Tautomerization of 2-nitroso-N-arylanilines by coordination as N,N'-chelate ligands to rhenium(I) complexes and the anticancer activity of newly synthesized oximine rhenium(I) complexes against human melanoma and leukemia cells in vitro.

JournalofInorganicBiochemistry104(2010)774–789 ContentslistsavailableatScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio Tautomerization of 2-nitroso-N-arylanilines by coordination as N,N′-chelate ligands to rhenium(I) complexes and the anticancer activity of newly synthesized oximine ☆ rhenium(I) complexes against human melanoma and leukemia cells in vitro Stefan Wirtha, Andreas U. Walleka, Anna Zernickela, Florian Feila, M. Sztiller-Sikorskab, K. Lesiak-Mieczkowskab, Christoph Bräuchlea, Ingo-Peter Lorenza,⁎ , M. Czyzb,⁎ aLudwig-MaximiliansUniversityMunich,DepartmentofChemistryandBiochemistry,Butenandtstr.5-13(HouseD),D-81377Munich,Germany bMedicalUniversityofLodz,DepartmentofMolecularBiologyofCancer,6/8Mazowiecka,92-215Lodz,Poland a r t i c l e i n f o a b s t r a c t Articlehistory: The synthesis, structural characterization and biological activity of eight ortho-quinone(N-aryl)-oximine Received6November2009 rhenium(I)complexesaredescribed.Thereactionofthehalogenidocomplexes(CO) 5 ReX(X=Cl(4),Br(5)) Receivedinrevisedform24March2010 with2-nitroso-N-arylanilines{(C H ClNO)NH(C H R)}(R=p-Cl,p-Me,o-Cl,H)(3a–d)intetrahydrofurane 6 3 6 4 Accepted26March2010 (THF) yields the complexes fac-(CO) XRe{(C H ClNO)NH(C H R)} (6a–d, 7a–d) with the tautomerized Availableonline1April2010 ligandactingasaN,N′-chelate.Thesub 3 stitutio 6 no 3 ftwocarbo 6 ny 4 lligandsleadstotheformationofanearly planar 5-membered metallacycle. During coordination the amino-proton is shifted to the oxygen of the Keywords: nitrosogroupwhichcanbeobservedinsolutionfor6and7by1HNMRspectroscopyandinsolidstateby Rhenium Oximine crystalstructureanalysis.Afterpurification,allcompoundshavebeenfullycharacterizedbytheir1Hand13C N,N′-chelates NMR,IR,UV/visible(UV/Vis)andmassspectra.TheX-raystructureanalysesrevealedadistortedoctahedral Invitroanticanceractivity coordination of the CO, X and N,N′-chelating ligands for all Re(I) complexes. Biological activity of four Melanoma oximine rhenium(I) complexes was assessed in vitro in two highly aggressive cancer cell lines: human Leukemia metastaticmelanomaA375andhumanchronicmyelogenousleukemiaK562.Chloridocomplexes(6aand 6c)weremoreefficientthanbromidocompounds(7dand7b)ininducingapoptoticcelldeathofbothtypes ofcancercells.Melanomacellsweremoresusceptibletotestedrhenium(I)complexesthanleukemiacells. Noneoftheligands(3a–d)showedanysignificantanticanceractivity. ©2010ElsevierInc.Allrightsreserved. 1.Introduction various biological metabolic processes [13–21] has also generated a renewedinterestinthisclassofcompounds. The chemistryof C-nitrosocompoundsstartedin 1874withthe Inthiscontexttheconvenientavailabilityof2-nitroso-N-arylani- synthesis of 4-nitroso-N,N′-dimethylaniline [1] and nitrosobenzene lines[22]hasdrawnourattentionfromN,O-bridging[23–25]and- [2]byA.von Baeyer. Their firstcoordination tometals (Cd(II)and chelating [26] to N,N′-chelating ligands. Before 2007 this class of Zn(II))wasreportedbyPickardandKenyonin1907[3].Sincethena compoundswasmostlyreportedasaby-product[27–29].Examples considerablevarietyofsyntheticroutestohigh-yieldpreparationsof are the photochemical cyclization of N-acyl-2-nitroarylanilines C-nitroso compounds has been developed. The most up to date [30,31]ortheFischer–Hepprearrangement[32].Onlytwocompara- reviewsonthistopichavebeenrecentlypublished[4,5].Notonlydue blecompoundswithadditionalfunctionalgroups(methyl6-hydroxy- to its rich coordination chemistry [6] the family of C-nitroso 4-methyl-3-nitroso-2-(phenylamino)benzoate [33] and 2-nitroso- compoundshasbeenextensivelyinvestigatedduringthelastdecades. 1,3,5-tris(phenylamino)benzene[34])wereobtainableingoodyields Its relevance in organic chemistry [7] was first proved 1899 by the earlier.Incoordinationchemistrythisligandsystemismentionedin Ehrlich–Sachs reaction [8]. Examples published in recent years are few binuclear Pd(II) complexes [35–38]. There, it is formed by the application in ene reactions [9] or hetero Diels–Alder reactions [10– reactionofatetranuclearPd(I)clusterwithnitrosoarenes.Contraryto 12].Thediscoveryof theimportantrolesofC-nitrosocompounds in these results, the reaction of 2-nitroso-N-arylanilines with Re(I) halogenido complexes of the type Re(CO) X (X=Cl, Br) leads to a 5 metal-induced tautomerization.Ano-quinoidsystemisformedand theamino-protonisshiftedtotheoxygenofthenitrosogroup.Inthis ☆ DedicatedtoProf.Dr.HubertSchmidbaurontheOccasionofhis75thBirthday. ⁎ Correspondingauthors.Fax:+4989218077867. reportwedescribethesynthesisandcharacterizationofeightRe(I) complexes showing this tautomeric behaviour and the results of E-mailaddresses:ipl@cup.uni-muenchen.de(I.-P.Lorenz), malgorzata.czyz@umed.lodz.pl(M.Czyz). testing for biological activity of four of these complexes. We have 0162-0134/$–seefrontmatter©2010ElsevierInc.Allrightsreserved. doi:10.1016/j.jinorgbio.2010.03.014 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 775 complexespresentedherearethefirstcombiningtheimineandthe oximefunctioninoneandthesameo-quinoidsystem. 2.Resultsanddiscussion 2.1.Synthesisandcharacterizationofligands3a–d Scheme1.SynthesisofC-nitrosocompounds3a–d. The 2-nitroso-N-arylaniline ligands 3a–d were synthesized in a one-potreactionfromanilines1a–dand1-chloro-4-nitrobenzene(2) selected6a,6c,7d,and7bforbiologicalstudies.Thecytotoxicityof withpotassium-tert-butoxideandaceticacidindimethylformamide the drugs was evaluated in A375, a human melanoma cell line (DMF)(Scheme1).Modificationsmadeonthisliteraturemethod[22] exhibiting high metastatic potential, and K562, a Bcr–Abl-positive aredescribedintheexperimentalsection. humanchronicmyelogenousleukemia(CML)celllinederivedfroma Thesynthesisyieldsligands3a–dasairstable,darkgreenorbrown patientinblastcrisis. powders,solubleforexampleindichloromethane,tetrahydrofurane Metal-induced tautomerization reactions have been examined or acetone and nearly insoluble in pentane or n-hexane. Mass especially in relation with Pt(II) pyrimindine [39] and adenine [40] spectrometric investigation in the direct electron-impact ionization model nucleobase interactions. In these examples a shift of the modewithdetectionofpositiveions(DEI+)showstheexpected[M+] equilibriumtothe“wrong”tautomercouldleadtobase-mispairingin peakandanassignablefragmentationpatternforallligands.Inthe1H nucleic acids. Moreover, metal-induced proton migration in com- NMR of 3a–d spectra a broad singlet in the range of 11.60 to plexes is an important attribute in connection with the design of 12.06ppm can be identified as the amino-proton signal. Another molecular electronic devices [41]. The capacity of intercalation into broadsingletatδ=8.64–8.66ppmcanbeassignedtotheprotonin DNA [42,43] and in general the strong metal–ligand π-interaction ortho-positionoftheNO-group.Theremainingsignalsoftheprotons havealsoattractedgreatinterestintoo-quinoneligandsystems.Much in meta-position and the second aromatic ring are observed at literatureonthistopicisconcernedwitho-quinoiddiimines[44–48], δ=6.93–7.52ppm. Exemplarily the 13C NMR spectrum of 3c is lesswithdioximes[49–52],buttothebestofourknowledgetheRe(I) depictedinFig.1. Fig.1.13CNMRspectraof3catA)25°CandB)−60°C. 776 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 ForeachligandoneC andoneC peakwas“missing”andavery q H broadsignalaround140ppmcouldbedetectedatroomtemperature (Fig. 1). Measurement at −60°C shows two “new” peaks at 130.8 (C 1)and142.2ppm(C 3)whichcanbeassignedtothecarbonsin q H ortho-positionofthenitrosogroup.