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Cytotoxic rhodium(III) and iridium(III) polypyridyl complexes: structure-activity relationships, antileukemic activity, and apoptosis induction.

PMID: 19101960
DOI:10.1002/cmdc.200800311 ACHTUNGTRENNUNG ACHTUNGTRENNUNG Cytotoxic Rhodium(III) and Iridium(III) Polypyridyl Complexes: Structure–Activity Relationships, Antileukemic Activity, and Apoptosis Induction Mara Dobroschke,[b] Yvonne Geldmacher,[a] Ingo Ott,[c] Melanie Harlos,[a] Lisa Kater,[b] Laura Wagner,[b] Ronald Gust,[c] William S. Sheldrick,*[a] and Aram Prokop[b] Meridional rhodiumACHTUNGTRENNUNG(III) polypyridyl complexes of the type mer- toxic than their facial IrIII polypyridyl counterparts: IC =20.3mm 50 [RhXACHTUNGTRENNUNG(DMSO)(pp)] (X=Cl, pp=phen 1, dpq 2, dppz 3; X=Br, for 7 and 4.6mm for fac-[IrClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 5 toward MCF-7 3 3 pp=phen 4) represent a promising class of potent cytostatic cells. The IC values for the complexes fac-[IrX(L)(pp)] 9–13 de- 50 3 agents for the treatment of lymphoma and leukemia. Exposure crease in the orders: a)Cl>Br for X and b)HO>DMSO for L. 2 of their DMSO solutions to light leads to slow isomerization to SpecificapoptoticcelldeathbyDNAfragmentationwasdetected mixtures of the mer and the generally less active fac isomers. As for leukemia (NALM-6) and lymphoma (BJAB) cells after incuba- a result, the IC values of 1 and 2 toward HT-29 cells increase tion with 2, 3, and 11 (X=Br, L=HO, pp=phen) for 72h. Loss 50 2 from 0.19 and 0.069mm on immediate use in the dark to 0.66 ofthemitochondrialmembranepotentialinlymphomacellsindi- and 0.312mm, respectively, after exposure of their DMSO stock cates that apoptosis is mediated via the intrinsic mitochondrial solutions to light for 7days. In striking contrast, the complexes pathway. LDH release assays after 1 or 3h demonstrate that ne- mer-[IrXACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] (X=Cl 7, Br 8) are significantly less cyto- croticdamageisnegligible. 3 Introduction Although the anticancer properties of dirhodiumACHTUNGTRENNUNG(II,II) carboxy- polypyridyl ligand: 1.9(cid:2)0.5, 0.19(cid:2)0.05, and 0.069(cid:2)0.021mm lates are well established,[1–3] relatively few investigations have against the HT-29 cell line respectively for pp=bpy, phen, beencarriedoutonthepossiblecytotoxicactivity ofrhodium- dpq.[10] A remarkable feature of these meridional complexes is ACHTUNGTRENNUNG(III) complexes. However, recent reports have suggested that their very high level of cellular uptake relative to other estab- octahedral chloridorhodiumACHTUNGTRENNUNG(III) complexes containing N-donor lished metallodrugs. For instance, mer-[RhClACHTUNGTRENNUNG(DMSO-kS)ACHTUNGTRENNUNG(dpq)] 3 ligands could offer considerable scope for the development of is accumulated some 102-fold in HT-29 cells (53.6ng Rh per anticancer agents. For instance, mer,cis-[RhClACHTUNGTRENNUNG(DMSO-kS)ACHTUNGTRENNUNG(NH)] mg cell protein) with respect to theexposure concentration of 3 2 3 exhibits remarkable cytotoxicity (IC =1.5(cid:2)0.4, 0.4(cid:2)0.2, and 1.0mm. In striking contrast, no significant cell uptake was ob- 50 9mm) toward the human cell lines A2780 (ovarian carcinoma), served for the same exposure concentration of the facial com- LoVo(colon carcinoma),andCalu (lung carcinoma),respective- plexes fac-[IrClACHTUNGTRENNUNG(DMSO-kS)(pp)] (pp=bpy, phen, dpq), which 3 ly.[4] High cytotoxic activity has also been established for fac- exhibit much lower IC values of >100, 4.6(cid:2)0.2, and 6.1(cid:2) 50 [RhCl([9]ane-NS)][5] ([9]ane-NS =1-aza-4,7-dithiacyclononane) 0.7mm toward HT-29 cells.[11] Although stable in aqueous and 3 2 2 and the terpyridine complexes[6] mer-[RhClACHTUNGTRENNUNG(tpy)] (tpy= methanol solutions, the facial iridiumACHTUNGTRENNUNG(III) complexes do rapidly 3 2,2’:6’,2’’-terpyridine) and [Rh(Im)ACHTUNGTRENNUNG(tpy)]Cl·3HO (Im=imida- isomerize to a mixture of fac and mer isomers in CHCl solu- 2 3 2 2 2 zole). tion on exposure to light. The meridional rhodiumACHTUNGTRENNUNG(III) com- We recently demonstrated that the cytotoxicities of organo- plexes mer-[RhClACHTUNGTRENNUNG(DMSO-kS)(pp)] are, in contrast, stable in 3 metallicRhIIIandIrIIIcomplexesofthetype[(h5-CMe)MCl(pp)]- CHCl solution but undergo photochemical isomerization in 5 5 2 2 ACHTUNGTRENNUNG(CFSO)(M=Rh, Ir)toward thehumancell linesMCF-7 (breast 3 3 cancer) and HT-29 (colon cancer) are directly correlated to the size of the polypyridyl ligand pp.[7,8] A similar dependence is [a] Y.Geldmacher,M.Harlos,Prof.Dr.W.S.Sheldrick observed for the analogous organoruthenium(II) complexes Lehrstuhlf(cid:2)rAnalytischeChemie Ruhr-Universit(cid:3)tBochum,44780Bochum(Germany) [(h6-CMe)RuCl(pp)]ACHTUNGTRENNUNG(CFSO).[9] These findings led us to investi- 6 6 3 3 Fax:(+49)234-3214420 gate the biological properties of the trichloridorhodiumACHTUNGTRENNUNG(III) E-mail:william.sheldrick@rub.de complexes mer-[RhCl 3 ACHTUNGTRENNUNG(DMSO-kS)(pp)] with the polypyridyl li- [b] M.Dobroschke,L.Kater,L.Wagner,Dr.A.Prokop gands pp=bpy, phen, dpq, dppz, dppn (bpy=2,2-bipyridine; DepartmentofPediatricOncology/Hematology phen=1,10-phenanthroline; dpq=dipyrido[3,2-f:2’,3’-h]qui- UniversityMedialCenterCharit(cid:4)Berlin,13353Berlin(Germany) [c] Dr.I.Ott,Prof.Dr.R.Gust noxaline; dppz=dipyrido[3,2-a:2’,3’-c]phenazine; dppn=ben- Institutf(cid:2)rPharmazie,FreieUniversit(cid:3)tBerlin zo[i]dipyrido[3,2-a:2’,3’-c]phenazine). Such complexes are ex- Kçnigin-Luise-Straße2–4,14195Berlin(Germany) tremely potent invitro cytotoxic agents and exhibit IC values 50 SupportinginformationforthisarticleisavailableontheWWWunder that are once again strongly dependent on the size of the http://dx.doi.org/10.1002/cmdc.200800311. ChemMedChem2009,4,177–187 (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim 177 MED W.S.Sheldricketal. polar solvents to an equilibrium mixture of fac and mer iso- DMSO solution relative to the signal of free DMSO at mers. 2.50ppm. Analogous low-field values in the range 3.75– Toestablishstructure–activityrelationships andto clarify the 3.84ppm were observed for the DMSO ligands in the nature of the active species, we have now studied the influ- trichloridorhodiumACHTUNGTRENNUNG(III)complexes1–3.