Cell-selective, apoptosis-inducing rhodium(III) crown thiaether complexes.

DOI:10.1002/cmdc.201000129 Cell-Selective, Apoptosis-inducing Rhodium(III) Crown Thiaether Complexes Ruth Bieda,[a] Mara Dobroschke,[b] Annika Triller,[c] Ingo Ott,[d] Marc Spehr,[e] Ronald Gust,[f] Aram Prokop,[b] and William S. Sheldrick*[a] Half-sandwichrhodium(III)polypyridyl (pp) complexes withthe storescytotoxicityasevidencedbyIC valuesintherange8.1- 50 metal atom capped by the facial crown thiaether 1,4,7-trithia- 11.6mm.Spectroscopic(CD,UV/Vis,NOESY)andviscositymeas- cyclononane[9]aneS representapromisingclassofapoptosis- urements indicate that the active dppz complex 8 (IC values: 3 50 inducing potent cytostatic agents. The necrotic damage 4.7–8.9mm) exhibits strong intercalative binding towards DNA caused by the complexes is negligible. In vitro cytotoxicity whereas the even more potent bipyrimidine complex 9 (IC 50 assays with the human cancer cell lines MCF-7 and HT-29 and values: 0.6–1.9mm) causes no alteration of the duplex B con- immortalized HEK-293 cells indicate that the dicationic k2N- formation. Weaker intercalative binding is observed for the (imino)complexes [([9]aneS)RhCl(pp)]2+ are much more active dpq complex 7. A comparative annexinV–propidium iodide 3 than monocationic complexes [([9]aneS)RhCl(L)]+ (L=imid- binding assay with lymphoma (BJAB) cells and healthy leuko- 3 2 azole, CHCN). Whereas the k2N(amino) complex [([9]aneS)- cytes demonstrates that the cytotoxic activity of complex 8 3 3 RhCl(piperazine)]2+ is inactive, replacing piperazine with the and particularly complex 9 is highly selective towards the ma- structurally analogous k2S (thiaether) ligand 1,4-dithiane re- lignantcells. Introduction Althoughavarietyofrhodiumcomplexeshavebeenshownto vitro cytotoxicity has also been recently reported for the com- exhibit significant anticancer activity, further exploitation has plex [([9]aneNS)RhCl] ([9]aneNS =1-aza-4,7-dithiacyclono- 2 3 2 been prevented by toxic side effects.[1] The majority of interest nane)againsttheovariancancercelllineNuTu-19.[14] has focused on dirhodium(II,II) tetracarboxylates of the type While the bpy complex [([9]aneS)RhCl(bpy)]Cl exhibits sig- 3 2 [RhL(RCOO)] (R=alkyl group; L=HO or another monoden- nificantly higher cytotoxicity towards MCF-7 and HT-29 cells 2 2 4 2 tateligand),[2–4]butrecentreportshavesuggestedthatoctahe- (IC values: 12.8(cid:2)0.2 and 4.4(cid:2)0.1mm, respectively) than its 50 dralchloridorhodium(III)complexes mayofferconsiderablepo- phen counterpart (IC values: 36.3(cid:2)6.0 and 72.2(cid:2)8.0mm), 50 tential as anticancer agents. For instance, mer,cis-[RhCl- theactivityofother([9]aneS)RhIIIcomplexesintheseriespp= 3 3 (DMSO)(NH)] exhibits promising cytotoxicity towards human phen, dpq, dppz, correlates directly to the size of the pp 2 3 ovarian (A2780) and colon carcinoma (LoVo) cell lines,[5] while ligand,asdocumentedbytherespectiveIC values:72.2(cid:2)8.0, 50 polypyridyl (pp) complexes of the type mer-[RhCl(DMSO)(pp)] 3 are potent antitumor agentsused in the treatment of lympho- [a] Dr.R.Bieda,Prof.Dr.W.S.Sheldrick ma and leukemia.[6] In vitro IC values of the latter type of Lehrstuhlf(cid:2)rAnalytischeChemie,Ruhr-Universit(cid:3)tBochum 50 compounds are strongly dependent on the size of the chelat- 44780Bochum(Germany) Fax:(+49)234-3214420 ing polypyridyl ligand within the series: pp=bpy, phen, dpq E-mail:william.sheldrick@rub.de (bpy=2,2’-bipyridyl; phen=1,10 phenanthroline; dpq=dipyri- [b] M.Dobroschke,Dr.A.Prokop do[3,2-f:2’,3’-h]quinoxaline).[7] High levels of cytotoxicity have DepartmentofPediatricOncology,CologneChildren’sHospital also been established for the similar meridional terpyridine Amsterdamerstraße59,50735Cologne(Germany) complexmer-[RhCl(2,2’:6’,2“-terpyridine)].