactordrivingresearchwiththistypeofmaterial. Inrecentyears, theeffectivenessofsomenanoconjugateshasbeendemonstratedagainstsomepathogenicmicrobes, Molecules2018,23,361;doi:10.3390/molecules23020361 www.mdpi.com/journal/molecules Molecules2018,23,361 2of17 showinggreatpotentialfornanomedicine. Thecombinationofantibiotics, antifungalagentssuch as azoles, metal complexes and nanoparticles could increase the efficiency of drugs, even against drug-resistantpathogens[3,4]. AgNPs have been used in recent years for the production of a new class of antimicrobials to combat a widespread range of pathogens. Even though there have been several studies showing thehighantibacterialeffectofAgNPs,theirmechanismofactionisnotfullyunderstood[5]. Thebroad activitMyolsecpuleesc 2t0r1u8, m23, xo FfORA PEgENR RPEsVIEaWg a inst morphologically and metabolically diverse2 omf 17i croorganisms could be related to a complex and multilayered mechanism of action. Several mechanisms have pathogenic microbes, showing great potential for nanomedicine. The combination of antibiotics, been proposed and include the alteration of cell walls and cytoplasm. Among these mechanisms antifungal agents such as azoles, metal complexes and nanoparticles could increase the efficiency of are thderumgse, mevbenr aagnaeinspt edrrmuge-raebsiisltiatnyt pianthcoregeansse [3a,4n].d respiration depletion, morphological changes of thecytoskAegleNtPosn h,atvhee bseeenp aurseadti ionn reocfentth yeeacrys tfoopr lthaes mpriocdumcteiomn borf aan neewfr oclmass tohfe ancteimlliwcroablila,lsa ntod plasmolysis combat a widespread range of pathogens. Even though there have been several studies showing the andinhibitionofbacterialDNAreplication[2]. Nevertheless,thehighbactericidalactivityisrelated high antibacterial effect of AgNPs, their mechanism of action is not fully understood [5]. The broad todosesover40µgmL−1,butthesedoseshavebeenreportedascytotoxicinmammaliancellsby activity spectrum of AgNPs against morphologically and metabolically diverse microorganisms Zhangcoeutlda lb.e[ r6e]laatendd tot ao xcoicmpolnexa aqnud amtuicltiolarygearendi smmecshasnuiscmh oafs acatliogna.e SebvyeraSla mssecehtanaisl.m[s7 h].avBe ebteteenr antibacterial activityprowpiotshedl oawnd AingclNudPe ltohea dalitnergastiocna nof bceella wchaliles vaendd cbyytotphlaesmad. Admitoionng tohfesceo mmepchoauninsmdss awrei tthhel owresistance membrane permeability increase and respiration depletion, morphological changes of the suchasmetalazolecomplexes,limitingthepotentialtoxicityofnanoparticles. Thesecompoundsare cytoskeleton, the separation of the cytoplasmic membrane from the cell wall, and plasmolysis and producedusingametallicsaltandanappropriateligandwithchelatingproperties. Theuseofazole inhibition of bacterial DNA replication [2]. Nevertheless, the high bactericidal activity is related to derivadtiovseess oavsera 40l iμgga mnLd−1i, sbuatn thiensete droessetsi hnagvea bpepenr oreapcohrtebde acsa cuystoetoaxzico ilne smhamavmealaiannt cieflulsn bgya Zlhaanngd antibacterial activiteiet sal.[ 8[6]]. aTndh etosxeic aocnt iavqiutaietisc oarrgeanriesmlast esudcht oast ahlegaire abyb iSlaistys etto alb. [i7n].d Berteteard ainlytibtaocteerniazl yacmtiveistya nd receptors inbiolwoigthic laowls AygsNtePm losa.diWnghs ecann tbhe eacahzieovleed lbiyg athned adidsitcioono ordf cionmapteoduntdos wCiothII loowr rCesuisItIa,ntchee ssuechc aosm plexeshave metal azole complexes, limiting the potential toxicity of nanoparticles. These compounds are higherantimicrobialactivitythanthefreeligand,andinsomecases,theyexceedthatofstandardtest produced using a metallic salt and an appropriate ligand with chelating properties. The use of azole substadnecreivsa[ti9v–e1s 2a]s. a ligand is an interesting approach because azoles have antifungal and antibacterial Thaceticvoitmiesb [i8n].e Tdhaescet iavcittivyitoiefsm areet raellactoemd tpo ltehxeeirs aabnildityn taon boinpda rretaicdlielys taog eaniznysmtebsa acntedr rieacehpatsorns oint beenassessed biological systems. When the azole ligand is coordinated to CoII or CuII, these complexes have higher yet and could be an interesting approach for utilizing lower azole concentrations for antibacterial antimicrobial activity than the free ligand, and in some cases, they exceed that of standard test activity. Inthiswork,wereportthesynthesisandcharacterizationofnewair-stablecobalt(II)and substances [9–12]. copper(II)Tchoem copmlebxinedde raicvtiavtitiyv eosf mofetaazl ocolemspalenxdes tahnedi rnaannotpibaraticctleesr iaaglaaincstti vbiatciteesriaa lhoans enoatn bdeeinn combination withAagssNesPsesd, wyeht icanhdr ecosuulldt ebde iann ininctreereasstiendg aacptpirvoiatcyh. for utilizing lower azole concentrations for antibacterial activity. In this work, we report the synthesis and characterization of new air-stable 2. Disccuobsaslit(oIIn) and copper(II) complex derivatives of azoles and their antibacterial activities alone and in combination with AgNPs, which resulted in increased activity. Complexes 