[10] ence of a)the geometry (mer vs. fac), b)the monodentate Solutions of 4 in DMSO isomerize to mixtures of the mer ligand L, and c)the anionic ligandsXin complexes ofthe type and fac isomers on exposure to light. A mer/fac ratio of 69:31 mer- and fac-[MX(L)(pp)] (M=Rh, Ir; X=Cl, Br; pp=phen, is observed in DMSO solution after exposure to light for 3 dpq) toward the cell lines MCF-7 and HT-29. The cytotoxic ac- 15min and an equilibrium ratio of 38:62 after 5days. A tivity of the previously characterized highly cytotoxic com- marked upfield shift from two separate doublets at 10.21 and plexes mer-[RhClACHTUNGTRENNUNG(DMSO-kS)(pp)] (pp=dpq 2, dppz 3) as well 10.26ppm to a common doublet at 9.89ppm is recorded for 3 as fac-[IrBrACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] 11 and mer-[IrClACHTUNGTRENNUNG(tpy)] 14 was also the H2 and H9 protons of 4 on isomerization to the C sym- 3 2 3 s evaluated invitro toward Burkitt-like lymphoma cells (BJAB). metricalfacisomerinwhichtheprotonsH2/H9,H3/H8,H4/H7, Further experiments were performed for these active com- andH5/H6aremagneticallyequivalent.Slightlymorerapidiso- plexes to assess the relative importance of necrosis and apop- merization was reported for the trichlorido analogue[10] mer- tosis for the observed cell death and whether the intrinsic mi- [RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 1, for which a mer/fac ratio of 60:40 is 3 tochondrialpathwayisinvolved. present in DMSO after 15min irradiation with ambient light and a lower percentage of the mer isomer in the equilibrium ratioof30:70after5days(Figure2). Results and Discussion A slower isomerization is observed for DMSO solutions of the dpqand dppz complexes 2and 3 in the presence of light. Synthesisandstructure For instance, whereas a 10mm solution of the dpq complex 2 We previously reported[10] the synthesis of the rhodiumACHTUNGTRENNUNG(III) exhibits a mer/fac ratio of about 75:25 after 24h, this slowly polypyridyl complexes mer-[RhClACHTUNGTRENNUNG(DMSO-kS)(pp)] (pp=phen, reverses to an equilibrium mixture at about 39:61 after 5days. 3 dpq, dppz, 1–3) (Figure1) as well as the analogous bpy and Valuesof63:37and50:50areobservedforthemer/facratioin a 2.5mm solution of the less soluble dppz complex 3 after 24h and 5days, respectively. Attempts to prepare facial trichloridorhodiumACHTUNGTRENNUNG(III) complexes of the type fac-[RhClACHTUNGTRENNUNG(DMSO- 3 kS)(pp)] in a manner similar to that employed for the analo- gous iridiumACHTUNGTRENNUNG(III) complexes[11] by stepwise reaction of RhCl·3HO with the appropriate polypyridyl ligand and DMSO 3 2 in aqueous or methanol solution in the dark led invariably to mixtures of the fac and mer isomers. The observed mer/fac ratios for the resulting mixtures in DMSO solution were similar to those recorded after irradiation of the mer complexes by daylight for 5days. Mixtures of fac and mer isomers were also obtained for the aqua precursors of the type [RhCl- Figure1.MeridionalrhodiumACHTUNGTRENNUNG(III)complexesmer-[RhXACHTUNGTRENNUNG(DMSO)(pp)]. 3 3 ACHTUNGTRENNUNG(HO)(pp)].[14] 2 Meridional iridiumACHTUNGTRENNUNG(III) complexes of the type mer-[IrX- 3 dppn derivatives by treatment of the precursor mer,cis-[RhCl- ACHTUNGTRENNUNG(DMSO-kS)ACHTUNGTRENNUNG(phen)] (X=Cl, 7; X=Br, 8) (Figure3) were prepared 3 ACHTUNGTRENNUNG(DMSO-kS)ACHTUNGTRENNUNG(DMSO-kO)][12,13] with an equivalent of the appropri- inananalogousmannertotheirrhodiumACHTUNGTRENNUNG(III)counterparts,and ate polypyridyl ligand in a solution of CHOH/HO (1:1 v/v). A facial iridiumACHTUNGTRENNUNG(III) complexes of the types fac-[IrClACHTUNGTRENNUNG(HO)(pp)] 3 2 3 2 similar strategy was employed to synthesize the (pp=phen, 9; pp=dpq, 10) and fac-[IrBr(L)ACHTUNGTRENNUNG(phen)] (L=HO, 3 2 tribromorhodiumACHTUNGTRENNUNG(III) complex mer-[RhBrACHTUNGTRENNUNG(DMSO-kS)ACHTUNGTRENNUNG(phen)] 4 11; L=1-MeIm, 1-methylimidazole, 12; L=1-MeBIm, 1-methyl- 3 reported herein (Figure1), in which the intermediate complex benzimidazole,13)(Figure4)byreactionofIrCl·3HOwiththe 3 2 [RhBrACHTUNGTRENNUNG(DMSO)] was produced by reaction of RhBr·3HO with appropriatepolypyridylligandinaqueoussolutionfollowedby 3 3 3 2 three equivalents of DMSO and subsequently treated without the ligand L where necessary (for 12 and 13). The respective further characterization with the appropriate polypyridyl meridional and facial ligand arrangements were confirmed by ligand. This phen complex was chosen to study the possible the 1HNMR spectra of the complexes in the dark. Figure5 de- influence of the anionic ligand X on the cytotoxicity of the pictsthearomaticregionofthe1HNMRspectrumofmer-[IrBr- 3 meridionalrhodiumACHTUNGTRENNUNG(III)polypyridylcompounds.Themeridional ACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 8, which together with its trichlorido counter- structure of 4 was confirmed by the significant downfield part 7 is indefinitely stable in CDCl solution in the dark. Very 3 shifts of protons H7 and H9 (0.05–0.07ppm in DMSO solution) slow isomerization to a mixture of mer and fac isomers is ob- of the pyridine ring situated trans to the kS DMSO ligand in servedfor7and8inDMSOsolutionwithexposuretoambient comparison with H2 and H4 of the pyridine ring in trans posi- light.After24h,respectivemer/facratiosof91:9and89:11are tion to a bromide ligand. Confirmation of the kS coordination recorded, which decrease to 83:17 and 81:19 after 5days. The of the DMSO ligand was provided by the pronounced down- preparation of both “mer” and “fac” complexes of the type field shift of its methyl 1HNMR resonance at 3.95ppm in [IrXACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] (X=Cl, Br) by solvent removal from aqueous 3 2 178 www.chemmedchem.org (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2009,4,177–187 CytotoxicRhodiumACHTUNGTRENNUNG(III)andIridiumACHTUNGTRENNUNG(III)PolypyridylComplexes Figure2.Aromaticregionofthe1HNMRspectrumofmer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)]1inDMSOafterexposuretoambientlightfor5days. 3 solutions of the [IrXACHTUNGTRENNUNG(phen)](cid:3) anions 4 with hydronium and ammonium counter-cations, respectively, was previously reported,[15] but the as- signments were based solely on IR dataandremaintentative. As reported previously for the complexes fac-[IrClACHTUNGTRENNUNG(DMSO)(pp)] Figure3.Meridional 3 (pp=bpy,phen,dpq,dppz,dppn),[11] iridiumACHTUNGTRENNUNG(III)complexesmer- [IrXACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)]. the facial iridiumACHTUNGTRENNUNG(III) complexes 9–13 3 are stable in DO or CDOD solution 2 3 in the presence of light, but slowly Figure4.FacialiridiumACHTUNGTRENNUNG(III)complexesfac-[IrX(L)(pp)]. isomerize to fac/mer mixtures in DMSO solution. Although we 3 were unsuccessful in obtaining single crystals of these com- pounds suitable for X-ray analysis, the facial arrangement and 15 are isostructural[17,18] and crystallize in the monoclinic couldbeconfirmedforfac-[IrClACHTUNGTRENNUNG(CHCN-kN)ACHTUNGTRENNUNG(phen)]9a.Suitable space group P2/n, in contrast to the triclinic compound mer- 3 3 1 crystals of this compound, the molecular structure of which is [RhClACHTUNGTRENNUNG(tpy)]·DMSO.[6] The molecular structure of 14 is depicted 3 depicted in Figure6, were isolated by slow evaporation of a in Figure7 and shows that the tridentate coordination mode solution of fac-[IrClACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] 9 in acetonitrile.