[8] [c] Dr.A.Triller 3 Organometallic half-sandwich complexes of the types [(h5- Lehrstuhlf(cid:2)rZellphysiologie,Ruhr-Universit(cid:3)tBochum 44780Bochum(Germany) CMe)MCl(pp)](CFSO) (M=Rh, Ir)[9,10] and [(h6-CMe)RuCl- 5 5 3 3 6 6 [d] Prof.Dr.I.Ott (pp)](CFSO),[11] with larger pp ligands such as dpq, dipyrido- 3 3 InstituteofPharmaceuticalChemistry,TechnischeUniversit(cid:3)tBraunschweig [3,2-a:2’,3’-c]phenazine (dppz), and benzo[i]dipyrido[3,2-a:2’,3’- Beethovenstraße55,38106Braunschweig(Germany) c]phenazine (dppn) also exhibit significant invitro cytotoxicity [e] Prof.Dr.M.Spehr towards the human HT-29 (colon cancer) and MCF-7 (breast RWTHAachen,AbteilungChemosensorik,Institutf(cid:2)rBiologieII cancer) cell lines. These anticancer properties are retained to a WorringerWeg1,52074Aachen(Germany) [f] Prof.Dr.R.Gust varying extent, or even augmented, for specific polypyridyl li- Institutf(cid:2)rPharmazie,FreieUniversit(cid:3)tBerlin gandswhenk3Ntrispyrazolylmethane(tpm)ork3S1,4,9-trithia- KçniginLuise-Straße2–4,14195Berlin(Germany) cyclononane ([9]aneS) are employed as alternative facial co- 3 SupportinginformationforthisarticleisavailableontheWWWunder ligands in half-sandwich RhIII complexes.[12,13] Considerable in http://dx.doi.org/10.1002/cmdc.201000129. ChemMedChem2010,5,1123–1133 (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim 1123 MED W.S.Sheldricketal. 20.9(cid:2)2.8, and 7.4(cid:2)2.2mM in HT-29 cells.[13] This suggests that Results and Discussion differentmechanisms-of-actionormoleculartargetsmaybein- volved, depending on pp ligand size within the ([9]aneS)RhIII 3 Synthesisandstructure complexes. As DNA fragmentation and mitochondrial permeability Compounds 1 and 2, of the type [([9]aneS)RhCl(LL)]Cl, where 3 2 measurements using Burkitt-like (BJAB) lymphoma cells have LL=piperazine (pip) or 1,4-dithiane, were synthesized by heat- demonstrated that ([9]aneS)RhIII complexes containing the ing solvent (S) complexes [RhCl(LL)(S)] with an equivalent of 3 3 dppz and 2,2’- bipyrimidine (bpm) ligands induce cell-specific 1,4,7-trithiacyclononane.Asmallamountofverysparinglysolu- apoptosis with only negligible necrosis;[13] such half-sandwich ble [([9]aneS)RhCl] was obtained as a side product in each 3 3 complexesrepresentapotentiallypromisingclassofanticancer caseandwasremovedbyfiltrationpriortotheisolationofthe agents. We have, therefore, further characterized their struc- desired products from the reaction solutions. Treatment of ture–activity relationships, with the aim of establishing lead RhCl·3HO with the appropriate bidentate ligand (LL) in water 3 2 substances, by studying the cell death-inducing properties of afforded the required intermediate complexes [RhCl(LL)(HO)] 3 2 novel types of ([9]aneS)RhIII complexes. These studies have in- insitu,whichwereusedwithoutseparationorcharacterization 3 cluded complexes with nonaromatic k2N and k2S bidentate for the preparation of 1 and 2. Complexes [([9]aneS)RhCl- 3 2 ligands 1 and 2, and two labile chloride ligands 3 and 4. The (Im)]Cl 3 and [([9]aneS)RhCl(CHCN)](CFSO) 4 were also ob- 3 2 3 3 3 tained in a two-step manner by reacting [9]aneS with in situ 3 complexes containing the monodentate ligands imidazole (Im) and acetonitrile, respectively. In the case of the acetonitrile complex 4, the in situ complex was prepared by reaction of RhCl·3HO with two equivalents of Ag(CFSO) in acetonitrile 3 2 3 3 followed by removal of the resulting AgCl precipitate (one equivalent). This inability of the excess Ag(CFSO) equivalent 3 3 tofacilitate substitution of asecond anionic chloride ligand by a neutral acetonitrile ligand is in accordance with the well- documented,limitedabilityofthiaetherstoneutralizethehigh positive charge of metal atoms by s donation.