1–4 and 9 and 10 (Scheme 1) have been reported previously (see the Materials and 2. Discussion MethodsandSupportingInformation). Thekeypointinthesynthesisofnewcomplexes5–8,11and 12wastheCcohmopicleexeosf 1a–4s uanitda b9 laendso 1l0v (eSnchtefmore e1a) chhavree baecetnio rnep.oTrtheed rperaevcitoiousnlys (wseeer tehec aMrraiteerdiaolsu atnidn solventsthat Methods and Supporting Information). The key point in the synthesis of new complexes 5–8, 11 and dissolvedboththeligandandtheMCl butdidnotsolubilizethefinalcomplex. Inthesynthesisof5, 12 was the choice of a suitable solvent f2or each reaction. The reactions were carried out in solvents 7and11,varioussolventsweretested,i.e.,ethanol,methanol(MeOH),acetone,tetrahydrofuran(THF) that dissolved both the ligand and the MCl2 but did not solubilize the final complex. In the synthesis andacoeft 5o,n 7i atrnidle 11(,C vHarioCuNs s)o,lvwenhtisc whedrei stessotelvde, id.e.t, hetehasntaorl,t minegthraenaocl t(aMnetOsH.H), aocwetoenvee, rt,etnraohryedarocftuiorann wasobserved 3 immed(iTaHteFl)y a.nTdh aeccetoobnaitlrtilce o(mCHp3lCeNxe),s wwheicrhe disisosloaltveedd athsea sitra-rsttinagb lreeabcltuanet,sn. oHno-wheyvgerr,o nsoc orpeaicctisoonl iwdass. Thesyntheses observed immediately. The cobalt complexes were isolated as air-stable blue, non-hygroscopic solids. ofthecoppercomplexeswerecarriedoutundermildconditionswithyieldsof44–91%. The syntheses of the copper complexes were carried out under mild conditions with yields of 44–91%. Scheme 1. Azole complexes. Scheme1.Azolecomplexes. Molecules2018,23,361 3of17 2.1. InfraredSpectroscopy Thenewcomplexeswereanalyzedbyinfraredspectroscopy,particularlylookingfortheband shifts due to metal coordination. Table 1 provides an overview of the main bands in the infrared spectra of the complexes. The bands were assigned considering previous reports of the ligands and similar complexes [8]. In complexes (1 and 2), the presence of the bands at 3344 cm−1 and 3265cm−1,whichareassignedtoN-Hvibrations,confirmedthecoordinationofthemetalstotheN2 of3,5-dimethylpyrazole. Wefoundashifttohigherwavenumberswithrespecttothefreeligand[8], indicatingtherigidityofthemoleculewhenthemetalwaspresent. In(3and4),theringvibrationsof (C=N)and(C-C)Pyrinthefreeligand(1035cm−1and970cm−1,respectively)alsoshowedtherigidity ofthestructureofthecomplex. Thiscasewasnotpresentincomplexes(5–8),wheretheringvibrations werenotaffectedbycoordinationlikelybecausetheligandwastoobig,andthecentralringswerenot influencedbytheelectrondensityofthemetal. Variationsintheinfraredspectraofeachcomplexwere observed,showingtheelectronicdifferencesofthecorrespondingmetals. Additionally,itwaspossible tofindM-Clvibrationswithverylowintensities. Table1.MainbandsintheFTIRspectraforthecomplexes.Pyr=pyrazole;Ind=indazole;Tol=toluene; vs=verystrong;s=strong;m=medium;w=weak;vw=veryweak. Compound Wavenumberν(cm−1) (N-H) (C-H) (C-H)Pyr (C-N) (M-Cl) 1 3344s 3142w 1568vs 1470m 427m 2 3265vs 3147vs 1570vs 1472m 431m (C-H) (C-CH3) (C=N) (C-C)Pyr (M-Cl) 3 3011w 1465s 1051m 1003m 493w 4 3027w 1467s 1044m 1001m 492w (C-H) (C-CH3) (C-C)Pyr (C-C-N) (M-Cl) 5 3127m 1468m 1608m 729m 419w 6 3137w 1469m 1608m 738m 420w (C-H) (C-C) (C-N) (C-H) (M-Cl) Ind Ind Ind 7 3094m 1628s 1519s 1478m 490w 8 3098m 1628s 1519s 1477m 497w (C-H) (H2C-N) (N-N) (C-N) (M-Cl) 9 3115w 1518s 1458m 1362w 507vw 10 3137s 1525s 1460m 1357w 503vw (C-H) (C-C) (N-N) (N=N) (M-Cl) Tol 11 2970w 1610m 1284m 1229s 419w 12 2968m 1610m 1288m 1233s 419w 2.2. UV-VisSpectroscopy The UV-Vis spectra were recorded to study the electronic properties of the complexes and to relatethepropertiestopossiblestructures. Mostofthecomplexesobtainedshowedintenseabsorption bands (200–300 nm). In complex 9, bands below 300 nm were not observed because the solvent showedahighabsorbanceinthiszone. Suchbandsareattributedtometaltoligandchargetransfer, andintraligandπ-π*transitions[13]. Thecobaltcomplexesshowedabluecolorinthesolidstate. WhendissolvedinDMSOorCH CN,thiscolorremained, whileiftheyweredissolvedinMeOH 3 (complex7)thecolorchangedtopalepink,acharacteristiccolorofoctahedralcobaltcomplexeswith coordinatedwater.Complexes1,9and11showedbandsat600–700nm,correspondingto4A ←4T 2g 2g and4A ←4T [14]. 2g 1g Complexes3and5showedathirdbandinthevisibleregionwithhigherenergy. Thisbandis relatedtoanequilibriumbetweentetrahedralandoctahedralgeometriesgivenbycoordinationwith solventsH OorCH CN.Incomplex7,abandat525nm(ε=17M−1cm−1)wasobserved,whichis 2 3 consistentwithCo(II)octahedralcomplexes[14]. Molecules2018,23,361 4of17 FortheCu(II)complexes, charge-transferbandswereobservedwithhighintensityintheUV regionofthespectrathatcouldberelatedwithtransitionsoftheorganicmoietyinthecomplexandto Molecules 2018, 23, x FOR PEER REVIEW 4 of 17 chargetransferfromthenonbondingorbitalofchloridetothevacantcopper(II)dorbitals[15]. AdditiFoonra tlhlye, Couv(eIIr) lcaopmppeldexseisg, nchaalsrgwe-terraensffoeru nbadnddsu weetroe oinbtseerrveeledc wtroithn ihcigrhe pinutlesnisointy. inIn thteh eUVca se of region of the spectra