[16] The forma- ofthetpyligandleadstosignificantdistortionsinthebasically 3 2 tionof9aisinaccordancewiththepreviouslyreported ability octahedral environment of the iridium atom Ir1. Whereas the of the facial trichloridoiridiumACHTUNGTRENNUNG(III) complexes to replace their bond angles of Cl1(cid:3)Ir1(cid:3)Cl2 and N2(cid:3)Ir1(cid:3)Cl3 (respectively neutral monodentate ligand L by softer alternatives, for exam- 179.51(3) and 177.34(8)8) are both close to the ideal angle of ple, DMSO by N-acetylmethio- nine in the case of fac-[IrCl- 3 ACHTUNGTRENNUNG(DMSO-kS)ACHTUNGTRENNUNG(dppz)].[11] The novel meridional iridium- ACHTUNGTRENNUNG(III) complexes mer-[IrXACHTUNGTRENNUNG(tpy)] 3 (X=Cl, 14; X=Br, 15) were pre- pared by reaction of IrX·3HO 3 2 with 2,2’:6’,2’’-terpyridine in methanol and were included in the investigations for compari- Figure5.Aromaticregionofthe1HNMRspectrumofmer-[IrBrACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)]8inCDCl inthedarkafter 3 3 son purposes. Compounds 14 3weeks. ChemMedChem2009,4,177–187 (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 179 MED W.S.Sheldricketal. 1808,thatofN1(cid:3)Ir1(cid:3)N3ismuchsmaller(161.34(12)8)owingto the participation of Ir1 in two strained five-membered chelate rings. The central Ir1(cid:3)N2 bond length of 1.927(3)(cid:3) is signifi- cantlyshorterthantheIr1(cid:3)N1andIr1(cid:3)N3distancesof2.044(3) and 2.049(3)(cid:3), and this leads to a lengthening of the trans sited Ir1(cid:3)Cl3 bond to 2.370(1) relative to the other Ir1(cid:3)Cl dis- tancesof2.356(1)(Ir1(cid:3)Cl1)and2.347(1)(cid:3)(Ir1(cid:3)Cl2). Cytotoxicityandstructure–activityrelationships Table1 lists the invitro cytotoxicity of the rhodiumACHTUNGTRENNUNG(III) com- plexes 1–4 against the human cancer cell lines MCF-7 and HT- 29afterrespectiveincubationperiodsof96and72h.Equilibri- um mixtures fac/mer-[RhClACHTUNGTRENNUNG(DMSO)(pp)] (pp=phen, 1a; dpq 3 2a; dppz 3a) predominantly containing the fac isomer were Figure6.Molecularstructureoffac-[IrClACHTUNGTRENNUNG(CHCN)ACHTUNGTRENNUNG(phen)]9a.Selectedbond obtained by exposing DMSO solutions of 1–3 to light for 3 3 distances((cid:3)):Ir1(cid:3)N12.042(4),Ir1(cid:3)N22.048(3),Ir1(cid:3)N32.005(4),Ir1(cid:3)Cl1 7days. The marked increases in the IC values for the irradiat- 50 2.352(1),Ir1(cid:3)Cl22.358(1),Ir1(cid:3)Cl32.340(1). ed DMSO solutions of 1a and 2a suggest that the fac isomers must exhibit a significantly lower cytotoxicity than their mer counterparts. An analogous, yet less pronounced, decrease in activity is also observed for the DMSO solutions of mer-[RhCl- 3 ACHTUNGTRENNUNG(DMSO)(pp)] (pp=bpy, dppn) after light exposure for a similar periodoftime.Surprisingly,nosignificantchangeincytotoxici- ty is detected for the equilibrium mixture fac/mer-[RhCl- 3 ACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dppz)] 3a in comparison with mer-[RhClACHTUNGTRENNUNG(DMSO)- 3 ACHTUNGTRENNUNG(dppz)] alone. The rate of the mer!fac isomerization and the value of the fac/mer ratio after 7days are somewhat lower for thetribromido complex4relative toits trichlorido analogue1. Therefore,itcanbepostulatedthattheincreasedkineticstabil- ity of the more active meridional isomer could be responsible for the slightly lower IC values of 0.203 and 0.091mm for 4 50 toward MCF-7 and HT-29 cells relative to those of 0.40 and 0.19mm recorded for 1. Isomerization of the trihalogenido complexes mer-[RhClACHTUNGTRENNUNG(DMSO-kS)(pp)] in DMSO can, in princi- 3 ple, proceed by either a multistep dissociation/coordination Figure7.Molecularstructureofmer-[IrClACHTUNGTRENNUNG(tpy)]14.Selectedbonddistances 3 process or by a concerted interchange pathway involving a 7- ((cid:3)):Ir1(cid:3)N12.044(3),Ir1(cid:3)N21.927(3),Ir(cid:3)N32.049(3),Ir1(cid:3)Cl12.356(1),Ir1(cid:3)Cl2 coordinated transition state. Second-order kinetics[19,20] have 2.347(1),Ir1(cid:3)Cl32.370(1). been reported for both water substitution in [(h5-CMe)Ir- 5 5 ACHTUNGTRENNUNG(phen)ACHTUNGTRENNUNG(HO)]2+ andchloridesub- 2 stitution in [(h6-CH)RuCl(en)]+. 6 6 Table1. InhibitoryactivityofrhodiumACHTUNGTRENNUNG(III)polypyridylcomplexestowardthehumancelllinesMCF-7andHT- Whereas activation parameters 29. supportaninterchangedissocia- tive pathway (I ) in the former IC [mm][a] d 50 case, density functional calcula- Complex MCF-7 HT-29 tions suggest that the inter- mer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(bpy)][10] 4.0(cid:2)0.5 1.9(cid:2)0.5 3 changepathwaywouldbemore 1[10] mer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 0.40(cid:2)0.06 0.19(cid:2)0.05 2[10] mer-[RhCl 3 ACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dpq)] 0.079(cid:2)0.012 0.069(cid:2)0.021 associative in the latter case. 3 3[10] mer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dppz)] 0.095(cid:2)0.020 0.073(cid:2)0.017 This shift in the I $I mechanis- 3 d a mer-[RhCl 3 ACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dppn)][10] 0.051(cid:2)0.012 0.070(cid:2)0.008 tic continuum is in accordance 4 mer-[RhBrACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 0.203(cid:2)0.033 0.091(cid:2)0.011 3 withthemarkeddecreaseinthe fac/mer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(bpy)] 5.1(cid:2)1.3 4.0(cid:2)1.1 1a fac/mer-ACHTUNGTRENNUNG[RhCl 3 3 ACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 1.1(cid:2)0.2 0.66(cid:2)0.02 transeffectoftheh6-C 6 H 6 ligand 2a fac/mer-ACHTUNGTRENNUNG[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dpq)] 0.474(cid:2)0.030 0.312(cid:2)0.032 in comparison with [h5-CMe](cid:3). 3 5 5 3a fac/mer-[RhCl 3 ACHTUNGTRENNUNG(DMO)ACHTUNGTRENNUNG(dppz)] 0.093(cid:2)0.05 0.057(cid:2)0.021 In view of the low trans effects fac/mer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dppn)] 0.15(cid:2)0.05 0.21(cid:2)0.04 3 of the halogenide and poly- [a]Values for 1–4 and mer-[RhClACHTUNGTRENNUNG(DMSO)(pp)] (pp=bpy, dppn)[10] are for freshly prepared DMSO solutions in pyridyl Ndonor ligands it seems 3 thedark,thosefor1a–3aandfac/mer-[RhCl 3 ACHTUNGTRENNUNG(DMSO)(pp)](pp=bpy,dppn)areforDMSOsolutionsofthecom- probablethatamoreassociative poundsafterambientlightirradiationfor7days. interchange pathway will also 180 www.chemmedchem.org (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2009,4,177–187 CytotoxicRhodiumACHTUNGTRENNUNG(III)andIridiumACHTUNGTRENNUNG(III)PolypyridylComplexes beadoptedbythecomplexesmer-[RhXACHTUNGTRENNUNG(DMSO)(pp)].Thepres- (HO<DMSO, Cl<Br) may be related to the extent of cellular 3 2 ence of the larger bromide ligands in 4 will increase steric uptake. crowding in the required transition state and could, therefore, Theinhibitionofcellproliferationbycomplexes2,3,11,and lead to the observed slowing in the rate of mer!fac isomeri- 14 was also evaluated invitro in BJAB cells (Burkitt-like lym- zation. phomacells).