“Freezing”rotationalongtheC–N bond leads to a significant splitting of the broad signal (Δ=11.4ppm).Thisisinducedofcoursebythedifferentsubstituents butinfactmoreduetothelargemagneticanisotropy[53]oftheNO- group. This effect of an asymmetric substituent upon the relative chemical shifts of the ortho-carbons is a known phenomenon and establishedforaromaticnitrosocompounds[54,55]. The IR spectra of ligand 3a–d (KBr pellets) show ν(C–H) absorptions between 3100 and 2900cm−1 but lack a pronounced ν(N–H)band,apparentlyasaconsequenceofthestrongintramolec- ularhydrogenbonding[32].Therefore,abroadandweakabsorption between 2900–2650cm−1 is observed due to N…H…O bonding. Surprisinglyaveryweakabsorptionforν(N–H)isdetectedinliquid phase IR spectra at higher wave numbers (3a: 3372cm−1; 3b: 3377cm−1; 3c: 3373cm−1; 3d: 3378cm−1 in CH Cl ). Nitroso 2 2 stretching absorptions are assigned according to the reports of Gowenlock et al. in the range of 1488–1513cm−1 for monomeric aromaticArNOcompounds[21,55,56].Theallocationofthesebandsis supportedduetothefactthattheydisappearaftercomplexation.This demonstrates the fundamental change in chemical character of the N―Obondwhentautomerizationfromnitrosotooximehappens. Fig. 2. Molecular structure of 5-Chloro-2-nitroso-N-p-tolylaniline (3b) with π–π Measurement of UV/Vis spectra of 3a–d in dichloromethane stacking(openbond)and3-centeredintermolecularhydrogenbond(dashedlines). The thermal ellipsoids are drawn at the 50% probability level [57]. Aromatic and revealed four intense absorptions for each ligand (Table 1). Three aliphatichydrogenatomsaswellaspartsoffurthermoleculesof3bareomittedfor arelocatedintheUVareaandareoriginatedfromπ–π*transitionsof clarity. thearomaticrings.Thefourthissituatedinthevisiblerangeandis identifiedasπ–π*NOtransition. Since no crystallographic information was available for this com- C―NbondlengthsofN(1)–C(1)andN(2)–C(2)arealwaysabitshorter poundclassintheliterature,molecularstructuresof3b(Fig.2)and3d thanN(1)–C(7).Inbothcasesthenitrosogroup,locatedintheplaneof havebeendeterminedbyX-raydiffractionanalysis.Singlecrystalswere theC(1)–C(6) ringand the amine, is stabilized by anintramolecular obtained by slow sublimation at 55°C and 1.0×10−3mbar. The hydrogenbond.N―Obondlengthsareintheexpectedrange(1.13– structureanalysisrevealedtwoplanararomaticrings(C(1)–C(6)and 1.29Å)[21]fornitrosoarenes. C(7)–C(12))whereasthefirstringshowssomequinoidcontribution. In both structures a secondary intermolecular interaction of the C―C bond lengths for C(3)−C(4) and C(5)–C(6) are noticeable amine proton forms a 3-centered hydrogen bond. In 3b the shortenedincomparisontotheremainingaromaticring.Furthermore intermolecular acceptor is chlorine (Cl(1)) (Fig. 2). For 3d the additional acceptor is a nitroso oxygen whereas the interacting Table1 molecule forms a second “back bonding” H-bridge. As further UV/Visabsorptiondataof3a–3dand6a–7d:inCH2Cl2 λ max[nm](ε[M−1cm−1])and intermolecular interaction a π–π stacking [58,59] of the C(1)–C(6) fluorescencedataof3cand6a–7dinCH2Cl2 λ max[nm]. ringoccurs(Fig.2).Intheunitcellof3bforexample,twoofthese UV Visible rings show absolutely coplanar arrangement with a plane-to-plane distanceof3.50Å.Aparalleldisplacementofonly1.00Åiscalculated 3a 253(13300),278(17100),312(14900) 461(8200) 3b 249(12900),269(12100),312(12700) 467(6900) for the centroids. For 3d a slight deviation from coplanarity is 3c 253(12000),275a(15500),312a 457(7200) observed. (13400) λ em 523b523b 3d 250(12000),274(12400),312(12400) 464(6800) 6a 321(4500) 471(4500),548a(9700) 2.2.SynthesisandcharacterizationofRe(I)complexes6a–7d λ em 607b 6b 320(4500) 474(5200),541a(10500) Thenoveloximinerhenium(I)complexes(6a–7d)areobtainedas λ em 573b illustratedinScheme2.RefluxingRe(CO) X(X=Cl(4),Br(5))indry 6c 322a(6400) 438(4500),470(4800),555a 5 THFleadstothesubstitutionoftwoCOligands.Completenessofthe (12600) λ em 510b 606b replacementcanbemonitoredwithliquidphaseIRspectroscopybya 6d 321(4900) 470(4800),544a(11200) shiftoftheν(CO)bands. λ em 612b Addition of one equivalent of 3a–d in dry THF and workup as 7 7 λ a b em 3 3 5 5 8 7 ( ( 6 5 2 4 0 0 0 0 ) ) 4 4 6 7 8 0 5 1 0 ( ( c 5 5 4 5 0 0 0 0 ) ) , , 5 5 4 4 9 4 a a ( ( 1 9 1 9 0 0 0 0 0 ) ) d qu es a c n r t i i b t e a d tiv i e n ly t . h C e om ex p p l e e r x i e m s e 6 n a t – a 7 l d se a c r t e io o n bt y a i i e n l e d d s a th s e da c r o k m g p re le e x n es (6 6 a a – – 7 7 b d ) λ em 586c ordarkpurple(7c,7d)powders.Theyareslowlydecomposingwhen 7c 362(6400) 441(3800),474(4400),557a exposedtomoistair,solubleindichloromethaneorchloroformand (11200) insolubleinpentane.Massspectrometricinvestigationsofcomplexes 7 λ d em 358(5600) 4 6 7 0 3 1 ( c 4700),546a(9800) 6a–7d(FAB+mode)exhibittheparentpeakaswellasacomparable λ em 608c fragmentation pattern resulting from successive loss of CO and a Excitationatthemarkedabsorptionpeak. halogenidoligands. b Accuracyofthemeasurement:3c,6a–d:±3nm. Comparison of the 1H NMR spectra of 6a–7d with the c Accuracyofthemeasurement:7a–d:±10nm. correspondingligandspectra(3a–d)showssimilartendencies.Inall S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 777 Scheme2.SynthesisoftheN,N′-chelatecomplexes6a–7d. cases the broad singlet of the acidic proton shows a large shift to strength. The stronger absorptions in the visible region arise from higher field due to migration from the amino group (3a–d: 11.60– π–π*transitionsoftheligand,mostlywithnodistinctmaxima,shifted 12.06ppm)tothenitrosooxygen(6a–7d:8.31–9.50ppm).Allthree therebyitstautomerizationintoano-quinoidform.Allspectrashow signals of the ortho-quinoid system are shifted to higher field after thisbathochromicshiftincomparisontothecorrespondingelectronic coordination.Theprotonsofthesecond,aromaticringofcomplexes transitions of 3a–d (Table 1). Compounds 6a–7d showed weak 6a–7dshownoconsistenttendenciescomparedtotheligandspectra. fluorescenceinthevisiblerangewithafluorescencequantumyieldof In the 13C NMR spectra of 6a–7d all signals for the CO ligands, the about10−5.Excitationattheabsorptionmaximaof6a–7dataround quaternary aromatic carbons and the aromatic C carbons are 550nmresultedinfluorescencemaximaataround610nm(Table1). H detected in the expected areas. Since rotation of the nitroso group Forbromidocomplexes7a–7dthesignaltonoiseratio(SNR)wasat along C(2)–N(2) is inhibited after coordination, all signals are thedetectionlimitandaboutoneorderofmagnitudelowerthanthe observable separately at room temperature. In comparison to the SNR of 6a–6d with chlorido ligand. Therefore the error of the starting materials 3a–d no general direction of the shifts can be fluorescence maxima position is significantly increased. That in identifiedinproducts6a–7d. consideration, the fluorescence maxima position of the chlorido IR spectra of 6a–7d in liquid phase show three intense ν(CO) complexcomparedwiththecorrespondingbromidocomplexseems absorptions. This is in accordance with C–symmetry of their facial tobesimilar,whilethedifferentligands(3a–d)haveasmallinfluence S arrangement. Surprisingly measurement of 7a–7d in KBr pellets onthemaxima.Itisnoteworthythattheligandinitsaromaticform exhibit up to five ν(CO) bands what apparently can be caused by (onlytestedfor3c)exhibitedalsoveryweakfluorescenceataround packingeffectsinsolidstate.Intheareaaround3000cm−1ν(C–H) 523nmwhenexcitingat275nmor312nm,butnotaround610nm. absorptionsaredetectedoverlappingwithabroadbandbetween3050 Thisindicatesthatthefluorescenceat510nmin6cisonlycausedby and 3200cm−1 attributed to ν(O―H). Comparison of IR data of therheniumcarbonylpartandthataround610nmresultsfromthe differenttransitionmetalcomplexescontaininganoximeoroximine ligandwhichhasbeenchangedintoitso-quinoidformshowinglower function [60–64] with spectra of 6a–7d leads to the following frequencies.Selectedfluorescencespectraareavailableassupporting assignment: absorptions at 1597–1606cm−1 to ν(C=N) of the information. iminemoiety,bandsfrom1543–1553cm−1toν(C=N)oftheoxime Themolecularstructuresofcomplexes6a–7dweredeterminedby moiety. The interval associated with ν(N―O) (1040–1060cm−1) X-raydiffractionanalysis.Singlecrystalswereobtainedbyisothermic showstwostrongabsorptionsveryclosetogetherforeachcomplex, diffusionofn-pentaneintoasolutionof6a–7dinCH Cl orCHCl .The 2 2 3 againcausedbypackingeffects. X-raystructureanalysisrevealedadistortedoctahedralcoordination The UV/Vis spectroscopic investigations of 6a–7d in CH 2 Cl 2 for all Re(I) complexes, consisting of the CO, halogenido and N,N′- revealedtypicallythreeandintwocases(6cand7c)fourabsorptions. chelatingligands3a–d. OneislocatedintheUVareaandiscausedbyad6-metal-to-ligand Crystaldataanddetailsofstructurerefinementforcompounds3b, charge-transfertransition,therestappearsinthevisiblerange.The 3dand6a–7daresummarizedinTable5.Selectedbondlengthsand difference of about 30m between chlorido (6a–6d) and bromido anglesarelistedinTable2.Exemplarily,only6aisdepictedinFig.3, complexes (7a–7d) may be caused by their different ligandfield ORTEP-plots of all other structures and hydrogen bond data are Table2 Selectedbondlengths(Å)andangles(°)ofcompounds3b,3dand6a–7d. Compound 3b 3d 6a 6b 6c 6d 7a 7b 7c 7d Re(1)–C(13)/C(14)6b – – 1.948(4) 1.943(4) 1.947(7) 1.904(12) 1.951(4) 1.936(5) 1.962(8) 1.927(7) Re(1)–C(14)/C(15)6b – – 1.921(4) 1.937(3) 1.937(7) 1.927(10) 1.937(4) 1.936(5) 1.912(8) 1.932(7) Re(1)–C(15)/C(16)6b – – 1.911(4) 1.897(4) 1.913(8) 1.904(12) 1.905(5) 1.899(6) 1.913(10) 1.901(8) Re(1)–Cl6/Br7 – – 2.481(1) 2.486(1) 2.480(2) 2.478(2) 2.621(1) 2.623(1) 2.614(1) 2.636(1) Re(1)–N(1) – – 2.120(3) 2.134(3) 2.129(5) 2.134(7) 2.133(3) 2.133(3) 2.128(6) 2.129(4) Re(1)–N(2) – – 2.141(3) 2.139(3) 2.136(5) 2.136(7) 2.120(3) 2.127(3) 2.137(5) 2.112(5) O(1)–N(2) 1.258(2) 1.259(2) 1.384(3) 1.374(3) 1.369(7) 1.379(8) 1.370(3) 1.375(4) 1.379(7) 1.309(6) N(2)–C(2) 1.388(2) 1.382(2) 1.298(4) 1.312(4) 1.308(8) 1.304(11) 1.310(4) 1.312(5) 1.303(8) 1.345(7) C(1)–C(2) 1.434(3) 1.430(2) 1.460(5) 1.461(4) 1.456(9) 1.468(12) 1.456(5) 1.456(6) 1.471(9) 1.447(7) N(1)–C(1) 1.350(2) 1.357(2) 1.310(4) 1.314(4) 1.313(8) 1.333(10) 1.315(4) 1.319(5) 1.314(9) 1.314(7) N(1)–C(7) 1.431(2) 1.420(2) 1.451(4) 1.439(4) 1.439(8) 1.441(11) 1.433(4) 1.437(5) 1.448(9) 1.431(7) N(1)–Re(1)–N(2) – – 73.27(10) 73.90(9) 73.8(2) 73.8(3) 73.95(11) 73.71(13) 73.7(2) 74.99(17) C(13)/C(14)6b–Re(1)–N(2) – –104.13(13) 103.04(11) 103.1(2) 103.1(3) 99.88(13) 100.67(16) 103.5(2) 96.4(2) C(14)/C(15)6b–Re(1)–N(1) – – 94.83(13) 95.15(11) 95.1(2) 96.5(3) 96.57(14) 97.54(16) 95.4(3) 96.5(2) N(2)–Re(1)–N(1)–C(1) – – 4.3(2) 8.9(2) 1.8(5) −7.1(6) −4.7(2) 5.1(3) 1.4(5) 1.4(4) C(1)–N(1)–C(7)–C(12) 74.4(3) 62.2(3) 69.8(4) 76.1(4) 96.4(8) −110.2(10) −103.9(4) 117.7(5) 99.7(8) 69.1(7) 778 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 Scheme3.Averagebondlengthsof6a–7dincomparisonwithaveragevaluesin3band 3d(inbrackets). dependence of this angle on the ligands was not identifiable. This Fig. 3. Molecular structure of Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-4-chlor- maybeaneffectofπ–π-interactionbetweenthearomaticorquinoid ophenyl)-oximine-N,N′}rhenium(I)(6a).Thethermalellipsoidsaredrawnatthe50% rings since in every structure an absolute coplanar arrangement of probabilitylevel[57].Aromatichydrogenatomsareomittedforclarity. thesemoietiesisobserved.Anexceptionis givenin 7dwhereonly some rings of the same type are coplanar. For complexes 6a–7c a availableassupportinginformation.BondlengthsRe–CO areinthe tricliniccrystalsysteminthespacegroupP-1isobserved,containing expected range for fac-Re(CO) 3 X-complexes with quinoid ligands twocomplexmoleculesintheunitcell.Anintermolecularhydrogen [46,65],justaswellastheRe–X(X=Cl,Br)bonds.TheRe―CO ax bond bondconnectstheoximefunctionofonemoleculeandthehalogenido intrans-positiontothehalogenidoligandisconsiderablyshorterthan ligandofanothermoleculeinthenextunitcellandviceversa.Thus theRe―CO eq bonds.Thisindicatesaπ-acceptingcharacterofthe N,N′- eachofthesepairsisconnectedthroughtwointermolecularhydrogen chelates.Re–Ndistancesin6a–7dshownorecognizablepreference bonds.Incomplex7damonocliniccrystalsysteminthespacegroup forashorterimineoroximebondingandareclosetobondlengths C2/c with eight molecules in one unit cell is observed. The only recentlyreportedforanortho-quinoiddiimineRe(I)complex[46]ora difference to 6a–7c is the lack of intermolecular hydrogen bonds Re(I) nitroso complex [66]. Only few structurally characterized betweencomplexmoleculesof7d.Insteadofthese,ahydrogenbond rhenium oximato complexes suitable for comparison are known. totheCH Cl enclosedinthecellisobserved.Thismaybethereason 2 2 Re–N bond lengths observed in 6a–7d are slightly longer than for the discrepancy. Apparently intermolecular hydrogen bonds reportedforanoximatoligandchelatingRe(V)(2.099(3)Å)[67]ora between the complex molecules are an integral factor in the dioxime ligand system chelating Re(III) (2.028(9)–2.108(9) [68] / arrangementwithintheunitcellofthesecompounds. 2.03(1)–2.17(1) [69]). Crystallographicinformation forRe(I) is only available for a monodentating, nonaromatic oximato ligand. This 2.3.Biologicalevaluation showsasomewhatlongerRe―Nbondlength(2.183(6)Å)[70]than 6a–7d. 2.3.1. Oximine rhenium(I) complexes inhibit melanoma adherent cell Aftercoordinationtheformeraromaticsystemof3a–dwithonly proliferationmoreefficientlythanproliferationofleukemiacells some quinoid contribution shows clearly quinoid topology. A Concentration–response and time course analyses were per- significantelongationoftheN―Obond(Scheme3)toanexpected formed using four rhenium(I) complexes (6a, 6c, 7d, and 7b). In scale (1.319(4) Å [67], 1.368Å [69], 1.396(9) Å [70]) confirms the someexperiments,the2-nitroso-N-arylanilineligands(3a–3d)were oximenatureoftheformernitrosogroup.ShorteningofN(1)–(C1) included.Tetrazoliumderivativereduction(MTT)assaywasusedto andN(2)–C(2)andexplicitlyalternatingC―Cbondlengthsindicate assesstheinfluenceofthedrugsonthemetabolicactivityofadherent theo-quinoidformoftheC(1)–C(6)ring.Thisisalsoinaccordanceto melanoma cells (A375) in relation to untreated control cells. Cell a recently published bond length pattern for o-quinoid ligands in proliferation of leukemic K562 cells cultured in suspension was differentoxidationstates[71].Theeffectofcoordinationtorhenium determinedusingTrypanbluedyeexclusionassay.First,IC values 50 ontheaveragebondlengthsinthesecondaromaticringisnegligible. wereestimatedforthecancercellinhibitionofproliferationasshown Thebidentateligands3a–dbindtorhenium(I)viaformationofa inFig.4.IC 50 valuesobtainedformelanomaA375cellstreatedwith nearlyplanarmetallacycle.Thisisconfirmedbytorsionangelscloseto 6a,6c,7d,and7bfor2dayswere0.9,0.7,1.3,and1.8μM,respectively 0° within the 5-membered ring. Distortion of the octahedral (Fig.4A). coordinationspherebecomesevidentbythesmallligandbiteangles Higher concentrations of oximine rhenium(I) complexes were N(1)–Re–N(2) around 74° and the angles between the chelating necessarytoreduceproliferationofK562cellsto50%comparedtothe nitrogen atoms and the equatorial carbonyls. Complexes 6a–7d control cells (Fig. 4B). But similarly to the results obtained for exhibit on the oxime-side (N(2)–Re(1)–CO eq ) an average angle of melanomacells,6aand6cweremoreefficientininductionofgrowth 101.7°.Theimine-side(N(1)–Re(1)–CO eq )showsvaluescloseto95°. arrest(IC 50 of3.4and3μM,respectively)than7dand7bwithIC 50 of The bridging nitrogen atom (N1) is trigonal planar in geometry 7.5μMand7.8μM,respectively.Ligand3creducedcellproliferation consistentwithitssp2nature,whereasthesumofsurroundingangles to96%and90%comparedtothecontrolcellswhenitwasusedatthe isalwaysverycloseto360°. concentrationsof4μMand8μM,respectively.Theotherligandswere ThesecondaromaticringconnectedtoN(1)ishighlyturnedoutof alsomuchlessefficientthantheirrespectiverhenium(I)complexes the plane defined by the metallacycle (69.1(7)°–117.7(5)°). A (notshown). S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 779 Fig.5.Oximinerhenium(I)complexesinhibitmelanoma(A)andleukemia(B)cell proliferation.Atimecourse.A375cellsweretreatedwith1.4μMandK562cellswith Fig. 4. Concentration–response analyses of the influence of oximine rhenium(I) 8μMoximinerhenium(I)complexesforupto3days.Viable,adherentA375melanoma complexesonmelanomaandleukemiacellproliferation.(A)MTTassaywasusedto cellswerequantifieddailybyMTTassayandviableK562leukemiacellsbyTrypanblue studycytostaticeffectsofoximinerhenium(I)complexesinhumanmelanomacellline exclusiontest.Thedataarethemean±SDofthreeindependentexperimentsdonein A375.Themeanoftheabsoluteabsorbancevaluesgivenbydrug-treatedcellswas triplicates(Pb0.05exceptfor7bonday1). dividedbythemeanoftheabsoluteabsorbanceofDMSO-treatedcontrolsampleand expressedasrelativenumberofviableadherentcells.