that could be related with transitions of the organic moiety in the complex and complexes4and6,itispossibletoassignthesignalsnear300nmtochargetransferbetweenchloride to charge transfer from the nonbonding orbital of chloride to the vacant copper(II) d orbitals [15]. andcopper. Inthevisibleregionnear500nm,theelectronictransitionoft ←e wasfound,whichis Additionally, overlapped signals were found due to interelectronic r2egpulsiogn. In the case of characteristicford9metals[16]. complexes 4 and 6, it is possible to assign the signals near 300 nm to charge transfer between chloride and copper. In the visible region near 500 nm, the electronic transition of t2g ← eg was found, which 2.3. TheoreticalCalculations is characteristic for d9 metals [16]. Despiteseveralattempts,nocrystalssuitableforX-raydiffractionanalysiswereobtained. Further 2.3. Theoretical Calculations computationalstudieswerecarriedoutforthenewcomplexestosupportthecharacterizedstructures. ThecalculaDteedspditaet aseavreeragl iavtetenmopnts,s unpo pcoryrtsitnalgs isnufitoarbmlea ftoior nX,-wrayh idchiffraagcrtieoens awneallylstios twheered iosbctuasinseedd. main Further computational studies were carried out for the new complexes to support the characterized bandsintheFTIRandUV-Visspectraandtheirassignation(seeabove). For5–8,11and12,adoublet structures. The calculated data are given on supporting information, which agrees well to the groundstatewasobtained(i.e.,oneunpairedelectron). discussed main bands in the FTIR and UV-Vis spectra and their assignation (see above). For 5–8, 11 Allthecomplexesexhibitaslightlydistortedtetrahedralgeometry. Thus,alltheseriescanbe and 12, a doublet ground state was obtained (i.e., one unpaired electron). ascribed asAtlel ttrhaeh ceodmrapllecxoesm epxhleibxiet sa oslfigChtoly(I Id)isatonrdtedC tue(trIaI)h.eMdraols gtleyomoeftrtyh.e Tuhupsa, iarleld theel esecrtireos ncarne sbied es in the -CoaCsclribaendd as- CteutrCahledfrraal gcmomepnltexaecsc oofr Cdoin(IgI) atondt hCeu(oIIb).t Maionsetdly sopf tihne- duepnaiirsetdy .elTechtreonr erseusildteins gin stthreu -ctures 2 2 supportCtohCel2e axnpde c-CteudCsl2y fsrtaegmmsenatc accocrodridninggt otot thhee eombtpailnoeyde dspliing-adnendi.stSyu. cThhes trreusuclttuinrges s,treuxchtuibrietss siuspopstorrut ctural the expected systems according to the employed ligand. Such structures, exhibits isostructural speciesbetweenCo(II)andCu(II),whichcanbeviewedasausefultemplateforfurtherevaluationof species between Co(II) and Cu(II), which can be viewed as a useful template for further evaluation of relationshipbetweendifferentmetalcentersandmolecularproperties. relationship between different metal centers and molecular properties. Moreover, the location of the -MCl fragment in relation to the central phenyl ring is shifted Moreover, the location of the -M2Cl2 fragment in relation to the central phenyl ring is shifted betweebnet5w–e8ena n5d–81 a1n,d1 211,,w 12h, icwhhiccahn cabne bae ua suesfeufulla abbiilliittyy ttoo ddeessigignn fufruthrtehr esrelseectlievcet icvaetalcyattica lsypteicciessp ecies (Schem(eSc2h).eme 2). Scheme2.Optimizedgeometriesfor5–8,11and12,fromcomputationalcalculationsinagreementto Scheme 2. Optimized geometries for 5–8, 11 and 12, from computational calculations in agreement to SchemeS1ch.eme 1. 2.4. The2r.m4.a TlhAernmaally Asinsalysis Thermogravimetric analyses were performed to study the different mass losses and to relate Thermogravimetric analyses were performed to study the different mass losses and to relate these losses to possible structures. The mass losses are based only on the percentages observed in the theselossestopossiblestructures. Themasslossesarebasedonlyonthepercentagesobservedin thermograms because the study was carried out without a detection system (TGA-MS). Complex 1 thethermogramsbecausethestudywascarriedoutwithoutadetectionsystem(TGA-MS).Complex showed high stability because a residue at the end of the analysis was observed (pyrazole-Co). 1 showCedomhpilgehx (s2t)a sbhioliwtyedb leocwa uthseermaarle sstiadbuilietya wtitthhe 75e%nd loossf otfh tehea nmaeltyasl iasndw 4a0s%o tbostaelr vloesds o(fp myraassz oatl e-Co). Comple6x00( 2°C).s Ihno 3w aendd l4o wraptihde trhmeramlaslt adbegilriatdyawtioinths w75e%re olobssserovfedth ine ma feetwa lsatenpds, 4in0d%icatotitnagl tlhoasts sotafbmle assat 600◦C.inInter3maenddiat4esr waperide gtehneerrmateadl. dCeogmrapdleaxteiso 5n asnwd e6r leosot bnseearrlvye 7d0%in oaf tfheew tostatel pmsa,sisn adt itchaet einndg othf tahtes table analysis, and complex 5 showed a very small loss (1.6%) at 150 °C, which could be related to the loss intermediates were generated. Complexes 5 and 6 lost nearly 70% of the total mass at the end of theanaloyf swisa,taenr dmcoolemcupleles xco5osrhdoinwateedd atov ceorbyasltm. Haollwloesvser(,1 t.h6e% I)Ra stp1e5c0tru◦Cm, dwidh incoht csohuowld bbaenrdesl areteladtetdo ttoh eloss H2O, so we assume that the mass loss corresponds to HCl. For complex 6, the total loss of the ligand