[15] In analogy, three equivalents of Ag(CFSO) are required to remove two 3 3 chloride ligands from RhCl·3HO to afford the bis-acetonitrile 3 2 complex [([9]aneS)RhCl(CHCN)](CFSO).[16] Complexes 1–4 3 3 2 3 32 were characterized by 1HNMR and positive ion LSIMS and gavesatisfactorymicroanalyses. PeptideandDNAbindingstudies cytotoxic activity of the complexes towards MCF-7 and HT-29 cells has also been compared with their activity towards im- Forthepreviouslycharacterizedseriesofppcompounds5–10, mortalized human embryonal kidney cells (HEK-293) and with the highest cytotoxicity towards MCF-7 and HT-29 cells is ex- the values obtained for these cell lines with the previously hibited by dppz complex 8 (IC values: 4.7(cid:2)0.5 and 7.4(cid:2) 50 characterized cations of polypyridyl (pp) complexes 5–10 of 2.2mm) and bpm complex 9 (IC values: 1.7(cid:2)0.5 and 1.9(cid:2) 50 the type [([9]aneS)RhCl(pp)]2+, where pp=bpy, phen, dpq, 0.1mm).[13] The implicit reversal of ligand-size dependence in 3 dppz,bpm, tap(5–10).[13]Themostactivesubstancesfromthe comparing the activity of the bpm complex to its phen ana- series 1–10 were selected for comparative cytotoxicity studies logue 6 (IC values: 36.3(cid:2)6.0 and 72.2(cid:2)8.0mm) suggests dif- 50 against BJAB lymphoma and leukocyte cells from healthy pa- ferent mechanisms-of-action may be involved for complexes 8 tients, to establish whether these lead complexes exhibit ade- and 9. This finding prompted us to study and compare the in- quatecellselectivitytojustifyfurtherinvestigation. teraction of selected compounds from the series 1–10 with suitablebiomolecules. Ourprevioustime-dependent1Hand31PNMRstudiesfor1:2 molar mixtures of the selected complex [([9]aneS)RhCl- 3 (dpq)]Cl (7) with guanosine-5’-monophosphate and N-acetyl- 2 methionine detected no additional signals over a 24h period at 378C corresponding to possible kN7 or kS coordinated products.[13] In contrast, slow hypochromic changes in the UV absorption spectrum of the complex in phosphate buffer at 208C were in accordance with slow Cl(cid:3)/HO and/or Cl(cid:3)/phos- 2 phate exchange. Of the members of the cobalt triad, RhIII is best suited electronically to accommodate both soft and hard ligands in its coordination sphere; many examples have been 1124 www.chemmedchem.org (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2010,5,1123–1133 Rhodium(III)CrownThiaetherComplexes reported formixedligand sets, although theyrepresent anap- kS coordination of the rhodium fragment to the cysteine side parent deviance from the general principle that metal atoms chain. This study indicates that soft donor atoms, such as the most often prefer either hard or soft ligands but not both at thiolate sulfur, are potential targets for the half-sandwich the same time.[17] For instance, dimethyl sulfoxide (DMSO) co- [([9]aneS)Rh]3+ fragment when it is coordinated by hard chlo- 3 ordinates RhIII through its oxygen atom, but IrIII through its ride ligands instead of two additional soft or borderline donor sulfur atom in half-sandwich cations of the type [(h5-CMe)M- atomsasinthe[([9]aneS)Rh(pp)]3+ fragments. 5 5 3 (DMSO)]2+ (M=Rh,Ir).[18] In thepresence of small molecules, characteristicchanges in 3 We have previously demonstrated that ESI tandem mass the circular dichroism (CD) spectra of DNA in the range 220– spectrometric techniques can be employed to identify binding 400nmprovideameansofmonitoringpossibleconformation- sites for the half-sandwich complex [(h6-cymene)RuCl(DMSO)] al changes for the double helix.[21] Calf thymus (CT) DNA is 2 with model peptides and with proteins in whole-cell sys- present in the Bform, for which a negative CD band at tems.