[21]Afteranincubationperiodof24h,theviability In striking contrast to the rhodiumACHTUNGTRENNUNG(III) complexes, facial and cell count were measured with a CASY Cell Counter and iridiumACHTUNGTRENNUNG(III) isomers are much more cytotoxic than their meri- Analyzer System, with the settings specifically defined for the dionalcounterparts7and8(Table2),whoseratherlowactivity requirements of the employed cells. The dose-dependent de- (16.8–45.9mm) precludes further discussion of their individual crease in cell proliferation is depicted for the highly potent structure–activity relationship. The IC values for fac-[IrCl- rhodiumACHTUNGTRENNUNG(III) complexes 2 and 3 in Figure8. ID values for 50 3 50 ACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] toward MCF-7 and HT-29 cells are respectively these complexes and the iridiumACHTUNGTRENNUNG(III) complexes 11 and 14 are 4.4- and 3.7-fold lower than for mer-[IrClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)]. listed in Table3. It is apparent that the meridional complexes 3 Whereas the latter isomer is some 51-fold less active toward 2, 3, and 14 are all effective at low-micromolar concentrations MCF-7 cells and 187 times less active toward HT-29 than mer- in inhibiting proliferation of the lymphoma cells (ID values: 50 [RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)], the facial IrIII and RhIII isomers appear to 0.4–0.8mm). The facial iridiumACHTUNGTRENNUNG(III) complex 11 is also effective 3 exhibit rather similar cytotoxicities. IC values of 4.6(cid:2)0.5 butatasignificantlyhigherdoselevel(ID =5mm). 50 50 (MCF-7) and 4.6(cid:2)0.2mm (HT-29) were measured for fac-[IrCl- 3 ACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)]and1.1(cid:2)0.2and0.66(cid:2)0.02mmfortheirradiat- Apoptosisinduction ed mixture fac/mer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 2a containing a fac/ 3 mer ratio of about 70:30 on the basis of 1HNMR studies. Two Necrotic cell death is characterized by the early release of lac- interesting structure–activity relationships can be established tate dehydrogenase (LDH), whereas apoptotic cells initially forthefacialiridiumACHTUNGTRENNUNG(III)complexesontakingtheIC valuesfor retain their membrane integrity and do not exhibit rapid re- 50 the compound pairs fac-[IrCl(L)ACHTUNGTRENNUNG(phen)] (L=DMSO, 5; HO, 9) lease of large intracellular proteins such as LDH. Figure9a de- 3 2 and fac-[IrXACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] (X=Cl, 9; X=Br, 11) into account. picts the LDH release established for BJAB cells after 1h incu- 3 2 Firstly, the DMSO complex is significantly more cytotoxic bationwithvariousconcentrationsoffac-[IrBrACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)](11) 3 2 (IC =4.6(cid:2)0.5 and 4.6(cid:2)0.2mm for MCF-7 and HT-29 cells) and mer-[IrClACHTUNGTRENNUNG(tpy)] (14). These results clearly indicate that the 50 3 than its aqua counterpart 9 (IC =14.8(cid:2)1.4, 12.6(cid:2)1.9mm) meridional tpy complex causes considerable unspecific 50 and secondly, a similar increase in activity is observed for the damagewithinashortperiodoftime,andisthereforeunsuita- aqua complexes 9 and 11 on going from X=Cl (9) to X=Br ble as an anticancer agent. In contrast, the facial complex 11 (11; IC =5.5(cid:2)0.3, 4.3(cid:2)0.2mm). The complexes fac-[IrBr(L)- has no significant unspecific cytotoxic effects on BJAB cells 50 3 ACHTUNGTRENNUNG(phen)] (L=1-MeIm 12, L=1-MeBIm 13) exhibit IC values in eventhoughtheconcentrationsused(2–10mm)wereanorder 50 the range 6.9–8.7mm toward MCF-7 and HT-29 cells and are ofmagnitudehigherthanfor14.Thiswasalsothecaseforthe therefore slightly less active than the aqua complex 11. The highly active rhodiumACHTUNGTRENNUNG(III) complexes mer-[RhClACHTUNGTRENNUNG(DMSO-kS)(pp)] 3 markedly lower activity of fac-[IrClACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] 9 relative to 2 and 3, for which concentrations in the range 0.4–1.0mm 3 2 theanalogousDMSOcomplex5indicatesthatanintermediate were employed for an incubation period of 3h (Figure9b). hydrolysis step may not be involved in the mechanism of These results indicate that necrosis does not have a significant actionof5.Indeed,no1HNMRevidenceforDMSO/HOsubsti- impactonthepotencyofthecomplexes2,3,and11. 2 tution was recorded for an aqueous solution of 5 over a Apoptosis, in contrast to unspecific necrosis, requires a con- period of 24h.It is possible that thedifferences in cytotoxicity trolled and regulated mechanism leading to cell death. DNA fragmentation (hypoploidy) is considered to be a typical effect Table2. InhibitoryactivityofiridiumACHTUNGTRENNUNG(III)polypyridylcomplexestowardthehumancelllinesMCF-7andHT-29. of apoptotic cell death, and we therefore quantified the induc- IC [mm][a] 50 tion of apoptosis for 2, 3, 11, Complex MCF-7 HT-29 and 14 by flow cytometric 5[11] fac-[IrClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 4.6(cid:2)0.5 4.6(cid:2)0.2 3 measurements of the DNA frag- 6[11] fac-[IrClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dpq)] 5.5(cid:2)0.9 6.1(cid:2)0.7 7 mer-[IrC 3 lACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 20.3(cid:2)0.5 16.8(cid:2)0.1 ments after incubating lympho- 3 8 mer-[IrBrACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] 32.3(cid:2)2.7 45.9(cid:2)5.4 ma cells (BJAB) and other cell 3 9 fac-[IrCl 3 ACHTUNGTRENNUNG(H 2 O)ACHTUNGTRENNUNG(phen)] 14.8(cid:2)1.4 12.6(cid:2)1.9 lines for 72h with the com- 10 fac-ACHTUNGTRENNUNG[IrCl 3 ACHTUNGTRENNUNG(H 2 O)ACHTUNGTRENNUNG(dpq)] 11.3(cid:2)0.1 10.6(cid:2)0.6 plexes.[22] The amounts of apop- 11 fac-ACHTUNGTRENNUNG[IrBrACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] 5.5(cid:2)0.3[b] 4.3(cid:2)0.2[b] 12 fac-ACHTUNGTRENNUNG[IrBr 3 ACHTUNGTRENNUNG(1- 2 MeIm)ACHTUNGTRENNUNG(phen)] 8.6(cid:2)2.2 8.7(cid:2)0.6 totic NALM-6 cells for various 3 13 fac-[IrBrACHTUNGTRENNUNG(1-MeBIm)ACHTUNGTRENNUNG(phen)] 8.4(cid:2)0.6[b] 6.9(cid:2)1.2[b] concentrations of 2 and 3 are il- 3 14 mer-[IrCl 3 ACHTUNGTRENNUNG(tpy)] 0.32(cid:2)0.07 0.13(cid:2)0.02 lustrated in Figure10a. Exten- 15 mer-[IrBrACHTUNGTRENNUNG(tpy)] 0.33(cid:2)0.02 0.26(cid:2)0.06 3 sive DNA fragmentation is ob- [a]Valuesfor5–8and14–15areforfreshlypreparedDMSOsolutions,thosefor9–13areforfreshlyprepared served even at very low concen- DMFsolutions.[b]Valuesof4.5(cid:2)0.3and3.9(cid:2)1.0mmwereobtainedforstocksolutionsof11inDMSOand7.3 trations (0.3, 0.8mm) used for and9.3mmforstocksolutionsof13inDMSO(MCF-7andHT-29cells,respectively). the cytotoxic complexes. The in- ChemMedChem2009,4,177–187 (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 181 MED W.S.Sheldricketal. mented. The cells appear to have undergone apoptosis by shrinkingandfragmentation. Acutelymphoblasticleukemia (ALL) is the most common ma- lignant disease in childhood. To determine whether apoptosis induction can also be found in primary human cells, we incu- bated 2 and 3 with leukemia cells taken from a patient with relapsed childhood ALL. The isolated primary lymphoblasts were treated with 2 and 3 at the ID concentrations estab- 50 lished for BJAB cells and with the cytostatic drugs daunorubi- cin, doxorubicin, and vincristine. As can be gauged from Figure11, complexes 2 and 3 appear to exhibit superior apoptosis induction relative to these standard drugs for the treatmentofchildhoodALL. Our investigations also clearly demonstrate that complexes 2 and 3 trigger the mitochondrial pathway of apoptosis. As illus- trated in Figure12a, dose-de- pendent loss of the mitochon- Figure8.InhibitionofcellproliferationinBurkitt-likelymphomacellsaftertreatmentwithcomplexesa)2and b)3for24hasmeasuredbyaCASYcellcounter(control=untreatedcells).N=numberofcellsinunitsof drial membrane potential was 105cellsmL(cid:3)1 (cid:2)ESD(n=3);I=inhibitionofcellproliferationwithvaluesgivenaspercentofcontrolvalues; observed for BJAB cells after 1(cid:4)105BJABcellsnormallygrowupto2.5(cid:4)105cellsmL(cid:3)1in24hintheabsenceofproliferationinhibitors. 