ofwatermoleculescoordinatedtocobalt. However,theIRspectrumdidnotshowbandsrelatedto was observed at 700 °C. Complexes 7 and 8 exhibited good thermal stability with retention of almost H2O,sohawlfe ofa tshseu imnietiatlh matasths aet m70a0s °sCl owsisthc oobrsreersvpeodn lodsssetos oHf tChel. iFndoarzcoolem ripnlge xan6d, 2thHeCtlo. Ftaolulro mssasosf lothsseesl igand wasobsweerrvee odbsaetr7v0ed0 f◦oCr .coCmopmlepxl e8,x weshi7cha nwder8e aenxahliybzietde ding tohroede dthifeferrmenatl sstteapbs:i lfiitryst,w a iltohssr oeft e2nHtCiol;n thoefna, lmost half of the initial mass at 700 ◦C with observed losses of the indazole ring and 2HCl. Four mass losseswereobservedforcomplex8,whichwereanalyzedinthreedifferentsteps: first,alossof2HCl; Molecules2018,23,361 5of17 then,alossofbenzene;andfinally,decompositionofoneindazolegroup. Themasslossesofcomplex 9wereassociatedwiththesplittingsoftheCH spacersandtriazoles. 2 2.5. CharacterizationofAgNPs 2.5.1. AtomMicoleFcuolersc 2e018M, 23i,c xr FoOsRc PoEpERy R(EAVIEFWM ) 5 of 17 a loss of benzene; and finally, decomposition of one indazole group. The mass losses of complex 9 Thenanoparticlesize,sizedistributionandshapeweremeasuredusingAFM.TheAgNPswere were associated with the splittings of the CH2 spacers and triazoles. coatedwithpolyvinylpyrrolidone(PVP)anddriedwithoutsonicationtomimicthemethodologyin theculturep2.l5a. tCehsa.raOcteurirzarteiosnu olf tAsgsNhPos wsphericalnanoparticles(Figure1A,B).TheAgNPsagglomerated onclustersupto50nm,duetothesurfacechargeofthenanoparticles.Nevertheless,theaverageheight 2.5.1. Atomic Force Microscopy (AFM) ofthesinglenanoparticleswas8.35nm,withaheightdistributionbetween5to13nm(Figure1B). The nanoparticle size, size distribution and shape were measured using AFM. The AgNPs were Theseresultssuggestthatthesizedofthetestednanoparticlesareintherangewiththemoreeffective coated with polyvinylpyrrolidone (PVP) and dried without sonication to mimic the methodology in antibacteriatlhpe cruolptuerret pielasteas.c Ocourr dreisnugltsw shiotwh stphheerliictaelr naatnuorpear[t1ic7le,1s 8(F]i.gure 1A,B). The AgNPs agglomerated on clusters up to 50 nm, due to the surface charge of the nanoparticles. Nevertheless, the average 2.5.2. DynamheiigchLt oigf thhte Ssicnagtlet enrainnogpa(rDticLleSs )was 8.35 nm, with a height distribution between 5 to 13 nm (Figure 1B). These results suggest that the sized of the tested nanoparticles are in the range with the more Wecalcefufelcattiveed atnhtiebaZct-eprioalt eprnotpiearlti(eδs )acfcroormdintgh weiethl ethcet rloiteprhatourree t[1ic7,1m8]o. bilitymeasurementsandtheHenry equationasshowninEquation(1): 2.5.2. Dynamic Light Scattering (DLS) 2εζ f(ka) We calculated the Z-potential (δ) froUmE th=e electro3pηhoretic mobility measurements and the Henry (1) equation as shown in Equation (1): whereUE istheelectrophoreticmobility,ζisthez2-εpζo(cid:1858)t(cid:4666)e(cid:1863)n(cid:1853)(cid:4667)tial,εdielectricconstant, f(ka)istheHenry’s functionandηisthesolutionviscosity[19].(cid:1847)O(cid:3006)u(cid:3404)rZ-3pηote ntialresultsevidencedthatth(1e) AgNPshad positivelychwahregree d(cid:1847)(cid:3006)s uisr ftahcee esleocftr1o8ph±ore3t.i6c mmoVbiilintyw, ζa tise rt,hae szs-phootewntniail,n εF digieulercetri1cD co.nTshtaentZ, -(cid:1858)p(cid:4666)o(cid:1863)(cid:1853)te(cid:4667) nist iathlei scorrelated totheimpacHteonrfyt’sh efubnciotio-inn atendra ηc tiiso tnhse osofluthtioenn vainscoopsitayr t[i1c9l]e. sO,usrp Ze-cpiofitecnatlilayl rwesiutlhts ceevlilduenlacerdu tphtaat kthee andprotein AgNPs had positively charged surfaces of 18 ± 3.6 mV in water, as shown in Figure 1D. The Z- adsorption. Nanoparticleswithapositivelychargedsurfacearemoresusceptibletoconformationsof potential is correlated to the impact of the bio-interactions of the nanoparticles, specifically with theproteincceollruolanr aupatnakde caondu lpdrobteeinm adosroerpetiaosni. lNyatnaokpeanrticinlesb wyitche all pso[s2it0iv]e.lPy ochsaitrgivede lsyurcfahcae ragree dmoprae rticleshave beenreportseudscetpotieblxee trot caonnftoirmmaictiroonbs ioaf ltheef fpercottesinb cyoraodnah aenrdin cgoutlod bGe rmaomre- neaesgilya ttiavkeen bina cbtye creilals [[2201]]. . Therefore, Positively charged particles have been reported to exert antimicrobial effects by adhering to Gram- weexpectedmoreofanantibacterialeffectfromthe10-nmAgNPsonGram-negativestrainsthanon negative bacteria [21]. Therefore, we expected more of an antibacterial effect from the 10-nm AgNPs Gram-positoivne Gsratrma-innegsa.tive strains than on Gram-positive strains. Figure 1. AgNPs characterization. (A) AFM image of the nanoparticles dispersed on silica glass; (B) Figure1.AgNPscharacterization.