Antibacterial Activities of Azole Complexes Combined with Silver Nanoparticles

17 Pages·2017·1.98 MB·English

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.:[phone](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