48h incubation with the meri- dional rhodiumACHTUNGTRENNUNG(III) compounds. This was also the case for the iridiumACHTUNGTRENNUNG(III)complexes 11and14 (Figure12b). After staining the cells with the dye JC-1 Table3. Biologicaldataforcomplexes2,3,11,and14inBJABcells. (5,5’,6,6’-tetrachloro-1,1,3,3’-tetraethylbenzimidazolylcarbocya- Compd ID [mm][a] AC [mM][b] c[mm][c] nine iodide), mitochondrial permeability was quantified by 50 50 flowcytometricdeterminationofthecellswithdecreasedfluo- 2 0.8 1.2 0.8 rescence, that is, with mitochondria displaying a lower mem- 3 0.4 1.0 0.6 11 5.0 40.0 25.0 branepotential. 14 0.5 0.6 4.0 [a]Inhibitionofproliferationafter24h.[b]Apoptosisinductionafter75h. Conclusions [c]Dissipation of mitochondrial membrane potential (Dy ), reported as m thecompoundconcentrationgiving50%cellswithlowDy m after48h. The meridional rhodiumACHTUNGTRENNUNG(III) polypyridyl complexes of the type mer-[RhXACHTUNGTRENNUNG(DMSO)(pp)] (X=Cl, Br) represent a highly promising 3 class of potent cytostatic agents. In extension of our previous invitro studies on breast cancer (MCF-7) and colon carcinoma duction of apoptosis in BJAB cells was also confirmed for the cells(HT-29),[10]thepresentworknowestablishestheconsider- meridional trichloridorhodiumACHTUNGTRENNUNG(III) complexes (Figure10b) and able potential of the polypyridyl complexes mer-[RhClACHTUNGTRENNUNG(DMSO)- 3 this was also the case for the doxorubicin-resistant cell line ACHTUNGTRENNUNG(dpq)] 2 and mer-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dppz)] 3 for the treatment of 3 7CCA when treated with the dpq complex 2 but not the dppz lymphoma and leukemia. Following the publication of our complex 3. After incubating BJAB cells with 3 for 12h, signifi- original results for the series mer-[RhClACHTUNGTRENNUNG(DMSO)(pp)] (pp=bpy, 3 cant changes could also be identified by fluorescence micro- phen, dpq, dppz, dppn), a report on the cytostatic activity of scopy. The cell integrity is damaged (Supporting Information, fac-[RhClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(bpy)]·DMSO and mer-[RhClACHTUNGTRENNUNG(DMSO)- 3 3 figureS1)andthenucleusoftheBJABcellsappearstobefrag- ACHTUNGTRENNUNG(phen)]·DMSO toward Caco-2 (colon adenocarcinoma) and 182 www.chemmedchem.org (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2009,4,177–187 CytotoxicRhodiumACHTUNGTRENNUNG(III)andIridiumACHTUNGTRENNUNG(III)PolypyridylComplexes ~600–800mm toward the cell lines MCF-7, SCG-7901 (gastric cancer), BEL-7402 (hepatic cancer), CNE-2 (nasopharyngeal carcinoma), and HELA (cervical cancer). The establishment of the structure–activity series Ru!Ir<Rh for complexes of the type mer- [MCl(L)(pp)] suggests that the rate of chloride and/ 3 orligandLsubstitutionmaybedecisiveindetermin- ing their cytotoxicity for a particular polypyridyl ligand. Whereas one or two of its chloride ions are rapidlyreplacedbyaqualigands,thisisnotthecase for the DMSO ligand in mer-[RuClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dpq)].[24] 3 The resulting cationic species not only intercalate into DNA but also react rapidly with bovine serum albumin,whichsuggeststhattheirverylowcytotox- icity may be due to rapid and strong covalent bind- ing to biomolecules other than the potential target molecules in the cell. In contrast, relatively rapid DMSO/HO exchange is observed for mer-[RhCl- 2 3 ACHTUNGTRENNUNG(DMSO)(pp)],[10] and the resulting neutral aqua com- plexesappeartoexhibitneitherintercalationnorco- valent binding to DNA. The iridiumACHTUNGTRENNUNG(III) complexes fac-[IrClACHTUNGTRENNUNG(DMSO)(pp)] are highly inert, and no signifi- 3 cant DMSO/HO or Cl(cid:3)/HO substitution is observed 2 2 for their aqueous solutions.[11] Like the meridional RhIII complexes, neither intercalative nor covalent in- teractions with DNA can be detected, but the soft IrIII compounds do react slowly with biomolecules containing S donor atoms, such as N-acetylmethio- nine. Taking all these facts into account, it can be postulated that the rate and nature of the ligand substitution reactions for the RhIII complexes may Figure9.a)DetectionofsignificantnecrosisforBJABcellsinthepresenceofcomplex14 indeed be favorable for high cytotoxicity, whereas relativetotheunspecificcytotoxiceffectsobservedforcomplex11.b)Cellviability valuesforcomplexes2and3;viabilitywasdeterminedusingtheLDHreleaseassayafter the lower activities of the analogous IrIII and RuIII anincubationperiodof1hforcomplexes11and14and3hforcomplexes2and3. compounds may be due to their respectively much higherormuchlowerkineticstabilities. The striking differences between the activities of A549 (lung adenocarcinoma) has also very recently ap- themeridionalandfacialisomersmayalsobetheresultofdif- peared.[23] In accordance with the trends listed in Table1, the feringkineticstabilitiesthatcouldinfluencetheextentofcellu- meridionalphencomplexismoreactivetowardthesecelllines lar uptake, which is much higher for mer-[RhClACHTUNGTRENNUNG(DMSO)(pp)][10] 3 (IC : 0.67(cid:2)0.05 and 0.38(cid:2)0.04mm) than the facial bpy com- than for fac-[IrClACHTUNGTRENNUNG(DMSO)(pp)].[11] Our present results indicate 50 3 plex(IC :1.19(cid:2)0.05and12.58(cid:2)0.50mm). that there is still much scope for tuning the activities of this 50 Our current studies demonstrate that both the meridional novel class of cytotoxic compounds [MCl(L)(pp)] (M=Rh, Ir) 3 rhodiumACHTUNGTRENNUNG(III) complexes mer-[RhClACHTUNGTRENNUNG(DMSO)(pp)] (pp=dpq 2, by varying both the coordination geometry (mer vs. fac) and 3 dppz3)andthefacialiridiumACHTUNGTRENNUNG(III)complexfac-[IrClACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] thecombinationofligands(X,L,pp). 3 2 11induceapoptosis viatheintrinsicmitochondrial pathway.In contrast to the likewise apoptosis-inducing complex mer-[IrCl- 3 ACHTUNGTRENNUNG(tpy)] 14, necrotic cell death appears to be negligible for the Experimental Section complexes of the general type [MCl(L)(pp)]. Complexes 2 and 3 Chemistry 3 exhibit superior apoptosis induction in comparison with the standard drugs daunorubicin, doxorubicin, and vincristine for Materials and instrumentation: UV/Vis spectra were recorded thetreatmentofchildhoodacutelymphoblasticleukemia. withanAnalytikJenaSPECORD200spectrometer.AJascoJ-715in- strument was used to measure CD spectra in the range 220– The synthesis and cytotoxicity studies of the analogous rutheniumACHTUNGTRENNUNG(III) complexes mer-[RuClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(dpq)] and mer- 500nm for 1:10 complex/DNA mixtures [complex=20mm, DNA 3 concentration in m(nucleotide)=200mm] in a 10mm phosphate [RuClACHTUNGTRENNUNG(CHCN)ACHTUNGTRENNUNG(dpq)] have also been recently reported.