(A)AFMimageofthenanoparticlesdispersedonsilicaglass;(B)3D 3D reconstruction of the AFM image; (C) Size distribution of the nanoparticles by height; (D) Z- reconstructipoontenotfiatlh meeAasFuMremiemntas gues;in(Cg )DSLiSz. eTdhiesster inbaunotipoanrtioclfest heexhnibainteod paa rstmicallel spbosyitihveeliyg hcht;ar(gDed) Z-potential measuremepnottsenutiasli onfg apDprLoxSim. Tatheley s1e8 ±n 3a.6n moVp.a Trhteircel ewsase axlshoi ab sietecodndaarys mpeaakll atp 1o0s0 imtiVv ecolyrrecsphoanrdginegd top otential of approximattehley fre1e8 P±VP 3c.o6atimngV. .Therewasalsoasecondarypeakat100mVcorrespondingtothefree PVPcoa ting. Molecules2018,23,361 6of17 2.6. BiologicalActivity ThebiologicalactivityoftheAgNPsalone,azolecomplexeswithoutAgNPsandtheazolecomplexes combinedwithAgNPswereevaluatedintriplicateagainsteightbacterialstrains,MRSA,S.typhimurium, E.aerogenes,B.cereus,E.faecalis,S.flexneri,S.aureusandE.coli,after8hand16hofexposure. Molecules 2018, 23, x FOR PEER REVIEW 6 of 17 2.6.1. Antibacte2r.6ia. BlioAlogcitciavl Aitcytivoityf theAgNPsOnly The biological activity of the AgNPs alone, azole complexes without AgNPs and the azole The antibacocmteprleixaels caocmtbivinietdi ewsitho AfgANPgs NwePres evhaaluvateedb ine etrniplirceatpe oagratiensdt ,eigahnt bdacitetrihala stsrabines,e MnRSsAh, own that their size, and conceSn. ttyrpahitmiuorniumd, Ee.fi aenroegetnhese, Bir. ceerefufse, cEt. ifvaeecanlise, sSs. .flexNnerai,n So. apuraerust iacnlde Es. cwolii, tahftesr i8z he asndf r1o6 mh of5 to 100 nm at exposure. aconcentrationof40µgmL−1havebeenreportedtohavebactericidaleffectsonmosttestedstrains[22]. Sizes under 302n.6m.1. Aanrtiebamcteorirale Aactcivtiitvy eof tthhea AngNbPisg Ognelyr nanoparticles, and it is thought that the activity is associated with anThee aasntiiebractreerilael aacsteivitoiefs osfi lAvgeNrPsi ohanvse bfereonm repothrteed,s amnda itl lhears bpeeanr sthicowlens th[a1t 7th]e.ir Asizte, the same time, and concentration define their effectiveness. Nanoparticles with sizes from 5 to 100 nm at a particleswithsphericalshapesshowbetterresults,almostfivetimesmoreeffectivethanparticleswith concentration of 40 μg mL−1 have been reported to have bactericidal effects on most tested strains different shape[s22[].2 S2iz,e2s3 u]n.dOer u30r nemx apree mriomre eacntitvse twhaenr beiglgiemr niatneodparttoiclsesp, ahnedr iitc isa tlhoAugghNt tPhast twhe iatchtivsitiyz es of 10 nm at 2µgmL−1concise anstsroacitaitoedn w.iAthd adn ietaisoienr arelleeaxsep oef rsiimlveer niotnsw friotmh nthae nsmoapllaerr tpiacrlteicsless i[z17e].s Abt ethtwe seamene t3im0ea, nd100nmdid particles with spherical shapes show better results, almost five times more effective than particles notshowbacterwiicthid daiflfeerefnfte schtasp,eds [a22t,a23n]. oOturs hexopweriemden.tsT wheerec loimnitceedn tot rspahtieoricnalo Afg2NPµs gwimth Lsiz−e1s owf 1a0s nmu sedtoevaluate at 2 μg mL−1 concentration. Additional experiment with nanoparticles sizes between 30 and 100 nm thesynergisticeffectwiththeazolecomplexes,sincetheliteraturereportsonlybacteriostaticeffects did not show bactericidal effects, data not showed. The concentration of 2 μg mL−1 was used to atthisconcentreavtailuoante [th2e, 2sy2n]e.rgTishtice erfefefcot wreit,ha thney azbolae ccotmerpilcexieds,a slinecef ftehec ltisterdateupree rnepdortos nontlyh beacAtergioNstaPti-cA zolecomplex synergyexcluseifvfeecltsy .at Itnhisf acocntc,enatlrlattiohne [2s,2t2r]a. iTnhesreifnoreo, uanry ebxacpteerriciimdale enfftesctss hdeopwende donb thaec tAegrNioP-sAtzaotliec effectsatthis complex synergy exclusively. In fact, all the strains in our experiments showed bacteriostatic effects concentration(Figure2). at this concentration (Figure 2). 90 d ate 80 ntre 70 o u 60 e t 50 v ati 40 el h r 30 wt 20 o of gr 10 % 0 Figure2.AntibaFicgtuerrei a2.l Aefnfteibcatcsteoriafl AefgfeNctsP osf aAlgoNnPes oalnonfeo ounr fGourra Gmram(+ ()+)a nandd ffoouurr GGrarma m(−) (s−tra)insst.r aThine s.TheAgNPs’s sizewere10nmAagtN2Psµ’s gsizme wLe−re1 1.0 nm at 2 μg mL−1. Bacteria were able to keep growing even in the presence of 2 μg mL−1 AgNPs. However, at the same time, none of the strains were able to grow at 100% compared to the untreated control. Some Bacteria were able to keep growing even in the presence of 2 µg mL−1 AgNPs. However, bacteria were more sensitive to the AgNPs, such as E. aerogenes, S. typhimurium, S. flexneri and E. coli. atthesametimInet,ernesotinngelyo, afntdh ine csontrtraaisnt wsitwh perreveioaubs lsetudtioes gthraot wrepoartte1d 0th0a%t AgcNoPms hpada rae mdajotro batchteericuidnet reatedcontrol. effect against gram-negative bacteria [21], we found that the AgNPs showed similar activity against SomebacteriaweremoresensitivetotheAgNPs,suchasE.aerogenes,S.typhimurium,S.flexneriand both Gram-positive and Gram-negative bacteria strains. This is most likely because Ag+ ions react E. coli. Interestsitnrognlgyly, awnithd biinolocgoicnalt rsaubssttawncietsh, supcrhe vasi opurosteisntsu, denizeysmetsh, aDtNrAe,p aonrdt eRdNAt,h dauteA tog NtheP s had a major interactions that occur with thiol, carboxylate, phosphate, hydroxyl, imidazole, indole or amine bactericideeffectagainstgram-negativebacteria[21],wefoundthattheAgNPsshowedsimilaractivity functional groups. These interactions can be produced simply or in combination, which can lead to a against both Gsrearimes -opf