(B)Trypanblueexclusiontest wasappliedtoassesscytostaticeffectsofthetesteddrugsinhumanCMLcelllineK562 concentrations of drugs in the range from 1μM to 100μM, 6c and asdescribedinMaterialsandMethods.Cellproliferationisexpressedasthepercentage 6areducedviabilityto50%ofthecontrolattheconcentrationsof7μM ofviablecellnumberinthecontrolculture.IC50valuesforeachcompoundineachcell linewerecalculated(seetextfortheresults).Thedataarethemean±SDofthree and 8μM, respectively. The same was observed for 7d and 7b at independentexperimentsdoneintriplicates. concentrations as high as 33μM and 29μM, respectively (Fig. 7B). Ligands3a–ddidnotsignificantlyaffectK562cellviabilityevenatthe Next,timecourseanalyseswereperformed.Viablemelanomacells concentrationof20μM(notshown). werequantifieddailybyMTTassay,leukemiacellswereevaluatedby Trypanbluestaining(Fig.5).Togetsimilarreductionofproliferation 2.3.3.InductionofapoptosisinA375melanomacellsandK562leukemic inbothcelllines,concentrationsofdrugsusedintheseexperiments cellsbyoximinerhenium(I)complexes were 1.4μM in A375 cell cultures (Fig. 5A) and 8μM in K562 cell Celldeathwasassessedinmelanomacellstreatedwithoximine cultures(Fig.5B).Asexpected6cand6aweremoreefficientthan7d rhenium(I)complexesbymorphologicalcharacteristicsofapoptotic and7binbothcelltypes. and necrotic cells in fluorescence microscopy after staining with acridine orange and ethidium bromide (AO/EB) (Fig. 8). More than 2.3.2.Effectsofoximinerhenium(I)complexesoncancercellviability 300 cells were analyzed and then the percentages of early/late ChangesinA375melanomacellviabilityinresponsetooximine apoptoticornecroticcellswerecalculated.Compound6c,whichmost rhenium(I) complexes were assessed by propidium iodide staining efficientlyreducedproliferationofadherentmelanomacells,induced andFACSanalysis(Fig.6).Ontheseconddayoftreatment,only6cat apoptosis in more than 80% of the cells when applied at the the concentration of 1μM significantly reduced the viability of concentrations of 1μM and 2μM (Table 3). For 6a and 7d, melanoma cells to 53% (47%±5 of cells were PI-positive; Pb0.05). concentration of 2μM was required to stimulate apoptosis to this Theotheroximinerhenium(I)complexes(6a,7b,and7d)appliedat extent.Compound7b,theleastefficientininhibitingproliferationof theconcentrationof1μMorligands(3a–d)usedattheconcentration adherentmelanomacells,wasalsotheleasteffectiveininducingcell of2μMdidnotinducetheappearanceofPI-permeablecells.When death.Noneofthetesteddrugs,includingligands,stimulatednecrosis concentrationofrhenium(I)complexeswasraisedto2μMviabilityof in melanoma cells. Therefore, we could assume that in the applied melanomacellswasdecreasedtothelevelbelow30%. conditions apoptotic cell death was the major cause of reduced TrypanblueexclusiontestwasusedtoassessviabilityofK562cells proliferationofmelanomacells. treatedwithoximinerhenium(I)complexes.Inthisassay,thenumber Bcr–Abl-expressing leukemic K562 cells are highly resistant to ofdeadcellstakingupTrypanbluewasexpressedaspercentageofthe apoptosisinducedbychemotherapeuticagents.Inthecurrentstudy, totalcellnumber(viableanddead)ineachexperimentalcondition. theywereassessedonday3fortheevidenceofapoptoticcelldeathby First,K562cellsweretreatedcontinuouslyfor3dayswith8μMofthe doublestainingwithAO/EB(Fig.9A).Inaddition,DAPIstainingwas oximine rhenium(I) complexes. On day 3, similar effectiveness was used to show the condensation and fragmentation of the nuclei observed for 6c and 6a, whereas 7d and 7b were still ineffective (Fig.9B). NeitherDAPIstaining nordoublestainingwithAO/EBfor (Fig. 7A). When the viability of K562 cells was tested at the earlyandlateapoptosisshowedaninductionofapoptosisafter3days 780 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 Fig.6.Effectsoftheoximinerhenium(I)complexesonmelanomacellviability.A375melanomacellsweretreatedfor2dayswith6c,6a,7d,and7batconcentrationsof1μMand 2μM,orwithligands3a–dattheconcentrationof2μM.ThepercentagesofcellswithPI-permeablemembranewereassessedbyFACSanalysisincombinedpopulationsofadherent andfloatingcells.Selectedhistogramsfromarepresentativeexperimentareshown.Thedataarethemean±SDoftwoindependentexperiments(Pb0.05)exceptfrom2μM oximinerhenium(I)complexeswhenonlyoneexperimentwasdone. oftreatmentwith8μM7dand7b.Atthesameconcentration,6cand Insummary,currentstudiesofanticanceractivityoffouroximine 6ainducedapoptoticcelldeathinthemajorityofK562 cells.More rhenium(I) complexes performed in melanoma and leukemia cell than90%ofcellswereeitherinearlyorlatestageofapoptosisafter lineshaverevealedthatintwoseriesofhalogenidoRe(I)complexes 3daysoftreatmentwiththesedrugs(Table4).Compound6cwasthe (X=Cl, Br), chlorido complexes were more efficient as anticancer mostefficientininductionofapoptosis.Thiswasclearlyvisibleinthe drugs in vitro. Chlorido complexes 6c and 6a possessed significant experiment showing fragmented nuclei which appeared in the cytostatic activity against leukemiaK562 cells and melanoma A375 presence of 6c at a concentration as low as 4μM (Fig. 9B). As cells.Thisactivitywasmuchhigherthanforbromidocompounds7d expected, ligands (3a–d) did not induce apoptotic cell death when and7b.LethaleffectsofchloridoRe(I)complexesinmelanomaand used at concentrations of 8μM and 20μM. Necrosis was also not leukemia cells were obtained at different concentrations of com- inducedintheseconditions. pounds.ChloridoRe(I)complexesattheconcentrationof1μMwere S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 781 partofthecytotoxicactivityofthesecompoundsmightbeconnected withBcr–Ablpathway. Melanomaisthemostaggressivetypeofskincancerandishighly resistanttoallcurrentlyusedchemotherapeutics[74].Thealkylating drug dacarbazine (DTIC), which is approved for treatment of metastatic melanoma, results in clinical responses of 5–10% of patients[75].HumanA375melanomacelllineusedinourstudy,is consideredashavinghighmetastaticpotentialandishighlyresistant to anticancer drugs. Therefore, the obtained results with oximine rhenium(I)complexes,especially thosechloridocompounds6cand 6a, are encouraging and need further investigation. One reason for highersusceptibility ofmelanomacellsthanleukemiacellstoRe(I) complexes could be the influence of the drugs on the adhesive potential of melanoma cells. In addition to clearly visible apoptosis induced by oximine rhenium(I) complexes, they might affect cell adhesion.By reducing thenumberofadherentcellsin culturethey could influence the relative number of cells able to proliferate. It shouldalsobetakenintoaccount,thatanysingle-agentchemother- apy tested in clinical setting for melanoma patients has resulted in response rates below 20%.[76,77] Therefore, instead of oximine rhenium(I)complexesalone,combinationwithotherdrugs,including those highly specific for deregulated pathways in melanoma cells, shouldbeconsideredforfutureevaluations. Taken together, our data demonstrated that albeit to different extent,newlysynthesizedoximinerhenium(I)complexesinvestigat- ed in this study could induce apoptotic cell death in leukemia and melanomacells.Toourknowledge,thisisthefirststudyshowingthe anticancer therapeutic potential of rhenium complexes against Fig.7.Effectsoftheoximinerhenium(I)complexesonleukemiacellviability.(A) AliquotsofK562cellculturestreatedwith8μMoximinerhenium(I)complexes6c,6a, melanoma and leukemia cells. Further studies are necessary to 7d,and7bwereremoveddailyforadeterminationofthenumberofviableanddead unraveltheexactmechanism(s)ofthecellularresponsesevokedby cells by Trypan blue exclusion test. The number of viable cells is expressed as these compounds as well to verify their effectiveness in in vivo percentageoftotalcellnumber.(B)K562cellsweretreatedwithoximinerhenium(I) models. complexesatdifferentconcentrationsandthenumberofviableanddeadcellswas assessedonday3byTrypanblueexclusiontest.Thedataarethemean±SDoftwo independentexperimentsdoneintriplicates(Pb0.05). 3.Experimental 3.1.General apparentlyeffectiveinstimulatingcelldeathonlyinmelanomacells, 6cwasthemostefficientinthisrespect.InleukemicK562cells,higher All experiments and manipulations were performed under dry concentrationswerenecessarytoachieveasimilarlevelofinhibition, argon atmosphere using Schlenk and vacuum-line techniques. Re but again compound 6c was the most effective. Comparison of (CO) X(X=Cl(4),Br(5))[78]werepreparedaccordingtoaliterature 5 cytotoxic and cytostatic effects revealed that apoptotic cell death, procedure. The published synthesis of 2-nitroso-N-arylanilines [22] defined as loss of cell membrane integrity, was the major cause of (3a–d) has to be modified to achieve good yields. Solvents were reduced proliferation in melanoma and leukemia cells. This was purifiedbystandardprocedures;dichloromethanewasdistilledfrom assessed in A375 melanoma cells as PI- and AO/EB-membrane calciumhydride,n-pentaneandn-heptaneweredistilledfromlithium permeabilityandinK562leukemiacellsasTrypanblue-andAO/EB- aluminiumhydrideandtetrahydrofuranewasdistilledfromsodium. membrane permeability, and shown as nuclei fragmentation. Bro- All solvents were stored under a dry argon atmosphere with 3Å mido compounds 7d and 7b were much less effective against molecular sieves (dichloromethane) respectively sodium pieces (n- melanoma and leukemia cells, however, 7d exerted some effect on pentane, n-heptane, THF). NMR spectra