[24] De- 3 3 buffer at pH7.2. LSIMS (liquid secondary ion mass spectrometry) spite their apparent structural analogy to the complexes 1–4 data were registered with a Fisons VG Autospec employing a and 7 and 8, these RuIII compounds can be regarded as effec- cesium ion gun (17kV) and 3-nitrobenzyl alcohol as the liquid tively inactive on the basis of their very high IC values of matrix. Bruker DPX 200 and DRX 400 spectrometers were used for 50 ChemMedChem2009,4,177–187 (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 183 MED W.S.Sheldricketal. 1HNMRspectroscopy,withchemicalshiftsreportedasd values relative to the signal of tetramethylsilane. Ele- mental analyses were performed on a Vario EL instru- ment (Elementar Analysensysteme, Jena, Germany). RhX·3HO and IrX·3HO (X=Cl, Br) were purchased 3 2 3 2 from Chempur, phen, tpy, 1-methylimidazole, and 1- methylbenzimidazole from Acros, and dimethyl sulfox- ide (DMSO) from J.T. Baker. The polypyridyl ligand dpq[25] was prepared inaccordance with published pro- cedures as were the complexes mer-[RhClACHTUNGTRENNUNG(DMSO)(pp)] 3 (pp=phen,dpq,dppz,1–3).[10] X-raystructuralanalyses:Intensitydatawerecollected with an Oxford Diffraction Xcalibur2 diffractometer equipped with a Sapphire-CCD using 18 wscans and Mo radiation (l=0.71073(cid:3)). The data were corrected Ka for absorption by the Gauss method and solved by direct methods with SHELX97. Refinement against F2 0 wasperformedbySHELXL97[26]withanisotropictemper- ature factors for non-hydrogen atoms, and protons as riding atoms at geometrically calculated positions. CCDC701113, 701114, and 701115 contain the supple- mentarycrystallographicdataforthispaper.Thesedata can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. mer-[RhBrACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] (4): RhBr·3HO (200.0mg, 3 3 2 0.54mmol) was heated in 1mL DMSO at 708C for 4h. After addition of cold EtOH the solution was left to standat(cid:3)188Ctoafford[RhBrACHTUNGTRENNUNG(DMSO)]asared-brown 3 3 precipitate, which was filtered off and dried under vacuum before further use. 1,10-Phenanthroline (34.5mg,0.17mmol)wasthenaddedto[RhBrACHTUNGTRENNUNG(DMSO)] Figure10.ApoptosisinductionasmeasuredbyDNAfragmentationina)leukemiacells 3 3 (100.0mg, 0.17mmol) in CHOH (10mL), and the reac- (NALM-6)andb)lymphomacells(regularBJABanddoxorubicin-resistantBJABcells=- 3 tion mixture was heated at 708C for 2h. The resulting Doxo7CCA)aftertreatmentfor72hwithvariousconcentrationsof2,3,anddoxorubicin (Dox).Dataaregiveninpercenthypoploidy(sub-G 1 ) (cid:2)ESD(n=3),whichreflectsthe solidwasfilteredoff,washedwithCH 3 OHandEt 2 Oand numberofapoptoticcells. driedinvacuum.Yield:49mg(48%);1HNMR(200MHz, [D]DMSO): d=3.95 (s, 6H, DMSO), 8.25–8.38 (m, 2H, 6 H3/H8), 8.42 (d, 2H, H5/H6), 9.01 (d, 1H, H4), 9.08 (d, 1H, H7), 10.21 (d, 1H, H2), 10.26ppm (d, 1H, H9); LSIMS: m/z (%): 522(69) [M(cid:3)Br]+, 443(36) [M(cid:3)Br(cid:3)DMSO]+, 364ACHTUNGTRENNUNG(100) [M(cid:3)2Br(cid:3)DMSO]+, 283(21) [M(cid:3)3Br(cid:3)DMSO]+; Anal. calcd for C H BrNORhS (M= 14 14 2 601.0): C 28.0%, H 2.3%, N 4.7%, S 5.3%, found: C 27.9%,H2.3%,N4.7%,S5.2%. mer-[IrClACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] (7): IrCl·3HO (176.3mg, 3 3 2 0.5mmol) and DMSO (36mL, 0.5mmol) were dissolved in CHOH (25mL) and heated for 2h at 808C. After sol- 3 ventremoval,N,N-dimethylformamide(DMF,25mL)and 1,10-phenanthroline (99.1mg, 0.5mmol) were added to the residue, and the reaction mixture was heated at 1208C for 2h. Following cooling to 258C and renewed solvent removal, the resulting solid was dissolved in CHOH (2mL), and the product was precipitated by ad- 3 dition of EtO and dried under vacuum. Yield: 210.2mg 2 (75%);1HNMR(200MHz,CDCl):d=3.78(s,6H,DMSO), 3 7.92(dd,1H,H3),8.01(dd,1H,H8),8.06(d,2H,H5/H6), 8.43 (d, 1H, H4), 8.56 (d, 1H, H7), 10.12 (d, 1H, H2), Figure11.ApoptosisinductionasmeasuredbyDNAfragmentationinprimaryleukemia 10.39ppm(d,1H,H9);LSIMS:m/z(%):521(10)[M(cid:3)Cl]+, cellsisolatedfromapatientwithrelapsedchildhoodALLaftertreatmentfor60hwith2, 480(7) [M(cid:3)DMSO]+, 445(37) [M(cid:3)Cl(cid:3)DMSO]+, 409(12) 3,andstandardcytostaticagentsinclinicaluse(Daun=daunorubicin,Dox=doxorubicin, Vcr=vincristine).AllcytostaticagentswereappliedattheLC 50 concentrationsdeter- [M(cid:3)2Cl(cid:3)DMSO]+; Anal. calcd for C 14 H 14 Cl 3 IrN 2 OS (M= minedforBJABcells.Dataaregiveninpercenthypoploidy(sub-G) (cid:2)ESD(n=3),which 556.9): C 30.2%, H 2.5%, N 5.0%, found: C 29.8%, H 1 reflectsthenumberofapoptoticcells. 2.5%,N5.1%. 184 www.chemmedchem.org (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2009,4,177–187 CytotoxicRhodiumACHTUNGTRENNUNG(III)andIridiumACHTUNGTRENNUNG(III)PolypyridylComplexes [M(cid:3)HO(cid:3)2Cl+Na]+; Anal. calcd 2 for C H ClIrNO (M=566.9): C 14 12 3 4 2 29.7%, H 2.1%, N 9.9%, found: C 29.3%,H2.1%,N10.2%. fac-[IrBrACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)]·2HO (11): 3 2 2 Preparation as for 9 with IrBr·3HO (291.6mg, 0.6mmol) 3 2 and o-phenanthroline (108.1mg, 0.6mmol). Yield: 315.3mg (83%); 1HNMR (200MHz, CDOD): d= 3 3.51 (s, 2H, HO), 7.79 (dd, 2H, 2 H3/H8), 7.97 (s, 2H, H5/H6), 8.49 (dd, 2H, H4/H7), 9.09ppm (dd, 2H, H2/H9); LSIMS: m/z (%): 648(3) [M(cid:3)HO]+, 568(1) 2 [M(cid:3)HO(cid:3)Br]+; Anal. calcd for 2 C H BrIrNO (M=666.2): C 12 14 3 2 3 21.6%, H 2.1%, N 4.2%, found: C 21.8%,H1.7%,N4.2%. fac-[IrBrACHTUNGTRENNUNG(1-MeIm)ACHTUNGTRENNUNG(phen)] (12): 1- 3 Methylimidazole (1-MeIm) (16mL, 0.2mmol)was addedtofac-[IrBr- 3 ACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)]·3HO 5 (123.8mg, 2 2 0.2mmol) in CHOH (10mL) and 3 the mixture was held at reflux for 2h. Following cooling to 258C and solvent removal under vacuum, HO/CHOH (1:1 v/v, 2 3 10mL)wasaddedtotheresulting solid,andthemixturewasheated for 72h at 808C. After renewed solvent removal, the product was Figure12.Mitochondrialpermeabilitytransitionasmeasuredbyflowcytometricanalysisinlymphomacells washedwithCH 3 OHandEt 2 Oand (BJAB)aftertreatmentwithvariousconcentrationsofa)2and3,andb)11and14for48h.Valuesofthemito- dried under vacuum. Yield: chondrialpermeabilitytransitionaregivenasthepercentageofcellswithlowDy (cid:2)ESD(n=3). 50.3mg (36%); 1HNMR (200MHz, m CDCl): d=3.89 (s, 3H, CH), 7.14 2 2 3 (d, 1H, 1-MeIm H), 7.38 (d, 1H, 1- MeIm H), 7.69 (dd, 2H, H3/H8) mer-[IrBrACHTUNGTRENNUNG(DMSO)ACHTUNGTRENNUNG(phen)] (8): Preparation as for 7 with 215.9mg 7.86 (s, 2H, H5/H6), 8.33 (dd, 2H, H4/H7), 8.52 (s, 1H, 1-MeIm H), 3 IrBr·3HO (0.5mmol). Yield: 223.4mg (65%); 1HNMR (200MHz, 9.18ppm (dd, 2H, H2/H9); LSIMS: m/z (%): 697(8) [M]+, 617(10) 3 2 CDCl): d=3.95 (s, 6H, DMSO), 7.90 (dd, 1H, H3), 8.00 (dd, 1H, [M(cid:3)Br]+;Anal.calcdforC H BrIrN (M=694.2):C27.7%,H2.0%, 2 2 16 14 3 4 H8),8.05(d,2H,H5/H6),8.44(d,1H,H4),8.54(d,1H,H7),10.28(d, N8.1%,found:C27.4%,H2.2%,N8.5%. 