eovsenittsi vtheat ainntedrfeGre rwaitmh v-intael gmaictriovbieal bpraoccetsesersi a[24s].t rIta hians sa.lsoT beheins reipsormtedo tshtat lsiiklveerl y because Ag+ ionsreactstronregalcyts wwiitthh thbei osullofhgyidcrayll (s-Su-Hb)s tgaronucpes so,ns ucecllh waalsls ptor ofotremin Rs-,S-eSn-Rz ybomndess, ,thDusN, bAlo,ckainngd RNA,dueto respiration and causing cell death [17]. theinteractionsthatoccurwiththiol,carboxylate,phosphate,hydroxyl,imidazole,indoleoramine functionalgroups. Theseinteractionscanbeproducedsimplyorincombination,whichcanleadto aseriesofeventsthatinterferewithvitalmicrobialprocesses[24]. Ithasalsobeenreportedthatsilver reactswiththesulfhydryl(-S-H)groupsoncellwallstoformR-S-S-Rbonds,thus,blockingrespiration andcausingcelldeath[17]. 2.6.2. AntibacterialActivityoftheAzoleComplexeswithandwithoutAgNPs The antibacterial activities of the ligands, its complexes and metal complexes combined with AgNPsagainstthebacterialstrainswerealsostudied. Thefreeligandsshowednoactivityagainst Molecules2018,23,361 7of17 any bacterium. This may be because azoles have higher anti-fungal activities since they inhibit theergosterolsynthesisinfungalcellwalls[25,26]. However,someazole-derivativecompoundshave exhibitedmoderateantimicrobialactivities[27–30],andithasbeenfoundthatazolesinbacteriaare inhibitors for enoyl acyl carrier protein reductase [31]. Manganese tricarbonyl coordinated to ketoconazole,miconazole,orclotrimazoleshowedhigherantibacterialactivitycomparedtothefree azoleonlyforGram-positivebacteria[32]. Westudiedtheantibacterialactivitiesoftwelvecomplexesderivedfromazole,whichcontained cobalt and copper. In previous studies carried out in our research group, it was found that metal complexescontainingcopperandcobaltinsteadofzincdisplayedbetterantibacterialeffectsagainst bacterial strains [8]. The invitro antibacterial results of complexes 3–5, 8 and 9 with and without AgNPsaresummarizedinTables2and3. As observed in Tables 2 and 3, complexes 3–5, 8 and 9 showed high antibacterial activities. The results suggest that these compounds at 8 and 16 h were effective against at least one of thestrainstestedinthisstudy. Thisfindingislikelyrelatedtothebettersolubility, bioavailability and lipophilicity of the complexes that reduced the permeability barriers of the cells and slowed thenormalcellularprocessesofthemicroorganisms,resultinginincreasedantimicrobialactivities orchelatingeffects[8,31–33]. At16h,bettersensitivitiestothebacteriaforalltheazolecomplexes wereobserved(Table3). However,thedosesrequiredtogeneratebactericidaleffectswereincreased forcomplexes5(S.aureusandMRSA)and3(MRSAandE.faecalis),whichisoneofthebacterium that is indicated by the CDC as a serious threat (Tables 2 and 3). The complexes inhibited higher activitiesagainstGram-negativestrainsthanGram-positivestrains. Ifwecomparecomplexes3and4, whereonlythemetalcenterdiffersintheirstructures,complex3showedhigherantimicrobialactivity. Thismightbeattributedtothepresenceofcobalt. Cobalthasbeenreportedtobeamoretoxicelement thancopper[34]. CohasalargeratomicradiusthanCu,andithasbeenreportedthatthebondlength betweenCoanditsligandcouldfavorantibacterialactivity[35]. Inaddition,copperisanessential traceelementformanybiologicalprocesses[36]. Asthedataanalysisfurtherindicates,complexes5, 8and9showedmoremoderateactivitiesthan3and4. Itappearsthatthepresenceofthepyrazole ringfavorsantibacterialactivitycomparedtocomplexescontainingindazoleortriazole. Somestudies haveshownthatpyrazolesincomplexescaninhibitDNAgyraseandtopoisomeraseIVinbacteriabut aremoreeffectiveagainstGram-positivebacteriathanGram-negativebacteria[37]. Thebindingof specificmetalionstoazolescontributestotheantibacterialactivity. Inaddition, thecombinationofAgNPswiththemetalcomplexesshowedbactericidaleffects at lower concentrations than in the absence of AgNPs for both Gram-positive and Gram-negative bacteriastrains. ThecombinationofmetalcomplexeswithAgNPsshowedbactericidalactivitieswhile thefreecomplexesandAgNPsalonejusthadbacteriostaticeffects. When complex 5 was combined with AgNPs, a 10-fold increase in antimicrobial activity was observed for S. aureus (Table 2), whereas in the case of S. flexneri, E aerogenes and S. typhymurium, weonlyobserveda2-foldincreaseinactivity. Interestingly,thiscomplexshowedbetterbiological resultsagainstGram-negativebacteria,probablyduetothepresenceoftoluene,whichisknownto induceincreasedfluidityinGram-negativecellmembranes[8]. Complex8inthepresenceofAgNPsshowed3-foldincreasedactivitiesagainstS.aureusand E. aerogenes (Table 2). Likewise, in the case of 4 with AgNPs, 2-fold increased activities against S. flexneriandE.faecaliswereevidenced. Compounds3and9showedsynergisticantibacterialeffectsbut inminorproportions. Theseresultsmaybeduetothefusionoftwoantimicrobialagentswithdistinct modesofaction,thechelatingeffectofthecomplexesandthepresenceofAg+ions(AgNPs),thatreact withDNAandenzymesofbacteria. Tothebestofourknowledge,theantibacterialactivitiesofAgNPs