were recorded with a Jeol proliferation of adherent melanoma cells. It is also interesting that Eclipse270,JeolEclipse400orJeolEX400spectrometeratambient amongchloridoandbromidocomplexes,thosehavingligandwithno temperatureunlessstatedotherwise.Allchemicalshiftsaregivenin substituent in para position (6c and 7d) showed higher cytotoxic ppmrelativetoTMS.Thesplittingofprotonresonancesinthereported activity than those with either methyl (7b) or chlorine (6a) 1HNMRspectraisdefinedass=singlet,d=doublet,dd=doubletof substituents. It is of note that ligands themselves (3a–d) did not doublets,ddd=doubletofdoubletsofdoublets,dddd=doubletof stimulate cellular death in melanoma and leukemia cells. The doublets of doublets of doublets, m = multiplet and br s = broad relationship between chemical structure and the vulnerability of singlet. IR spectra were measured in the range of 4000–400cm−1 cancercellstodrug-inducedapoptosisneedstobefurtherexplored. usingaPerkinElmerSpectrumOneFT-IRspectrometer.Theintensity K562celllinederivedfromapatientinblastcrisisiscommonly ofreportedIRsignalsisdefinedasvs=verystrong,s=strong,m= accepted as a cellular modelof advanced phase of chronic myelog- mediumandw=weak.UV/visible(UV/Vis)datawasrecordedwitha enousleukemia(CML)[72,73].Itwasreportedbymanygroupsthat VarianCary50UV/Visspectrophotometer.Theemissionspectrawere commonlyusedanticancerdrugsdonotefficientlyinduceapoptosis recordedonaFS900fluorescencespectrometer(EdinburghAnalytical in K562 cells, mainly due to the constitutive Bcr–Abl activity and a Instruments).MassspectrawereobtainedbyaJeolMStationJMS-700 negligiblelevelofp53.Onlyimatinib,bydirectlytargetingactivityof in direct electron-impact ionizationmode (DEI)or positive ion fast fusionkinaseBcr–Abl,inducesmitochondria-dependentapoptosisin atombombardmentionizationmode(FAB+)(3-nitrobenzylalcohol K562 cell line [73]. Therefore, the results obtained for the newly (NBA)matrix).Multi-isotopecontainingfragmentsrefertotheisotope synthesizedoximinerhenium(I)complexessuggestedthatatleasta withthehighestabundance.Elementalanalyseswereperformedwith 782 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 Fig.8.Theoximinerhenium(I)complexesinduceapoptosisbutnotnecrosisinmelanomacells.DoublestainingofA375melanomacellsfollowing2dayexposuretooximine rhenium(I)complexesattheconcentrationsof1μMand2μM.Cellswerestainedwithnucleicacidselectivefluorochromes:membrane-permeableacridineorangeandimpermeable ethidiumbromide.Representativemicroscopicfieldsareshown.Viablecellshadbrightgreenchromatinwithorganizedstructure.Inearlyapoptoticcells,thechromatinwas condensedorfragmentedbutstillstainedgreen.Inlateapoptoticcells,itwascondensedorfragmentedandstainedorange.Necroticcellshadbrightorangechromatinwith organizedstructure.Tendifferentfieldswererandomlyselectedforcounting300cellsandthepercentagesofearlyandlateapoptoticcells,andnecroticcellswerecalculated. QuantitativedataarepresentedinTable3. aHeraeuselementarvarioELbytheMicoanalyticalLaboratoryofthe cooled mixture of conc. AcOH (6mL) in DMF (6mL) was added DepartmentofChemistryandBiochemistry,LMU. (changeofcolourtobrown).Thesolutionwasallowedtoreachroom temperature,pouredintowaterandextractedthreetimeswithEtOAc. 3.2.Synthesisofligands3a–d The combined organic layers were washed three times with water, onetimewithbrine,thenasolutionofNaHCO andwateragain,then 3 driedwithNa SO .Afterevaporationthecrudeproductwaspurified A solution of tBuOK (24mmol, 2.69g) in 8mL DMF was cooled 2 4 (acetone/dry ice/−78°C) till it was nearly freezing. First a cooled bycolumnchromatographyonsilicagel(dichloromethane–pentane). solutionofaniline1a–d(8mmol)in4mLDMF(changeofcolourto 3.2.1.5-Chloro-N-(4-chlorophenyl)-2-nitrosoaniline(3a) light yellow or green), then a cooled solution of 1-chloro-4- Reagents: 1.02g (8.00mmol) 1a, 1.26g (8.00mmol) 2. Yield: nitrobenzene(2)(8mmol)in4mLDMFwasaddeddropwise(change ofcolourtopurple).Afterstirringatthistemperaturefor5–10mina 1.01g (3.78mmol, 47%), brown powder. — 1H NMR (400MHz, CDCl ):δ=6.99(dd,3J =8.7Hz,4J =1.7Hz,1H,H4),7.05(d, 3 H,H H,H 4J =1.9Hz,1H,H6),7.18–7.23(m,2H,H8+H12),7.39–7.44(m, H,H Table3 2H,H9+H11),8.65(brs,1H,H3),11.81(brs,1H,NH)ppm.13CNMR Dualstainingwithacridineorangeandethidiumbromideindicatesaninductionof (100MHz,CDCl ):δ=114.3(C6),119.3(C4),126.3(C8+C12),130.2 apoptosisbut notnecrosis after 2days oftreatment ofmelanoma A375cells with 3 indicatedconcentrationsoftestedcompounds. (C9+C11),132.5(C10),135.2(C7),141.2(br,C1+C3),145.0(C5), 155.2(C2)ppm.IR(KBr,cm−1):ν̃=2963(w),2925(w),2854(w), [µM] Earlyapoptosis Lateapoptosis Necrosis 1612(m),1592(s),1560(s),1504ν(N=O)(m),1489(m),1462(m), Control – 1 6 2 1338(m),1262(s),1154(vs),1106(vs),1092(vs),1012(m),942 6a 1 11 23 1 (m), 809 (s), 798 (vs), 560 (m). UV/Vis (CH Cl ): λ (ε)=253 2 17 73 1 2 2 max 6c 1 29 50 1 (13,300),278(17,100),312(14,900),461nm(8200M−1cm−1).MS 2 14 70 9 (DEI):m/z(%)=266.2(13)[M+],249.2(100)[M+–H–O],235.2(29) 7d 1 3 10 1 [M+–H–NO],231.2(16)[M+–Cl],214.2(4)[M+–Cl–OH],201.2(28) 2 26 54 4 [M+–Cl–NO],166.2(16)[M+–2Cl–NO].C H Cl N O(267.11gmol−1): 7b 1 3 11 1 12 8 2 2 calcd.C53.96,H3.02,N10.49;foundC53.88,H3.13,N10.38. 2 15 43 3 3c 2 2 2 5 3.2.2.5-Chloro-2-nitroso-N-p-tolylaniline(3b) Aminimumof300cellswascountedandallfourcellularstateswererecorded.Then, thepercentagesofearlyorlateapoptoticornecroticcellswerecalculated.Bolddata Reagents: 857mg (8.00mmol) 1b, 1.26g (8.00mmol) 2. Yield: pointsindicatesignificantdifferences(Pb0.05)fromthecontroldatapoints. 873mg (3.54mmol, 44%), dark green powder. Green crystals were S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 783 Fig.9.Theoximinerhenium(I)complexesinduceapoptosisbutnotnecrosisinleukemiacells.(A)DoublestainingofK562leukemiacellsfollowing3dayexposuretooximine rhenium(I)complexesattheindicatedconcentrations.ThemicroscopicfieldsobtainedforK562cellsexposedtoligands3a–careincluded.Quantitativedataarepresentedin Table4.(B)K562cellswerestainedwithDAPIfornuclearfragmentationandanalyzedbyfluorescencemicroscopy.Arrowsindicatesomeexamplesofcellswithfragmentednuclei. obtainedbyslowsublimationof3bat55°Cand1.0×10−3mbar.—1H 2H,H8+H12),7.21–7.27(m,2H,H9+H11),8.65(brs,1H,H3),12.06 NMR(270MHz,CDCl ):δ=2.39(s,3H,CH ),6.93(dd,3J =8.8Hz, (brs,1H,NH)ppm.13CNMR(100MHz,CDCl ):δ=21.2(CH ),114.6 3 3 H,H 3 3 4J =1.9Hz,1H,H4),7.05(d,4J =2.0Hz,1H,H6),7.10–7.16(m, (C6), 118.7 (C4), 125.1 (C8+C12), 130.6 (C9+C11), 133.7 (C7), H,H H,H 784 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 Table4 (267.11gmol−1):calcd.C53.96,H3.02,N10.49;foundC53.93,H Dualstainingwithacridineorangeandethidiumbromideindicatesaninductionof 2.90, N 10.36. apoptosis but not necrosis after 3days of treatment of K562 cells with indicated concentrationsoftesteddrugs. [µM] Earlyapoptosis Lateapoptosis Necrosis 3.2.4.5-Chloro-2-nitroso-N-phenylaniline(3d) Reagents: 745mg (8.00mmol) 1d, 1.26g (8.00mmol) 2. Yield: Control − 0.2 0.8 1.3 DMSO – 0.3 0.7 1.0 775mg (3.33mmol, 42%), dark green powder. Green crystals were 6a 4 2.4 1.8 1.0 obtainedbyslowsublimationof3dat55°Cand1.0×10−3mbar.—1H 8 45.0 51.6 0.5 NMR (270MHz, CDCl ): δ=6.96 (dd, 3J =8.8Hz, 4J =2.0Hz, 3 H,H H,H 6c 2 0.0 1.5 1.2 1H,H4),7.11(d,4J =2.0Hz,1H,H6),7.23–7.28(m,2H,H8+H12), 4 30.1 12.9 1.2 7.28–7.34(m,1H, H H ,H 10),7.40–7.49(m,2H,H9+H11),8.66(brs,1H, 8 42.5 49.5 1.0 7d 4 0.2 0.7 0.7 H3),12.02(brs,1H,NH)ppm.13CNMR(100MHz,CDCl 3 ):δ=114.5 8 2.4 2.5 0.5 (C6), 119.0 (C4), 125.1 (C8+C12), 127.0 (C10), 130.0 (C9+C11), 20 51.2 45.1 0.9 136.5(C7),141.4(br,C1+C3),144.9(C5),155.2(C2)ppm.IR(KBr, 7b 8 0.2 2.2 0.7 cm−1):ν̃=3084(w),3021(w),2924(w),1608(m),1587(s),1559 10 3.0 0.9 1.1 (vs), 1498 ν(N=O) (s), 1457 (m), 1354 (m), 1333 (m), 1160 (m), 20 58.3 39.1 0.2 3a 4 0.2 0.2 1.7 1153(m),1098(vs),1081(m),1073(m),941(m),796(m),534(s). 8 0.6 0.9 1.6 UV/Vis(CH Cl ):λ (ε)=250(12000),274(12400),312(12400), 2 2 max 3c 4 0.0 1.1 1.4 464nm (6800M−1cm−1). MS (DEI): m/z (%)=231.3 (9) [M+–H], 8 0.2 0.9 2.1 215.3(100)[M+–H–O],201.3(25)[M+–H–NO],167.3(23)[M+–H– 3d 4 0.2 0.2 1.6 8 0.1 0.8 1.0 NO–Cl].C 12 H 9 ClN 2 O(232.67gmol−1):calcd.C61.95,H3.90,N12.04; 20 0.1 1.3 2.0 foundC62.06,H3.88,N11.82. 