1H, H2), 10.39ppm (d, 1H, H9); LSIMS: m/z (%): 690(5) [M]+, 611(15)[M(cid:3)Br]+,531(17)[M(cid:3)2Br]+;Anal.CalcdforC H BrIrNOS fac-[IrBrACHTUNGTRENNUNG(1-MeBIm)ACHTUNGTRENNUNG(phen)]·HO (13): Preparation as for 12 with 1- 14 14 3 2 3 2 (M=690.3): C 24.4%, H 2.0%, N 4.1%, found: C 24.0%, H 2.1%, N methylbenzimidazole (26.4mg, 0.2mmol). Yield: 59.5mg (39%); 4.1%. 1HNMR (200MHz, CDCl): d=1.24 (s, 3H, CH), 7.51 (m, 3H, 2 2 3 MeBIm H), 7.69 (dd, 2H, H3/H8) 7.83 (s, 2H, H5/H6), 7.97 (d, 1H, fac-[IrClACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(phen)] (9): IrCl·3HO (105.8mg, 0.3mmol) and 3 2 3 2 MeBIm H), 8.31 (dd, 2H, H4/H7), 9.23 (dd, 2H, H2/H9), 9.57 (s, 1H, 1,10-phenanthroline (54.0mg, 0.3mmol) were dissolved in 10mL MeBIm H); LSIMS: m/z (%): 665(6) [M(cid:3)Br]+; Anal. calcd for HO and heated for 4h at 1108C. Following cooling to 258C and 2 C H BrIrNO (M=762.3): C 31.5%, H 2.4%, N 7.3%, found: C solvent removal under vacuum, the resulting solid was washed 20 18 3 4 31.4%,H2.3%,N7.3%. with CHOH and EtO and dried under vacuum. Yield: 78.9mg 3 2 (53%); 1HNMR (200MHz, CDOD): d=3.49 (s, 2H, HO), 7.76 (dd, 3 2 mer-[IrClACHTUNGTRENNUNG(tpy)]·3HO (14): IrCl·3HO (70.5mg, 0.2mmol) and 2H, H3/H8), 7.94 (s, 2H, H5/H6), 8.46 (dd, 2H, H4/H7), 9.05ppm 3 2 3 2 2,2’:6’,2’’-terpyridine (46.6mg, 0.2mmol) were heated for 2h at (dd, 2H, H2/H9); LSIMS: m/z (%): 478(1) [M(cid:3)HO]+, 443(7) 2 808CinCHOH(10mL).Aftercoolingto258Candsolventremoval [M(cid:3)HO(cid:3)Cl]+; Anal. calcd for C H ClIrNO (M=496.8): C 29.0%, 3 2 12 10 3 2 under vacuum, the resulting solid was washed with CHOH and H2.0%,N5.6%,found:C28.6%,H2.0%,N5.6%. 3 EtO and dried under vacuum. Yield: 98.0mg (92%); 1HNMR 2 fac-[IrClACHTUNGTRENNUNG(HO)ACHTUNGTRENNUNG(dpq)]·HO (10): Preparation as for 9 with dipyri- (400MHz, [D]DMSO): d=7.59 (dd, 2H, H2/H2’), 8.11 (dd, 2H, H3/ 3 2 2 6 do[3,2-f:2’,3’-h]quinoxaline (69.6mg, 0.3mmol). Yield: 76.5mg H3’), 8.15 (t, 1H, H6), 8.49 (d, 2H, H5/H5’) 8.77 (d, 2H, H1/H1’); (45%); 1HNMR (200MHz [D]DMSO): d=7.97 (m, 2H, H3/H8), 9.19 LSIMS: m/z (%): 496(5) [M(cid:3)Cl]+, 461(6) [M(cid:3)2Cl]+; Anal. calcd for 6 (s, 2H, H11/H12), 9.25 (d, 2H, H4/H7), 9.45ppm (d, 2H, H2/H9); C H ClIrN (M=531.8): C 30.7%, H 2.9%, N 7.2%, found: C 15 11 3 3 LSIMS: m/z (%): 517(1) [M(cid:3)HO(cid:3)Cl+Na]+, 482(3) 30.3%,H2.7%,N6.8%. 2 ChemMedChem2009,4,177–187 (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 185 MED W.S.Sheldricketal. mer-[IrBrACHTUNGTRENNUNG(tpy)]·3HO (15): Preparation as for 14 with IrBr·3HO concentrations that cause 50% inhibition of cell proliferation. Re- 3 2 3 2 (97.2mg, 0.2mmol). Yield: 108.4mg (81%); 1HNMR (400MHz, sultswerecalculatedfrom2–3independentexperiments. [D]DMSO):d=7.50(dd,2H,H2/H2’),8.01(dd,2H,H3/H3’),8.11(t, 6 1H,H6),8.45(d,2H,H5/H5’)8.73ppm(d,2H,H1/H1’);Anal.calcd The cytotoxicity of 2, 3, 11, and 14 toward BJAB cells was mea- for C H BrIrN (M=665.2): C 25.1%, H 2.4%, N 5.8%, found: C suredbyreleaseoflactatedehydrogenase(LDH)asdescribedpre- 25.2% 15 ,H 11 2.2 3 %, 3 N5.8%. viously.[29]Afterincubationwithvariousconcentrationsofthecom- plexesfor1or3hat378C,LDHactivityreleasedbyBJABcellswas measured in the cell culture supernatants using the Cytotoxicity Biologicalinvestigations Detection Kit from Boehringer Mannheim (Mannheim, Germany). Thesupernatantswerecentrifugedat1500rpmfor5min.Cell-free Materials: RNaseA was obtained from Qiagen (Hilden, Germany), supernatants (20mL) were diluted with phosphate-buffered saline propidium iodide from Serva (Heidelberg, Germany), and JC-1 (PBS, 80mL), and a reaction mixture containing 2-[4-iodophenyl]-3- fromMolecularProbes,Invitrogen(Karlsruhe,Germany). [4-nitrophenyl]-5-phenyltetrazolium chloride (INT), sodium lactate, Cell cultures: MCF-7 breast cancer and HT-29 human colon carci- oxidized nicotinamide adenine dinucleotide (NAD+) and diaphor- noma cells were maintained in 10% (v/v) fetal calf serum (FCS) ase(100mL)wasadded.Time-dependentformationofthereaction containingcellculturemedium[minimumessentialEagle’ssupple- product was the quantified photometrically at 490nm. The maxi- mented with NaHCO (2.2g), sodium pyruvate (110mgL(cid:3)1), and mumamountofLDHactivityreleasedbythecellswasdetermined 3 gentamicin sulfate (50mgL(cid:3)1) adjusted to pH7.4] at 378C under bylysisofthecellswith0.1%TritonX-100inculturemedium,and 5% CO, and were passaged twice a week according to standard wassetas100%celldeath. 2 procedures. BJAB (Burkitt-like lymphoma) and NALM-6 (human Determination of cell viability: Cell viability was determined by Bcell precursor leukemia) cells were maintained at 378C in RPMI using the CASY Cell Counter and Analyzer System from Innovatis 1640(GIBCO,Invitrogen)supplementedwith10%heat-inactivated (Bielefeld, Germany). Settings were specifically defined for the re- FCS, penicillin (100000UL(cid:3)1), streptomycin (0.1gL(cid:3)1), and l-gluta- quirements of the cells used. With this system, the cell concentra- mine (0.56gL(cid:3)1). The cells were subcultured every 3–4days by di- tioncan beanalyzedsimultaneously inthree different sizeranges: lution of the cells to a concentration of 1(cid:4)105cellsmL(cid:3)1. Twenty- celldebris,deadcells,andviablecells.Cellswereseededataden- fourhoursbeforetheassaysetup,cellswereculturedataconcen- sityof1(cid:4)105cellsmL(cid:3)1andtreatedwithvariousconcentrationsof trationof3(cid:4)105cellsmL(cid:3)1toascertainstandardizedgrowthcondi- 2, 3, 11, and 14; non-treated cells served as controls. After a 24h tions. For apoptosis assays, the cells were then diluted to a con- incubation period at 378C, cells were resuspended properly and centration of 1(cid:4)105cellsmL(cid:3)1 immediately before addition of the 100mL of each well was diluted in 10mL CASYton (ready-to-use various complexes. To generate 7CCA (doxorubicin-resistant BJAB) isotonic saline solution) for an immediate automated count of the cells, BJAB cells were exposed to an initial concentration of cells. 0.1mgL(cid:3)1 doxorubicin and then treated with doxorubicin up to 1mgL(cid:3)1,wheneverthevitalityofthecellswas>85%. Measurement of DNA fragmentation: Apoptotic cell death was Patients:Primarylymphoblastswereobtainedbybonemarrowas- determined by a modified cell-cycle analysis, which detects DNA piration of patients with relapsed acute lymphoblastic leukemia fragmentation at the single-cell level as described previously.[22] (ALL). The diagnosis was established by immunophenotyping of Cells were seeded at a density of 1(cid:4)105cellsmL(cid:3)1 and treated leukemiacellsaccordingtoB(cid:5)n(cid:5)etal.