combinedwithazolecomplexeshavenotbeenpreviouslyreported. Additionally,theantibacterial activityofAgNPs(10nm)ataconcentrationof2µgmL−1hasnotbeenreported. Molecules2018,23,361 8of17 Table2.Minimuminhibitoryconcentrations(MIC)(¶)andminimumbactericidalconcentrations(MBC,µgmL−1)ofbacterialstrainsexposedtoazolecomplexesand 10-nmAgNPs(2µgmL−1)for8h. *AgNPswitha10-nmdiameterwereaddedatthesameconcentrationof2µgmL−1;¶Bacteriaexhibitedonlyinhibitoryactivities. Table3.Minimuminhibitoryconcentrations(MIC)(¶)andminimumbactericidalconcentrations(MBC,µgmL−1)ofbacterialstrainsexposedtoazolecomplexesand 10-nmAgNPs(2µgmL−1)for16h. *AgNPswitha10-nmdiameterwereaddedatthesameconcentrationof2µgmL−1;¶Bacteriaexhibitedonlyinhibitoryactivities. Molecules2018,23,361 9of17 Molecules 2018, 23, x FOR PEER REVIEW 9 of 17 22..77.. PPhhyyssiiccoocchheemmiiccaall CChhaarraacctteerriizzaattiioonn ooff tthhee CCoollllooiiddss IInn FFiigguurree 33,, iitt iiss ccaann bbee seseenen thtahta At AgNgNPsP hsahvaev tehethire hirighhigeshte asbtsaobrsboarnbcaen bceetbweetewne 4e1n74 n1m7 namnda 6n7d8 6n7m8,n wmh,iwle hciolemcpolmexp 9le hxa9s haa msaaxmimaxuimm uambsoarbbsaonrbcea nact e51a6t 5n1m6.n m. FFiigguurree 33.. UUVV//VViiss ssppeeccttrraa ttoo sshhooww tthhee iinntteerraaccttiioonn bbeettwweeeenn ccoommpplleexx 99 aanndd AAggNNPPss.. The spectrum of the solution with complex 9 and the AgNPs seems to be the sum of the Thespectrumofthesolutionwithcomplex9andtheAgNPsseemstobethesumoftheabsorbance absorbance of each component, with a slightly change in 650–750 nm range. However, when the of each component, with a slightly change in 650–750 nm range. However, when the spectra was spectra was recorded 24 h later, the absorption due to nanoparticles decreased significantly, probably recorded 24 h later, the absorption due to nanoparticles decreased significantly, probably because because the complex molecules were adsorbed over the surfaces of the AgNPs. Additionally, there is thecomplexmoleculeswereadsorbedoverthesurfacesoftheAgNPs. Additionally,thereisnochange no change in the absorbance wavelength of the components, suggesting that there is no change in intheabsorbancewavelengthofthecomponents,suggestingthatthereisnochangeinnanoparticle nanoparticle size and that the metal centers of the complexes have no effect on absorbance. These size and that the metal centers of the complexes have no effect on absorbance. These suggest suggest that the interaction between the azol complexes and AgNPs are just physical, without a that the interaction between the azol complexes and AgNPs are just physical, without a change change in the structure of the coordination compound or the nanoparticles. inthestructureofthecoordinationcompoundorthenanoparticles. The results suggest that the presence of AgNPs could effectively improve the antibacterial The results suggest that the presence of AgNPs could effectively improve the antibacterial activities of some azole complexes exhibiting broad-spectrum biocidal activities toward many activitiesofsomeazolecomplexesexhibitingbroad-spectrumbiocidalactivitiestowardmanydifferent different microbial strains. The antibacterial efficacies could be related to the azolyl ring, the metal microbialstrains. Theantibacterialefficaciescouldberelatedtotheazolylring,themetalcenterand center and the AgNPs diffusion, but further studies are necessary to elucidate the antibacterial theAgNPsdiffusion,butfurtherstudiesarenecessarytoelucidatetheantibacterialmechanismwith mechanism with combination of AgNPs with metal complexes. combinationofAgNPswithmetalcomplexes. 2.8. Cytotoxicity 2.8. Cytotoxicity Mammalian cells exposed to AgNPs alone did not evidence signs of cytotoxic effects. The MammaliancellsexposedtoAgNPsalonedidnotevidencesignsofcytotoxiceffects. Theviability viability assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reported assaysusing3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)reportedviabilities viabilities higher than 90%. However, the combination of most azole complexes with AgNPs had a higherthan90%. However,thecombinationofmostazolecomplexeswithAgNPshadadeleterious deleterious effect, except for compounds 4 and 8 (Figure 4). effect,exceptforcompounds4and8(Figure4). The fibroblast viability was always higher than 60% for all complexes in combination with Thefibroblastviabilitywasalwayshigherthan60%forallcomplexesincombinationwithAgNPs; tAhgesNePvsa; ltuheessseu vgagleusetsa stuogxgicesetf fae cttoaxtic5 0efµfegctm aLt −510. μAglt mhoLu−g1.h Aloltwhoeurgdho sleoswceoru dldosbees ecmouplldo ybeed eimnpanloimyeadl in animal studies without losing antibacterial effect, these studies are required to identify the studieswithoutlosingantibacterialeffect,thesestudiesarerequiredtoidentifythepotentialLD of 50 tphoetecnotmiapl oLuDn5d0 sofw tihteh cAogmNpPosu[n1d7s]. with AgNPs [17]. Molecules2018,23,361 10of17 Molecules 2018, 23, x FOR PEER REVIEW 10 of 17 FigureFi4g.uCrey 4t.o Ctyotxoitcoxeifcf eefcftesctos foaf zazoolele ccoommpplleexxees s(5(05 μ0gµ mgLm−1)L a−lo1n)ea alondn