3b 4 0.0 0.3 0.9 8 0.1 0.9 1.5 20 0.0 1.7 2.3 3.3.Synthesisofcomplexes6a–dand7a–d Aminimumof300cellswascountedandallfourcellularstateswererecorded.Then, thepercentagesofearlyorlateapoptoticornecroticcellswerecalculated.Bolddata Re(CO) X(X=Cl,Br)(4and5)wasdissolvedin20mLdryTHFand pointsindicatesignificantdifferences(Pb0.05)fromthecontroldatapoints. refluxed fo 5 r 20h. The elimination of two CO ligands leading to the intermediates 4′ and 5′ could be observed by liquid phase IR 137.1(C10),141.7(br,C1+C3),144.8(C5),155.2(C2)ppm.IR(KBr, spectroscopy in THF. The resulting pale-yellow mixture was added cm−1):ν̃=3084(w),3034(w),2913(w),1605(m),1554(s),1509 to a solution of one equivalent of ligand 3a–d in 10mL dry THF. ν(N=O)(s),1488(s),1436(m),1350(m),1333(s),1143(s),1100 Stirringatroomtemperatureresultedinachangeofcolourfromred (vs),1175(s),1018(m),945(m),938(m),813(m),800(s),509(m). browntodarkviolet.Fullconversiontothedesiredcomplexes6a–d UV/Vis(CH 2 Cl 2 ):λ max (ε)=249(12900),269(12100),312(12700), and7a–dwasagainmonitoredbyliquidphaseIRspectroscopyafter 467nm (6900M−1cm−1). MS (DEI): m/z (%)=245.3 (6) [M+–H], statedreactiontime.AfterevaporationofnearlyallTHF(rest2–3mL) 229.3(100)[M+–H–O],214.3(19)[M+–H–NO],180.3(17)[M+–H– 30–40mLofdryn-heptanewasaddedandsubsequentlythesolvent NO–Cl],166.3(5)[M+–H–NO–Cl–Me].C 13 H 11 ClN 2 O(246.69g mol−1): wasagainremovedtillcomplexes6a–dand7a–dprecipitateasdark calcd.C63.29,H4.49,N11.36;foundC63.39,H4.27,N11.32. green or purple solids. After filtration the solids were washed four timeswith5mLdryn-heptaneanddriedinvacuo. 3.2.3.5-Chloro-N-(2-chlorophenyl)-2-nitrosoaniline(3c) Reagents: 1.02g (8.00mmol) 1c, 1.26g (8.00mmol) 2. Yield: 812mg (3.04mmol, 38%), brown powder. — 1H NMR (270MHz, 3.3.1. Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-4-chlorophenyl)- CDCl ): δ=7.02 (dd, 3J =9.2Hz, 4J =2.0Hz, 1 H, H4), 7.02 oximine-N,N′}rhenium(I)(6a) 3 H,H H,H (d,4J =1.9Hz,1H,H6),7.24(ddd,3J =7.9Hz,3J =7.4Hz, Reagents: 135mg (0.373mmol) 4, 100mg (0.373mmol) 3a, H,H H,H H,H 4J =1.7Hz, 1H, H10), 7.34 (ddd, 3J =7.9Hz, 3J =7.4Hz, reaction time: 20h. Yield: 196mg (0.342mmol, 92%), dark green H,H H,H H,H 4J =1.6Hz, 1H, H11), 7.46 (ddd, 3J =8.0Hz, 4J =1.7Hz, powder.Darkredcrystalswereobtainedbyslowisothermicdiffusion H,H H,H H,H 5J =0.3Hz, 1H, H12), 7.52 (ddd, 3J =7.9Hz, 4J =1.6Hz, of n-pentane into a solution of 6a in dichloromethane. — 1H NMR H,H H,H H,H 5J =0.4Hz, 1 H, H9), 8.64 (br s, 1H, H3), 11.60 (br s, 1H, NH) (270MHz,CDCl ):δ=6.70(dd,4J =1.8Hz,5J =0.7Hz,1H,H6), H,H 3 H,H H,H ppm. 1H NMR (400MHz, CDCl , −60°C): δ=7.02 (d, 4J = 6.86(dd,3J =10.0Hz,4J =1.8Hz,1H,H4),6.99–7.10(m,1H,H8 3 H,H H,H H,H 1.8Hz, 1H, H6), 7.06 (dd, 3J =8.7Hz, 4J =1.8Hz, 1H, H4), or12),7.32–7.42(m,1H,H8or12),7.52(dd,3J =9.9Hz,5J = H,H H,H H,H H,H 7.25 (dd, 3J =7.6Hz,3J =7.6Hz, 1H, H10), 7.34 (dd, 3J = 0.7Hz,1H,H3),7.52(dd,3J =7.6Hz,4J =1.5Hz,2H,H9+H11), H,H H,H H,H H,H H,H 7.6Hz, 3J =7.6Hz, 1H, H11), 7.45 (d, 3J =7.8Hz, 1H, H12), 9.43(brs,1H,NOH)ppm.13CNMR(100MHz,CDCl ):δ=117.4(C6), H,H H,H 3 7.51 (d, 3J =7.9Hz, 1H, H9), 8.88 (d, 3J =8.7Hz, 1H, H3), 118.2(C3),123.6(C8or12),124.7(C8or12),130.1(C9or11),130.4 H,H H,H 12.03 (br s, 1H, NH) ppm. 13C NMR (68MHz, CDCl ): δ=114.5 (C9or11),131.1(C4),134.4(C10),143.6(C5),147.8(C7),155.3(C2), 3 (C6), 119.5 (C4), 126.2 (C12), 127.7 (C10), 127.9 (C11), 129.4 163.7(C1),176.8(CO),193.7(CO),194.5(CO)ppm.IR(KBr,cm−1): (C8),130.9(C9),134.4(C7),138.9(br,C1+C3),144.8(C5),155.4 ν̃=3098 (w), 3084 (w), 2033 ν(CO) (vs), 1950 ν(CO) (vs), 1930 (C2) ppm. 13C NMR (100MHz, CD Cl , −60°C): δ=113.6 (C6), ν(CO) (vs), 1604 ν(C=N) (w), 1553 ν(C=N) (w), 1483 (m), 1421 2 2 118.9 (C4), 125.8 (C12), 127.4 (C10), 127.5 (C11), 128.1 (C8), (m),1398(w),1303(m),1189(m),1161(w),1093(w),1054ν(N– 130.0 (C9), 130.8 (C1), 133.1 (C7), 142.2 (C3), 144.0 (C5), 154.6 O)(m),1045ν(N–O)(m),1013(w),931(w),829(w),803(w),586 (C2) ppm. IR (KBr, cm−1): ν̃=3086 (w), 2963 (w), 2923 (w), (w)543(w),518(w),444(w).IR(THF,cm−1):ν̃=2029ν(CO)(vs), 1605 (s), 1589 (s), 1581 (s), 1565 (vs), 1506 ν(N=O) (m), 1476 1952ν(CO)(m),1918ν(CO)(m).IR(CH Cl ,cm−1):ν̃=2033ν(CO) 2 2 (m), 1349 (m), 1339 (m), 1158 (s), 1105 (vs), 1079 (s), 946 (s), (vs),1954ν(CO)m),1926ν(CO)(m).UV/Vis(CH Cl ):λ (ε)= 2 2 max 804 (m), 750 (vs), 549 (m). UV/Vis (CH Cl ): λ (ε)=253 321(4500),471(4500),548nm(9700M−1cm−1).MS(FAB+):m/z 2 2 max (12000), 275 (15500), 312 (13400), 457nm (7200M−1cm−1). (%)=572.0 (76) [M+], 544.1 (50) [M+–CO], 537.1 (100) [M+–Cl], MS (DEI): m/z (%)=266.1 (6) [M+], 249.1 (26) [M+–H–O], 235.1 488.1 (78) [M+–3CO], 453.1 (78) [M+–3CO–Cl]. C H Cl N O Re 15 8 3 2 4 (14) [M+–H–NO], 231.2 (100) [M+−Cl], 216.2 (10) [M+–Cl–O], (572.80gmol−1):calcd.C31.45,H1.41,N4.89;foundC31.28,H1.39, 201.2 (13) [M+–Cl–NO], 166.2 (14) [M+–2Cl–NO]. C H Cl N O N4.72. 12 8 2 2 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 785 3.3.2. Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-p-tolyl)-oximine-N, 1H, H8 or 12), 7.34–7.40 (m, 1H, H8 or 12), 7.43 (dddd, 3J = H,H N′}rhenium(I)(6b) 7.5Hz, 3J =7.5Hz, 4J =1.2Hz, 4J =1.2Hz, 1H, H10), 7.50 H,H H,H H,H Reagents: 133mg (0.368mmol) 4, 91mg (0.368mmol) 3b, (dd, 3J =10.0Hz, 5J =0.5Hz, 1H, H3), 7.56 (ddd, 3J = H,H H,H H,H reaction time: 22h. Yield: 170mg (0.308mmol, 84%), dark green 7.5Hz,3J =7.5Hz, 4J =1.3Hz, 2H, H9+H11), 8.94 (br s, 1H, H,H H,H powder.Blackcrystalswereobtainedbyslowisothermicdiffusionof NOH)ppm.13CNMR(100MHz,CD Cl ):δ=117.8(C3),117.9(C6), 2 2 n-pentaneintoasolutionof6binchloroform.—1HNMR(270MHz, 122.2 (C8 or 12), 123.1 (C8 or 12), 128.4 (C10), 129.8 (C9), 129.8 CDCl ):δ=2.45(s,3H,CH ),6.75(dd,4J =1.8Hz,5J =0.7Hz, (C11),131.1(C4),143.1(C5),149.5(C7),155.5(C2),163.4(C1),177.6 3 3 H,H H,H 1H, H6), 6.84 (dd, 3J =9.9Hz, 4J =1.8Hz, 1H, H4), 6.93–7.05 (CO),194.6(CO),195.6(CO)ppm.IR(KBr,cm−1):ν̃=3054(w),3032 H,H H,H (m,1H,H8or12),7.29–7.35(m,3H,H9+H11+H8or12),7.49(dd, (w), 2935 (w), 2032 ν(CO) (vs), 1950 ν(CO) (vs), 1938 ν(CO) (vs), 3J =9.9Hz,5J =0.7Hz,1H,H3),9.09(brs,1H,NOH)ppm.13C 1606ν(C=N)(m),1590(w),1548ν(C=N)(w),1451(w),1421(m), H,H H,H NMR(100MHz,CDCl ):δ=21.4(CH ),117.9(C3),118.0(C6),122.0 1398 (w), 1294 (s), 1187 (m), 1056 ν(N–O) (s), 1047 ν(N–O) (s), 3 3 (C8or12),123.2(C8or12),130.3(C9or11),130.6(C9or11),131.3 940(w),856(w),802(m),703(m),626(w),587(w)544(w).IR(THF, (C4), 138.8 (C10), 142.7 (C5), 147.1 (C7), 155.3 (C2), 163.2 (C1), cm−1): ν̃=2029 ν(CO) (vs), 1951 ν(CO) (m), 1917 ν(CO) (m). IR 177.2 (CO), 193.9 (CO), 195.0 (CO) ppm. IR (KBr, cm−1): ν̃=3049 (CH Cl ,cm−1):ν̃=2032ν(CO)(vs),1952ν(CO)(s),1925ν(CO)(s). 2 2 (w), 3025 (w), 2927 (w), 2032 ν(CO) (vs), 1955 ν(CO) (vs), 1937 UV/Vis (CH Cl ): λ (ε)=321 (4900), 470 (4800), 544nm 2 2 max ν(CO) (vs), 1605 ν(C=N)(m), 1599 (m), 1548 ν(C=N) (m), 1501 (11200 M−1cm−1). MS (FAB+): m/z (%)=538.0 (72) [M+], 510.0 (m),1421(m),1397(m),1296(s),1189(m),1161(m),1054ν(N–O) (45)[M+–CO],503.0(100)[M+–Cl],454.0(72)[M+–3CO],419.0(68) (s),1048ν(N–O)(s),1017(w),931(w),803(m),588(w),543(w), [M+–Cl–3CO].C H Cl N O Re(538.36gmol−1):calcd.C33.46,H1.69, 15 9 2 2 4 456(w).IR(THF,cm−1):ν̃=2028ν(CO)(vs),1950ν(CO)(m),1916 N5.20;foundC33.69,H1.76,N5.02. ν(CO)(m).IR(CH Cl ,cm−1):ν̃=2031ν(CO)(vs),1951ν(CO)(s), 2 2 1924ν(CO)(m).UV/Vis(CH Cl ):λ (ε)=320(4500),474(5200), 3.3.5. Bromido-tricarbonyl-{4-chloro-o-quinone-(N-4-chlorophenyl)- 2 2 max 541nm(10500M−1cm−1).MS(FAB+):m/z(%)=552.1(71)[M+], oximine-N,N′}rhenium(I)(7a) 524.1(49)[M+–CO],517.1 (100)[M+–Cl],568.1.0(84)[M+–3CO], Reagents: 128mg (0.315mmol) 5, 84mg (0.315mmol) 3a, 433.1(78)[M+–Cl–3CO].C H Cl N O Re(552.38gmol−1):calcd.C reaction time: 22h. Yield: 169mg (0.274mmol, 87%), dark green 16 11 2 2 4 34.79,H2.01,N5.07;foundC35.05,H2.12,N5.15. powder.Browncrystalswereobtainedbyslowisothermicdiffusionof n-pentane into a solution of 7a in dichloromethane. — 1H NMR 3.3.3. Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-2-chlorophenyl)- (400MHz,CDCl ):δ=6.74(dd,4J =1.8Hz,5J =0.7Hz,1H,H6), 3 H,H H,H oximine-N,N′}rhenium(I)(6c) 6.89 (dd, 3J =9.9Hz, 4J =1.8Hz, 1H, H4), 7.04 (br d, 3J = H,H H,H H,H Reagents: 136mg (0.376mmol) 4, 100mg (0.376mmol) 3c, 7.6Hz,1H,H8or12),7.42(brd,3J =7.6Hz,1H,H8or12),7.52(dd, H,H reaction time: 28h. Yield: 194mg (0.339mmol, 90%), dark green 3J =7.6Hz,4J =1.3Hz,2H,H9+H11),7.55(dd,3J =9.9Hz, H,H H,H H,H powder. Brown crystals were obtained by slow isothermic diffu- 5J =0.7Hz,1H,H3),8.94(brs,1H,NOH)ppm.13CNMR(100MHz, H,H sion of n-pentane into a solution of 6c in dichloromethane. — 1H CDCl ):δ=117.3(C6),117.7(C3),123.5(C8or12),125.0(C8or12), 3 NMR(400MHz,CD Cl ):δ=6.57(dd,4J =1.8Hz,5J =0.7Hz, 130.1(C9or11),130.4(C9or11),130.9(C4),134.4(C10),143.1(C5), 2 2 H,H H,H 1H, H6), 6.91 (dd, 3J =10.0Hz, 4J =1.9Hz, 1H, H4), 7.39 147.9(C7),154.8(C2),163.0(C1),176.3(CO),192.9(CO),194.2(CO) H,H H,H (ddd, 3J =8.0Hz, 3J =7.3Hz,4J =1.9Hz, 1H, H10), 7.48 ppm.IR(KBr,cm−1):ν̃=3101(w),3084(w),2037ν(CO)(vs),1953 H,H H,H H,H (ddd, 3J =7.9Hz, 3J =7.2Hz,4J =1.4Hz, 1H, H11), 7.53 ν(CO) (vs), 1940 ν(CO) (vs), 1930 ν(CO) (vs), 1604 ν(C=N) (m), H,H H,H H,H (ddd, 3J =7.9Hz, 4J =1.9Hz,5J =0.4Hz, 1H, H12), 7.54 1544ν(C=N)(w),1483(m),1423(m),1264(s),1193(m),1153(w), H,H H,H H,H (dd, 3J =9.9Hz, 5J =0.7Hz, 1 H, H3), 7.60 (ddd, 3J = 1056ν(N―O)(s),1047ν(N―O)(m),1014(m),932(m),824(w), H,H H,H H,H 8.1Hz, 4J =1.4Hz,5J =0.4Hz, 1H, H9), 9.05 (br s, 1H, NOH) 798(w),585(w)516(m),443(w).IR(THF,cm−1):ν̃=2030ν(CO) H,H H,H ppm. 13C NMR (100MHz, CD Cl ): δ=117.6 (C6), 117.7 (C3), (vs),1954ν(CO)(m),1921ν(CO)(m).IR(CH Cl ,cm−1):ν̃=2034 2 2 2 2 125.2 (C12), 126.1 (C8), 128.3 (C11), 129.4 (C10), 130.5 (C9), ν(CO)(vs),1955ν(CO)(m),1928ν(CO)(m).UV/Vis(CH Cl ):λ 2 2 max 131.1 (C4), 143.9 (C5), 146.0 (C7), 154.7 (C2), 164.5 (C1), 177.2 (ε)=358 (6200), 475 (5400), 549nm (11000M−1cm−1). MS (CO), 194.2 (CO), 195.1 (CO) ppm. IR (KBr, cm−1): ν̃=3110 (w), (FAB+): m/z (%)=617.8 (86) [M+], 589.8 (47) [M+–CO], 537.0 3070 (w), 2031 ν(CO) (vs), 1960 ν(CO) (vs), 1936 ν(CO) (vs), (100) [M+–Br], 533.9 (81) [M+–3CO], 453.0 (73) [M+–3CO–Br]. 1605 ν(C=N) (m), 1548 ν(C=N) (w), 1469 (m), 1421 (m), 1401 C H BrCl N O Re(617.25gmol−1):calcd.C29.19,H1.31,N4.54; 15 8 2 2 4 (w), 1297 (m), 1191 (w), 1161 (w), 1055 ν(N―O) (m), 1046 found C 29.03, H 1.29, N 4.39. ν(N―O) (m), 935 (w), 800 (w), 763 (w), 740 (w) 623 (w), 588 (w),547(w),456(w).IR(THF,cm−1):ν̃=2029ν(CO)(vs),1953 3.3.6. Bromido-tricarbonyl-{4-chloro-o-quinone-(N-p-tolyl)-oximine-N, ν(CO) (m), 1924 ν(CO) (m). IR (CH Cl , cm−1): ν̃=2033 ν(CO) N′}rhenium(I)(7b) 2 2 (vs),1953ν(CO)(m),1932ν(CO)(m).UV/Vis(CH Cl ):λ (ε)= Reagents:129mg(0.318mmol)5,78mg(0.318mmol)3b,reaction 2 2 max 322 (6400), 438 (4500), 470 (4800), 555nm (12600M−1 cm−1). time:23h.Yield:167mg(0.280mmol,88%),darkgreenpowder.Dark MS(FAB+):m/z(%)=571.9(75)[M+],544.0(32)[M+–CO],537.1 redcrystalswereobtainedbyslowisothermicdiffusionofn-pentane (100) [M+–Cl], 509.1 (13) [M+–Cl–CO], 488.0 (26) [M+–3CO], intoasolutionof7bindichloromethane.—1HNMR(270MHz,CDCl ): 3 481.1(10)[M+–Cl–2CO],453.1(54)[M+–Cl–3CO],452.1(54)[M+– δ=2.45(s,3H,CH ),6.79(dd, 4J =1.8Hz,5J =0.7Hz,1H,H6), 3 H,H H,H Cl–3CO–H], 416.1 (18) [M+–2Cl–3CO–H]. C H Cl N O Re 6.87(dd,3J =9.9Hz,4J =1.8Hz,1H,H4),6.93–7.03(m,1H,H8 15 8 3 2 4 H,H H,H (572.80gmol−1):calcd.C31.45,H1.41,N4.89;foundC31.52,H or12),7.29–7.35(m,3H,H9+H11+H8or12),7.51(dd,3J =9.9Hz, H,H 1.51,N4.81. 5J =0.7Hz,1H,H3),8.31(brs,1H,NOH)ppm.13CNMR(100MHz, H,H CDCl ):δ=21.3(CH ),117.5(C3),117.7(C6),121.9(C8or12),123.4 3 3 3.3.4.Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-phenyl)-oximine-N, (C8or12),130.3(C9or11),130.6(C9or11),131.1(C4),138.8(C10), N′}rhenium(I)(6d) 142.2(C5),147.2(C7),154.8(C2),162.5(C1),176.6(CO),193.1(CO), ReagentsReagents:147mg(0.406mmol)4,94mg(0.406mmol) 194.5(CO)ppm.IR(KBr,cm−1):ν̃=3097(w),3089(w),3024(w), 3d,reactiontime:22h.Yield:192mg(0.357mmol,88%),darkgreen 2919(w),2034ν(CO)(vs),1957ν(CO)(vs),1951ν(CO)(vs),1939 powder.Redcrystalswereobtainedbyslowisothermicdiffusionofn- ν(CO)(vs),1925 ν(CO)(vs),1604 ν(C=N)(m), 1544ν(C=N)(w), pentane into a solution of 6d in dichloromethane. — 1H NMR 1502(m),1454(w),1423(m),1267(s),1240(m),1193(m),1153(m), (400MHz, CD Cl ): δ=6.75 (dd, 4J =1.7Hz, 5J =0.5Hz, 1H, 1055ν(N–O)(s),932(w),795(m),623(w)586(w),514(w).IR(THF, 2 2 H,H H,H H6),6.89(dd,3J =10.0Hz,4J =1.8Hz,1H,H4),7.10–7.16(m, cm−1): ν̃=2030 ν(CO) (vs), 1952 ν(CO) (m), 1919 ν(CO) (m). IR H,H H,H 786 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 (CH Cl ,cm−1):ν̃=2032ν(CO)(vs),1953ν(CO)(s),1926ν(CO)(s). 3.4.Cellcultureconditionsanddrugtreatment 2 2 UV/Vis (CH Cl ): λ (ε)=357 (5400), 481 (5500), 544nm 2 2 max (9900M−1cm−1). MS (FAB+): m/z (%)=596.0 (83) [M+], 568.0 Testedoximinerhenium(I)complexesandligandswerediluted (44)[M+–CO],517.2(100)[M+–Br],512.1.0(84)[M+–3CO],433.1 inDMSOandstoredat−20ºC.Workingsolutionswerepreparedin (75) [M+–Br–3CO]. C H BrClN O Re (596.83gmol−1): calcd. C RPMI1640mediumimmediatelybeforeuse.ThehumanK562cell 16 11 2 4 32.20,H1.86,N4.69;foundC32.53,H1.89,N4.57. line was a gift of Prof. Jean Claude D'Halluin (INSERM 125, Lille, France).ThisBcr–Abl-positivecelllinewasmaintainedasdescribed previously [79]. For experiments, K562 cells were seeded at the 3 ox .3 im .7. ine B - r N o , m N i ′ d }r o h - e tr n i i c u a m rb ( o I) ny (7 l- c { ) 4-chloro-o-quinone-(N-2-chlorophenyl)- density3.5×104mL−1,22hlaterthetestedrhenium(I)complexes orligandsatindicatedconcentrationswereaddedtocellscultured Reagents: 118mg (0.291mmol) 5, 78mg (0.291mmol) 3c, in suspension. A375, a human melanoma adherent cell line with reaction time: 22h. Yield: 154mg (0.250mmol, 86%), dark pur- high metastatic potential, derived from a 54year old female with ple powder.Blackcrystalswereobtainedbyslowisothermicdiffusion malignant melanoma (a gift of Prof. Piotr Laidler, Jagiellonian of n-pentane into a solution of 7c in dichloromethane. — 1H NMR University,Poland)wasmaintainedinRPMI1640mediumsupple- (270MHz,CDCl ):δ=6.54(dd,4J =1.8Hz,5J =0.7Hz,1H,H6), 3 H,H H,H mented with 10% FBS and antibiotics. For drug exposure experi- 6.89(dd,3J =9.9Hz,4J =1.8Hz,1H,H4),7.37(ddd,3J =7.9Hz, H,H H,H H,H ments, culture medium was substituted with fresh medium 3J =7.5Hz,4J =1.8Hz,1H,H10),7.46(ddd,3J =7.8Hz,3J = H,H H,H H,H H,H containing0.5%FBSandtestedcompoundsatindicatedconcentra- 7.5Hz,4J =1.5Hz,1H,H11),7.58(dd,3J =9.9Hz,5J =0.7Hz, H,H H,H H,H tions. Equivalent final concentration of DMSO was used in the 1H,H3),7.58(ddd,3J =7.9Hz,4J =1.5Hz,5J =0.4Hz,1H,H9), H,H H,H H,H controlcultures. 7.64 (ddd, 3J =7.8Hz, 4J =1.8Hz,5J =0.4Hz, 1H, H12), 9.50 H,H H,H H,H (brs,1H,NOH)ppm.13CNMR(100MHz,CDCl ):δ=117.3(C6),117.6 3 3.5.Proliferationandviabilityofcancercells (C3), 125.7 (C12), 126.1 (C8), 128.4 (C11), 129.5 (C10), 130.6 (C4), 130.6(C9),143.2(C5),146.2(C7),154.3(C2),163.8(C1),176.3(CO), 193.0(CO),194.2(CO)ppm.IR(KBr,cm−1):ν̃=3097(w),3086(w), 3.5.1.Leukemiacellviabilityandproliferationassay CellproliferationandviabilityweredeterminedbyusingTrypan 2025ν(CO)(vs),1956ν(CO)(vs),1947ν(CO)(vs),1934ν(CO)(vs), blue dye exclusion assay (Sigma-Aldrich, St. Louis, MO, USA). K562 1926ν(CO)(vs),1602ν(C=N)(m),1544ν(C=N)(m),1468(m),1453 cellswereseededatthedensity4×104mL−1,22hlatertheoximine (w),1425(m),1391(w),1278(s),1198(w),1149(w),1060ν(N–O) (s),1047ν(N–O)(m),948(w),935(w),806(m),625(w),586(w),518 rhenium(I)complexesorligandswereaddedatindicatedconcentra- (w),452(w).IR(THF,cm−1):ν̃=2031ν(CO)(vs),1954ν(CO)(m), tions.Non-treatedcellswerecultivatedascontrol.Proliferationrate 1926ν(CO)(m).IR(CH Cl ,cm−1):ν̃=2034ν(CO)(vs),1955ν(CO) or cell viability was evaluated as described previously [79]. Briefly, 2 2 treatmentwithdrugswascarriedoutupto3daysandaliquotswere (m), 1935 ν(CO) (m). UV/Vis (CH Cl ): λ (ε)=362 (6400), 441 (3800),474(4400),557nm(11200 2 M− 2 1cm m − a 1 x ).MS(FAB+):m/z(%)= removeddailyfordeterminationofdeadandviablecellnumberusing Trypanbluedyeexclusiontest.Inproliferationassay,onlyviablecells 617.9(92)[M+],589.8(33)[M+–CO],537.0(100)[M+–Br],534.0 were counted that did not take up Trypan blue. Comparison was (21) [M+–3CO],509.0(13)[M+–Br–CO],496.0(37)[M+–3CO–H–Cl], made relative to values obtained for the untreated control and 481.0(12)[M+–Br–2CO],453.0(58)[M+–Br–3CO],452.0(54)[M+–Br– expressedaspercentageofthecontrol.Inviabilityassay,thenumber 3CO–H], 417.1 (16) [M+–Br–3CO–H–Cl]. C H BrCl N O Re (617.25gmol−1):calcd.C29.19,H1.31,N4.54;fou 1 n 5 dC 8 29.2 2 6,H 2 1 4 .45, ofviablecellsnottakingupTrypanblueisexpressedaspercentage of the total cell number (viable and dead) in each experimental N4.55. condition.Themediumwasnotchangedduringtheinductionperiod. Each experiment was conducted in triplicate and repeated three 3.3.8.Bromido-tricarbonyl-{4-chloro-o-quinone-(N-phenyl)-oximine-N, times. N′}rhenium(I)(7d) Reagents: 141mg (0.347mmol) 5, 81mg (0.347mmol) 3d, 3.5.2.Melanomacellproliferationassay reaction time: 20h. Yield: 178mg (0.305mmol, 88%), dark purple Cell proliferation was measured by a colorimetric MTT (3-(4,5- powder.Blackcrystalswereobtainedbyslowisothermicdiffusionof Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. n-pentane into a solution of 7d in dichloromethane. — 1H NMR Briefly, cells were seeded in 24-well plates and allowed to adhere (270MHz,CDCl ):δ=6.75(dd,4J =1.8Hz,5J =0.7Hz,1H,H6), for6hinmediumcontaining10%fetalbovineserum(FBS).Next,they 3 H,H H,H 6.88 (dd, 3J =9.9Hz, 