[27]Lymphoblastsandmono- withvariousconcentrationsof2,3,11,and14.Aftera72hincuba- nuclear cells were separated by centrifugation over Biocoll (Bio- tion period at 378C, cells were collected by centrifugation at chrom KG, Berlin, Germany). After separation, the percentage of 1500rpm for 5min, washed with PBS at 48C, and fixed in PBS/ leukemia cells was >95%. The leukemia cells were immediately formaldehyde (2% v/v) on ice for 30min. After fixation, cells were seededatadensityof3(cid:4)105cellsmL(cid:3)1inRPMI1640completecell pelleted, incubated with EtOH/PBS (2:1 v/v) for 15min, pelleted, culturemediumandincubatedfor60hwithdaunorubicin,doxoru- and resuspended in PBS containing 40mgmL(cid:3)1 RNaseA. RNA was bicin, and vincristine, as well as with complexes 1 and 2 at their digested for 30min at 378C, after which the cells were pelleted LD concentrationsinBJABcells.Theuseofthecellsisinaccord- once again and finally resuspended in PBS containing 50mgmL(cid:3)1 50 ance with the ethical standards of the Responsible Committee on propidium iodide. Nuclear DNA fragmentation was quantified by Human Experimentation and the Helsinki Declaration as revised in flow cytometric determination of hypodiploid DNA (fluorescence- 2000. It is also in accordance with the positive vote of the ethics activated cell sorting, FACS). Data were collected and analyzed committeefromDecember14,2000 fortheALL-REZ-BFM studyin using a FACScan (Becton Dickinson, Heidelberg, Germany) appara- 2002. Informed signed consent was obtained from either the pa- tusequipped withCELLQuestsoftware. Dataare giveninpercent tientorfromtheirnextofkin. hypoploidy (sub-G 1 ), which reflects the number of apoptotic cells. Cytotoxicity measurements: The antiproliferative effects of com- Measurementofthemitochondrialpermeabilitytransition:After plexes1–15towardMCF-7andHT-29cellsweredeterminedbyan an incubation period of 48h with various concentrations of 2, 3, established procedure.[28] Cells were suspended in cell culture 11,and14,cellswerecollectedbycentrifugationat1500rpm,48C medium (MCF-7: 10000cellsmL(cid:3)1; HT-29: 2850cellsmL(cid:3)1), and ali- for 5min. The mitochondrial permeability transition was then de- quotsthereofwereplatedin96-wellplatesandincubatedat378C termined by staining the cells with 5,5’,6,6’-tetrachloro-1,1’,3,3’-tet- under 5% CO for 72h (MCF-7) or 48h (HT-29). Stock solutions of raethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes, 2 thecompoundsinDMSOorDMFwerefreshlyprepared anddilut- Leiden, The Netherlands). 1(cid:4)105 cells were resuspended in 500mL ed with cell culture medium to the desired concentrations (final phenol-red-free RPMI 1640 without supplements, and JC-1 was DMSOorDMFconcentration:0.1%v/v).Themediumintheplates added to give a final concentration of 2.5mgmL(cid:3)1. The cells were was replaced with the medium containing the compounds in incubatedfor30minat378Cwithmoderateshaking.Controlcells graded concentrations (six replicates). After further incubation for werelikewiseincubatedintheabsenceofJC-1dye.Thecellswere 96h (MCF-7) or 72h (HT-29), the cell biomass was determined by harvested by centrifugation at 1500rpm, 48C for 5min, washed crystal violet staining, and the IC values were established as the with ice-cold PBS, and resuspended in 200mL PBS at 48C. Mito- 50 186 www.chemmedchem.org (cid:2)2009Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2009,4,177–187 CytotoxicRhodiumACHTUNGTRENNUNG(III)andIridiumACHTUNGTRENNUNG(III)PolypyridylComplexes chondrialpermeabilitytransitionwasthenquantifiedbyflowcyto- [11] M.A.Scharwitz, I. Ott, R. Gust, A.Kromm, W.S. Sheldrick, J.Inorg.Bio- metricdeterminationofthecellswithdecreasedfluorescence,that chem.2008,102,1623–1630. is,withmitochondriadisplayingalowermembranepotential.Data [12] B.R.James,R.H.Morris,Can.J.Chem.1980,58,399–408. [13] E. Alessio, P. Faleschini, A. SessantioSanti, G. Mestroni, M. Calligaris, were collected and analyzed using a FACScan (Becton Dickinson, Inorg.Chem.1993,32,5756–5761. Heidelberg, Germany) apparatus equipped with CELL Quest soft- [14] E.D.McKenzie,R.A.Plowman,J.Inorg.Nucl.Chem.1970,32,199–212. ware. Data are given in percentage of cells with low DY , which m [15] J.A.Broomhead,W.Grumley,Inorg.Chem.1971,10,2002–2009. reflectsthenumberofcellsundergoingmitochondrialapoptosis. [16] X-ray structural analysis of fac-[IrClACHTUNGTRENNUNG(CHCN)ACHTUNGTRENNUNG(phen)] 9a: M=560.86, tri- 3 3 clinic P1¯, a=7.159(1), b=10.234(1), c=12.860(1)(cid:3), a=92.48(1), b= Fluorescence microscopy: Microscopic work was performed with 98.46(1), g=110.25(1)8, V=869.74(6)(cid:3)3, T=120K, Z=4, d = an Axioskop2 with the fluorescence option from Zeiss, equipped calcd 2.142gcm(cid:3)3, m=8.142mm(cid:3)1. A total of 6952 reflections were mea- with the set of filters 09, with l =450–490nm and l =515nm. ex em sured,of which 3040 wereunique (R =0.026) and2664had I>2s(I). BJABcellswereincubatedwith3 for12hat378Candsubsequent- Final residuals were R =0.022 (forI> in 2 t s(I)),wR =0.0470(for all data), 1 2 ly fixed with formaldehyde. The fixed cells (20mL) were applied to andGOOF=0.956,withalargestresidualpeak1.36eA(cid:3)3,andalargest aslidewithpoly-l-lysine(Sigma)intoapaintedcircle(Kisker).After residualholeof(cid:3)1.00eA(cid:3)3. a careful drying process, the fixed cells were washed twice with [17] X-ray structural analysis for mer-[IrClACHTUNGTRENNUNG(tpy)] 14: M=531.82, monoclinic 3 PBS. Afterward the BisBenzimidH 33258 (Sigma) coloring agent P2 1 /n, a=8.264(1), b=14.020(1), c=13.647(1)(cid:3), b=105.07(1), V= wasappliedinadilutedconcentrationof0.25mgmL(cid:3)1PBS.BisBen- 1526.74(7)(cid:3)3, T=120K, Z=4, d calcd =2.314gcm(cid:3)3, m=9.267mm(cid:3)1; 10600 measured reflections with 2678 unique (R =0.016) and 2270 zimidcolorsanddisplaysthenucleioftreatedcells.Beforethemi- int withI>2s(I).FinalresidualswereR =0.016 forI>2s(I),wR =0.033(all croscopystudies,theslidesweretreatedwithglycerin(Sigma)and 1 2 data)andGOOF=1.020,withalargestresidualpeakof1.31eA(cid:3)3,and keptinthedarkat48C. alargestresidualholeof(cid:3)0.62eA(cid:3)3. [18] X-ray structural analysis for mer-[IrBrACHTUNGTRENNUNG(tpy)] 15: M=665.20, monoclinic 3 P2/n, a=8.438(1), b=14.363(1), c=13.957(1)(cid:3), b=106.42(1), V= Acknowledgements 1 1622.5(2)(cid:3)3, T=120K, Z=4, d =2.723gcm(cid:3)3, m=15.615mm(cid:3)1; calcd 7036measuredreflectionswith2830unique(R =0.117)and1187with int Financial support for this work in Berlin and Bochum by the I>2s(I). Final residuals were R 1 =0.047 for I>2s(I), wR 2 =0.099 (all data)andGOOF=0.558,withalargestresidualpeakof1.89eA(cid:3)3,and Deutsche Forschungsgemeinschaft (DFG) within the research alargestresidualholeof(cid:3)1.07eA(cid:3)3. group FOR630 “Biological function of organometallic com- [19] T. Poth, H. Paulus, H. Elias, C. D(cid:7)cker-Benfer, R. vanEldik, Eur. J. Inorg. pounds” is gratefully acknowledged. We also thank Corazon Chem.2001,1361–1369. Frias, Heike Scheffler, and Manuela Winter for excellent technical [20] F. Wang, A. Habtemariam, E.P.L. vander Geer, R. Fernandez, M. 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