[19] The [(h6-cymene)Ru]2+ fragment forms chelate com- 246nm,causedbythehelical conformation,and apositive CD plexeswithpeptidesthatarestableenoughtosurviveMSioni- band at 275nm, due to base stacking, are observed.[22] Aspre- zation and subsequent collision-induced dissociation to a viously reported forbpm and tap complexes 9 and 10, nosig- series of b+- and y+-type fragment ions.[20] Amino acid resi- nificantchangesintheCTDNAspectrumweredetectedinthe duescontainingN-or O-donor atomsin theirside chains were presence of complexes 2–4 at r=[complex]/[DNA]=0.2 for established as ruthenium binding sites. As the [([9]aneS)Rh]3+ [DNA]=m (base pairs).[13] In contrast, a significant decrease 3 fragment of complexes 3 and 4 were predicted to be similarly (D[q]/[q]) of (cid:3)43% in the molar ellipticity for the positive CD capable of affording stable chelate complexes with suitable band at approximately 275nm was observed for the complex peptides, we were interested in employing MS/MS techniques 1/CT DNA mixture (figureS1 in the Supporting Information). to test this hypothesis and determine any apparent binding Similar decreases were previously reported[13] for the bpy and preference. To this end, ESI-MS/MS experiments were per- phen complexes 5 and 6 and may be indicative of adistortion formed on 1:1 mixtures of the acetonitrile complex 4 with the of the BDNA conformation, due to either groove binding or modelpeptidesH-AGXVGAGLIK-OH(X=C,D,H,M)followinga covalentbinding.Thedetectionoftwonegativeinduced-circu- 24h incubation period at 378C. The pH of the interaction mix- lar dichroism (ICD) signals for the 1:5 [([9]aneS)RhCl(dpq)]Cl/ 3 2 tures was reduced to approximately 4.0 prior to ESI-MS/MS CT DNA mixture indicated the presence of two competing ionization. The presence of a complex/peptide adduct could modes of DNA binding.[13] As illustrated in Figure2a, results of only be established for the cysteine-containing decapeptide, the CD titration performed in this work further suggest that whoseMSspectrum containedan[M+H]+ peakatm/z=1168 themodesmaybeconcentrationdependent.Probablebinding as well as a peak for [M+2H]2+ at m/z=585, both with ade- interactions include groove binding or intercalation, although quate relative intensities. Cysteine was confirmed as the rhodi- covalent binding following chloride exchange cannot be ruled um binding site by the MS/MS analysis of the peak at m/z= out. The CD spectrum of the analogous dppz complex[13] with 585 (Figure1), with a total of six b+ and three y+ ions able to CT DNA at r=0.2 contained the strongly negative ICD at be assigned. The observation of the b + ([AGC@ - 2H]+), y + 294nm, characteristic of an intercalative binding mode, which 3 8 ([C@VGAGLIKOH - H]+) and y + ([H-VGAGLIKOH]+) fragment has previously been observed for other half-sandwich dppz 7 2 ions, where @=[([9]aneS)Rh]3+, is clearly in accordance with complexes.[9,11,23] The ICD shifted to 285nm and greatly in- 3 creased in magnitude for r=0.4 (Figure2b). Total loss of BDNA conformation and the possible adoption of an A conformation at this higher ratio are indicated by the disappearance of the negative band at approximately 246nm. These interesting findings prompted us to further investi- gate the binding modes of the dpqanddppzcomplexestoaB- type double helix by 1HNMR spectroscopy. The palindromic octanucleotide d(5’-CGCTAGCG- 3’), which has previously been 2 employed to characterize the binding interaction of the bis-in- tercalator TOTO,[24] was selected forthisaim.Whileaminimumof Figure1.MS/MSspectrumofthemolecularion[AGC@VGAGLIK-OH]2+atm/z=585. six base pairs is necessary to ChemMedChem2010,5,1123–1133 (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 1125 MED W.S.Sheldricketal. enabletheformationofanormalB-typedoublehelix with a minor and major groove, the presence of more than 12 base pairs causes unnecessary peak overlapping and associated problems with data anal- ysis.