4J =1.8Hz, 1H, H4), 7.08 (br d, 3J were treated for 2days with tested compounds at indicated H,H H,H H, =7.2Hz, 1H, H8 or 12), 7.42 (dddd, 3J =7.3Hz, 3J =7.3Hz, concentrations in the culture medium containing 0.5% FBS. MTT H H,H H,H 4J =1.4Hz,4J =1.4Hz,1H,H10),7.43–7.48(m,1H,H8or12), reagent (thiazolyl blue tetrazolium bromide; Sigma-Aldrich; H,H H,H 7.50–7.58(m,2H,H9+H11),7.53(dd,3J =9.9Hz,5J =0.7Hz, 0.84mgmL−1in PBS)wasaddedandleftin theculturesfor3hat H,H H,H 1H, H3), 8.53 (br s, 1H, NOH) ppm. 13C NMR (100MHz, CDCl ): 37ºC prior to addition of 800μL solubilization reagent (DMSO in 3 δ=117.6(C3),117.6(C6),122.0(C8or12),123.4(C8or12),128.6 opticalgrade).ThereductionofatetrazoliumcomponentintoDMSO- (C10),129.8(C9or11),130.1(C9or11),131.0(C4),142.5(C5),149.6 solubleformazanproductwasmonitoredatawavelengthof540nm (C7),154.8(C2),162.7(C1),176.4(CO),193.0(CO),194.4(CO)ppm. using a spectrophotometer. The mean of the absolute absorbance IR(KBr,cm−1):ν̃=3106(w),3083(w),2038ν(CO)(vs),1959ν(CO) values given by drug-treated cells was divided by the mean of the (vs), 1925 ν(CO) (vs), 1908 ν(CO) (vs), 1597 ν(C=N) (m), 1545 absoluteabsorbanceofDMSO-treatedcontrolsampleandexpressed ν(C=N)(m),1451(m),1418(w),1392(m),1285(s),1184(w),1151 asrelativenumberofviableadherentcells.Datashowthemeanofat (w),1051ν(N–O)(s),1042ν(N–O)(m),938(w),803(m),763(w), least three independent experiments±SD. IC was calculated. For 50 703(m),623(w),579(w)540(w).IR(THF,cm−1):ν̃=2030ν(CO) time course, the number of viable, adherent melanoma cells was (vs),1953ν(CO)(m),1920ν(CO)(m).IR(CH Cl ,cm−1):ν̃=2033 estimated each day by MTT assay after incubation with tested 2 2 ν(CO)(vs),1954ν(CO)(m),1927ν(CO)(m).UV/Vis(CH Cl ):λ compoundsattheconcentrationof1.4µM. 2 2 max (ε)=358(5600),473(4700),546nm(9800M−1cm−1).MS(FAB+): m/z(%)=582.0(87)[M+],554.0(46)[M+–CO],503.1(100)[M+– 3.5.3.Flowcytometricanalysisofmelanomacellviability Br],498.1(74)[M+–3CO],419.2(67)[M+–Br–3CO].C H BrClN O Thecytotoxicityoftestedcompoundsonculturedmelanomacells 15 9 2 4- Re(582.81gmol−1):calcd.C30.91,H1.56,N4.81;foundC31.24,H was detected by propidium iodide (PI; Sigma) staining and FACS 1.64,N4.74. analysis. Cells were treated with tested drugs at indicated Table5 Crystaldataanddetailsofstructuralrefinementon3b,3dand6a–7d. 3b 3d 6a 6b 6c 6d 7a 7b 7c 7d Formula C13H11ClN2O C12H9ClN2O C15H8Cl3N2O4Re C16H11Cl2N2O4Re C16H10Cl5N2O4Re C15H9Cl2N2O4Re C15H8BrCl2N2O4Re C21H23BrClN2O4Re C16H10BrCl4N2O4Re C16H11BrCl3N2O4Re FW[gmol−1] 246.692 232.665 572.800 552.382 657.732 538.355 617.251 668.982 702.183 667.739 Temperature[K] 200(2) 200(2) 200(2) 200(2) 200(2) 200(2) 200(2) 200(2) 200(2) 200(2) Wavelength[Å] 0.71073 0.71073 0.71073 0.71073 0.71073 0.71073 0.71073 0.71073 0.71073 0.71073 Crystalsystem Monoclinic Monoclinic Triclinic Triclinic Triclinic Triclinic Triclinic Triclinic Triclinic Monoclinic Spacegroup P21/c P21/c P-1 P-1 P-1 P-1 P-1 P-1 P-1 C2/c a[Å] 9.957(2) 10.599(2) 8.862(2) 8.754(2) 9.273(2) 9.027(2) 6.769(2) 6.777(2) 9.344(2) 25.708(5) b[Å] 15.364(3) 6.658(2) 10.524(2) 10.732(2) 10.129(2) 9.796(2) 11.821(2) 11.949(2) 10.198(2) 10.095(2) c[Å] 8.390(2) 15.413(3) 11.074(2) 11.525(2) 11.110(2) 10.499(2) 12.065(2) 15.573(3) 11.318(2) 17.501(3) α(°) 90 90 71.76(3) 116.22(3) 86.19(3) 109.38(3) 93.42(3) 69.53(3) 86.08(3) 90 β(°) 112.38(3) 99.59(3) 66.85(3) 101.44(3) 85.36(3) 101.12(3) 105.14(3) 88.26(3) 85.79(3) 102.45(3) γ(°) 90 90 80.71(3) 101.02(3) 79.82(3) 99.30(3) 102.05(3) 77.92(3) 80.49(3) 90 V[Å3] 1186.9(5) 1072.4(4) 901.0(4) 902.9(5) 1022.2(4) 833.5(4) 904.7(3) 1153.9(5) 1059.1(4) 4435.1(15) Z 4 4 2 2 2 2 2 2 2 8 ρ calc.[gcm−3] 1.3806 1.4411 2.1114 2.0318 2.1370 2.1451 2.2659 1.9254 2.2019 2.0001 µ[mm−1] 0.305 0.333 7.210 7.048 6.623 7.631 9.242 7.141 8.152 7.665 F(000) 512 480 540 524 624 508 576 644 660 2512 Crystalsize[mm] 0.16×0.13×0.02 0.33×0.18×0.16 0.30×0.17×0.12 0.11×0.10×0.09 0.23×0.22×0.14 0.17×0.08×0.02 0.21×0.12×0.10 0.19×0.16×0.13 0.24×0.18×0.15 0.16×0.08×0.03 θrange[°] 3.45–25.37 3.90–26.34 3.88–26.35 3.28–27.52 3.17–27.56 3.70–25.50 3.20–26.41 3.73–26.35 3.70–26.33 3.16–27.00 Indexranges −11≤h≤11 −13≤h≤10 −11≤h≤11 −11≤h≤11 −12≤h≤12 −10≤h≤10 −8≤h≤8 −8≤h≤8 −10≤h≤11 −32≤h≤32 −18≤k≤17 −8≤k≤6 −13≤k≤13 −13≤k≤13 −13≤k≤12 −10≤k≤11 −14≤k≤14 −14≤k≤14 −12≤k≤12 −12≤k≤12 −10≤l≤10 −19≤l≤19 −13≤l≤13 −14≤l≤14 −14≤l≤14 −12≤l≤12 −15≤l≤15 −19≤l≤19 −13≤l≤14 −21≤l≤22 Reflnscollected 7202 4750 10164 19819 22333 5763 17978 9553 10588 40519 Independentreflns 2151 2166 3644 4122 4681 3068 3700 4655 4281 4832 Rint 0.0420 0.0284 0.0304 0.0389 0.0610 0.0587 0.0328 0.0282 0.0249 0.0474 Completeness to θ 99.0 99.0 99.3 99.4 99.2 99.0 99.5 98.9 99.2 99.6 [%] Refinement Full-matrix Full-matrix Full-matrix Full-matrix Full-matrix Full-matrix Full-matrix Full-matrix Full-matrix Full-matrix method least-squaresonF2 least-squaresonF2 least-squaresonF2 least-squaresonF2 least-squaresonF2 least-squaresonF2 least-squaresonF2 least-squaresonF2 least-squaresonF2 least-squaresonF2 Data/restraints/ 2151/0/159 2166/0/149 3644/0/227 4122/0/228 4681/2⁎/239 3068/0/219 3700/0/227 4655/0/226 4281/2⁎/239 4832/10⁎/264 parameters Goodness-of-fit 1.039 0.876 0.964 1.036 1.046 0.909 1.153 1.023 1.056 1.060 onF2 FinalRindices R1=0.0374 R1=0.0355 R1=0.0207 R1=0.0216 R1=0.0399 R1=0.0442 R1=0.0213 R1=0.0239 R1=0.0359 R1=0.0371 [IN2σ(I)] wR2=0.0872 wR2=0.0766 wR2=0.0389 wR2=0.0447 wR2=0.1021 wR2=0.0818 wR2=0.0478 wR2=0.0535 wR2=0.1001 wR2=0.0906 Rindices R1=0.0580 R1=0.0631 R1=0.0270 R1=0.0257 R1=0.0471 R1=0.0707 R1=0.0236 R1=0.0331 R1=0.0459 R1=0.0505 (alldata) wR2=0.0979 wR2=0.0813 wR2=0.0395 wR2=0.0458 wR2=0.1065 wR2=0.0876 wR2=0.0487 wR2=0.0567 wR2=0.1033 wR2=0.0993 Largest diff. peak/ 0.208and−0.218 0.176and−0.205 1.291and−0.710 0.805and−0.897 2.051and−2.413 2.348and−1.723 0.586and−0.851 1.328and−0.740 2.548and−1.803 1.672and−1.518 hole[eÅ−3] CCDCnumber 730545 730546 730547 730548 730549 730550 730551 730552 730553 730554 *Restraintsonlyusedforrefinementofsolventmolecules. S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 787 788 S.Wirthetal./JournalofInorganicBiochemistry104(2010)774–789 concentrationsfor2days.Aftertreatment,bothattached(harvested containthesupplementarycrystallographicdataforthispaper.These by trypsinization) and floating cells were collected, centrifuged at data can be obtained free of charge from The Cambridge Crystallo- 400×gfor5minandstainedwithPI(8μgmL−1)for10minatroom graphicDataCentreviahttp://www.ccdc.cam.ac.uk/data_request/cif. temperature in the dark. PI-positive cells were identified on a FACSCalibur (Becton Dickinson). 10,000 events were analyzed for 4.Conclusions eachsampleandresultswereprocessedbyusingCellQuestsoftware (BectonDickinson). Thepresentpaperhasreportedthesynthesisofthefirsto-quinoid oximine complexes. Two series of halogenido Re(I) compounds 3.6.Fluorescencemicroscopy (X=Cl,Br)havebeensynthesizedandsubjectedtofullspectroscopic characterisation to ensure a systematic approach to this new 3.6.1.Acridineorange/ethidiumbromidestaining configuration. Two ligands known from literature and two new Cell death was studied morphologically using fluorescent dyes: ligandshavebeenemployedforthisstudy.Structuresfromallnovel acridine orange (AO) and ethidium bromide (EB). Briefly, the complexes (6a–7d) and from two of the ligands (3b and 3d) have melanoma cells were cultured for 2days and leukemia cells for beenconfirmedbysinglecrystalX-raycrystallography,sothereisno 3dayswithorwithouttestedagentsatindicatedconcentrations.Cells doubt about the reported oximine character and the proton shift. (1×105)werecollectedbycentrifugationandresuspendedin20μLof Biologicalstudieshaverevealedthatthenewlysynthesizedoximine stainingsolutionmixtureof100µgmL−1ofEBand100µgmL−1of rhenium(I)complexescouldinduceapoptoticcelldeathinleukemia AO (1:1) (Sigma Chemical Co.). Then, they were examined by and melanoma cells, thus reducing proliferation of drug-treated ultraviolet fluorescence microscopy (Olympus BX 41). In each cancercells.Chloridocomplexes(6aand6c)weremoreefficientthan experiment,morethan300cellswereanalyzedandthenpercentages bromidocompounds(7dand 7b)in stimulating apoptosis. Noneof ofearly/lateapoptoticornecroticcellswerecalculated.Thecellswith thetestedligands(3a–d)showedanysignificantanticanceractivity. bright green chromatin with organized structure were counted as Assystematicinvestigationsinthisligandsystemjuststarted,work viable. 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