eina cnodmbinincaotimonb winitaht iAognNwPsi t(h10A- gNPs (10-nmnmd idaimameeteterr aatt 22 μµgg mmLL−1−).1 T).hTe hHeFFH cFeFllsc welelsrew exepreoseexdp toos tehde ctoomthpelecxoesm apnlde xAegsNaPnsd foAr g24N hP. sfor24h. 3. Materials and Methods 3. MaterialsandMethods 3.1. General Information 3.1. GeneralInformation All manipulations were routinely performed in an inert atmosphere (nitrogen) using standard ASlclhmlenakn-itpuubela tteiochnnsiqwueerse. Arollu trienaegleyntp-gerrafdoer msoeldveinntsa nwienree rdtraitemd, odsipshtilelered,( naintdro gsteonre)du suinndgers taa ndard Schlennkit-rtougbeen teactmhnoisqpuheerse.. ATlhler esatgaertnint-gg rcaodmepsooulnvdesn twsewree rperdepriaerded, dbisatsieldle do,na nthde sltioterreadtuuren:d beirs(a3,n5i-trogen atmosdpimheerteh.yl-1T-hpeyrasztaorlytiln)mgetchoamnep [o3u8]n, dbiss(w1,2e,r4e-trpiarzeopl-a1r-eydl)mbeathseande o[3n9]t, h3e,5-lbitise(r3a,5t-udriem:etbhiysl(p3y,5ra-dzoiml- ethyl- 1-ylmethyl)toluene [40], 3,5-bis(benzotriazol-1-ylmethyl)toluene [41] and 1,3-bis(indazol-1- 1-pyrazolyl)methane [38], bis(1,2,4-triazol-1-yl)methane [39], 3,5-bis(3,5-dimethylpyrazol-1- ylmethyl)benzene (L). The synthetic protocols for the previously reported complexes ylmethyl)toluene[40],3,5-bis(benzotriazol-1-ylmethyl)toluene[41]and1,3-bis(indazol-1-ylmethyl) dichloro[bis(3,5-dimethylpirazol-NN)]cobalt(II) (1), dichloro[bis(3,5-dimethylpirazol-NN)]- benzene (L). The synthetic protocols for the previously reported complexes dichloro[bis(3,5- copper(II) (2), dichloro[bis(3,5-dimethyl-1-pyrazolyl)methane-NN]cobalt(II) (3), dichloro[bis(3,5- dimetdhiymlpetihrayzl-o1l--pNyrNaz)o]clyolb)malett(hIIa)n(e1-)N,Ndi]cchoplopreor[(IbIi)s (3,(54-),d imdeitchhylolrpoi[rbaizs(o1l,-2N,4-Ntr)ia]-zcool-p1p-yelr)m(IIe)th(2a)n,ed-NicNh]l-oro[bis (3,5-dicmobeatlht(yIIl)- 1(-9p) yarnadz odliyclh)lmoreot[hbaisn(1e,-2N,4N-tr]icaozobla-1lt-(yIlI)) m(3e),thdainceh-lNoNro][cboipsp(3e,r5(I-Id) im(10e)t hayrel- 1p-rpoyvirdaezdo liynl )tmhee thane- NN]coSuppppelre(mIIe)n(t4a)r,yd Inicfohrlmoraoti[obni s[8(1]., E2,l4em-treinatzalo aln-1a-lyysl)ems (eCt,h Ha naned-N NN) w]-ecroeb paelrtf(oIrIm)(e9d) wainthd ad FiLcAhlSoHro 2[0b0i0s (1,2,4- triazoCl-H1-NySl)/Om Aentahlyazneer- (NThNer]cmoop Fpisehre(IrI S)ci(e1n0ti)fica, rWealpthroamvi,d MeAd, UinSAt)h. FeouSruieprp-tlreamnsefonrtma riynfrIanrefdo r(mFTaIRti)o n [8]. spectra were recorded on a Thermo Nicolet NEXUS FTIR spectrophotometer (Thermo Fisher Elementalanalyses(C,HandN)wereperformedwithaFLASH2000CHNS/OAnalyzer(Thermo Scientific) using KBr pellets Melting points were determined on a Mel-Temp® 1101D apparatus FisherScientific,Waltham,MA,USA).Fourier-transforminfrared(FTIR)spectrawererecordedon (Eletrothermal, Staffordshire, UK) in open capillary tubes, and they are reported uncorrected. Ultraviolet- aThermoNicoletNEXUSFTIRspectrophotometer(ThermoFisherScientific)usingKBrpelletsMelting visible (UV-Vis) spectra were recorded on a Cary 100 spectrophotometer (Agilent Technologies, Kansas pointsweredeterminedonaMel-Temp®1101Dapparatus(Eletrothermal,Staffordshire,UK)inopen City, KS, USA). Thermogravimetric analyses (TGA) of the complexes were obtained on a NETZSCH STA capilla4r0y9 PtuCb/PeGs, faronmd 8th toe y10a mregr oefp tohret ceodmupnlecxoersr ienc nteitdro.gUenlt mraevdiioal. eTth-ve issaimblpele(sU wVe-rVe issu)bsjpecetecdtr tao wdyenreamreicc orded onaChaeraytin1g0 0ovsepr eac ttermoppheroattuorme eratenrge( Aofg 3i0le–n70t0T °eCc hwnitohl oag hieeast,inKga rnastea sofC 1i0t y°,CK mSi,nU−1.S TAh)e. TTGh ecrumrvoesg wraevreim etric analysaensal(yTzGedA to) goifveth theec poemrcpenletaxgees mwaessr eloossb atsa ain feudncotionna oNf thEeT tZemSCpeHratSuTreA. 409PC/PGfrom8to10mgof thecomplexesinnitrogenmedia. Thesamplesweresubjectedtodynamicheatingoveratemperature 3.2. Synthesis of the Complexes range of 30–700 ◦C with a heating rate of 10 ◦C min−1. The TG curves were analyzed to give thepe3r.c2e.1n. tDaigcehlmoraos[3s,5lo-bsiss(a3s,5a-dfiumnecthtiyolnpyorfazthole-1t-eymlmpeethraytl)utorelu.ene-NN]cobalt(II) (5) A solution of 3,5-bis(3,5-dimethylpyrazol-1-ylmethyl)toluene (0.33 mmol; 101,4 mg) in 3.2. SynthesisoftheComplexes acetonitrile (CH3CN, 2 mL) was added to a solution of CoCl2 (0.38 mmol; 49.8 mg) in CH3CN (5 mL). 3.2.1.TDhiec hrleoarcoti[o3n, 5m-bixistu(3re,5 w-daism reeftlhuyxlepdy froarz 1o2l -h1 -aynldm dertiheydl )utnodluere nveac-NuuNm].c Aob paultr(eI Ib)lu(5e) compound was obtained by crystallization from dichloromethane (DCM). Yield: 128 mg (89%). M.p.: 192–193 °C. IR Asolutionof3,5-bis(3,5-dimethylpyrazol-1-ylmethyl)toluene(0.33mmol;101,4mg)inacetonitrile (CH CN,2mL)wasaddedtoasolutionofCoCl (0.38mmol;49.8mg)inCH CN(5mL).Thereaction 3 2 3 mixturewasrefluxedfor12handdriedundervacuum. Apurebluecompoundwasobtainedby crystallizationfromdichloromethane(DCM).Yield: 128mg(89%). M.p.: 192–193◦C.IR(KBr)ν/cm−1: 2921m,1553s,1468s,1421s,1368s,1271w,1047m,799m,729s. Anal. calc. forC H N CoCl : C,52.07; 19 24 4 2
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