[25] The chosen oligonucleotide offers both a cen- tral TA/AT sequence for a possible 1:1 intercalation mode and two CG/TA sequences for either 1:1 or symmetrical2:1intercalationmodes. 1HNMR analysis of changes in the exchange kinet- ics of the hydrogen-bonded nucleobase imino pro- tons on metal complex binding can often provide useful insightsintothenature of interaction withthe DNAdoublehelix.[26,27]Upfieldshiftsarecharacteristic ofcompoundsthatintercalateintotheduplex,[28]and small upfield shifts were indeed observed for reso- nances of the G2, T4 and G6 imino protons for mix- tures of [([9]aneS)RhCl(dppz)]Cl and d(5’-CGCTAG- 3 2 CG-3’) at r=0.1 and 0.5. These signals broadened 2 significantly at low metal complex/octanucleotide ratios, then effectively disappeared at r=1.0 (Figure3).Similartrendswereobservedfortheimino protons in [([9]aneS)RhCl(dpq)]Cl/d(5’-CGCTAGCG- 3 2 3’) mixtures, although the broadening was less pro- 2 nounced at lower values of r=1.0 and 2.0 before fi- nallydisappearingatr=3.0(figureS2intheSupport- ing Information). The broadening of the imino protonsignalsinbothcasescorrespondstorapidsol- vent exchange due to weakening or interruption of the Watson–Crick hydrogen bonds. Both the broader signals and the upfield shifts of the resonances are clearly indicative of the adoption of an intercalative bindingmodebythedppzanddpqcomplexes. Figure2.CDspectraofCTDNAandmixturesofa)thedpqcomplex7andb)thedppz Addition of the dppz complex to the octanucleo- complex8withCTDNAatvariousratiosrina10mmphosphatebuffer(pH7.2)afterin- cubationperiodsof60min.Molarellipticities[q]aregivenindegcm2dmol(cid:3)1(cid:3)10(cid:3)3. tide induced extreme broadening of the aromatic H6/8resonancesofthecentralC3,T4,A5andG6nu- cleobases, even at low complex/ octanucleotide ratios of r=0.1 and 0.5 (Figure4). Although the extent of line broadening pre- vented a detailed two-dimen- sional NOESY analysis of the binding interaction, 1HNMR spectra did provide useful infor- mation concerning the preferred intercalation site(s). The extent of peak widening for the C3–G6 nucleobases suggests that the metal complex intercalates at more than one of the possible sites in the central sequence of theoligonucleotide,andthatthe resulting binding forms display intermediate exchange kinetics on the NMR time scale. A possi- ble preference for intercalation Figure3.Changesintheiminoprotonregion(12.0–14.0ppm)ofthe1HNMRspectrumofd(5’-CGCTAGCG-3’) at between the C3/G6’ and T4/A5’ 2 293Kuponadditionofthecomplex[([9]aneS)RhCl(dppz)]Cl (8)atr=0.1,0.5and1.0. base pairs, and their palindromic 3 2 1126 www.chemmedchem.org (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2010,5,1123–1133 Rhodium(III)CrownThiaetherComplexes thoughalackofcrosspeakspre- vented definite identification of the H4/7 resonances, NOEs be- tween H2/9 and [9]aneS pro- 3 tons in the 3.4–3.9ppm range (figureS3 in the Supporting In- formation) did allow the assign- ment of the broad resonance at 9.21pm to this proton pair. Only one dppz···nucleotide NOE could be detected in the two-dimen- sional NOESY spectrum at r=2. Although it could not be as- signed with certainty, it seems probable that the identity is cor- rect, due to a short contact be- tween dppz H2/9 or H4/H7 pro- tonsandthesugarH2’protonof T4. The presence of several reso- nances for both the H2/9 and H4/7 protons at r=2.0 and the fact that the NOE could not be Figure4.One-dimensional1HNMRspectraoftheoctanucleotided(5’-CGCTAGCG-3’) itselfandmixturesof 2 [([9]aneS)RhCl(dppz)]Cl (8)withtheoctanucleotideinPBScontaining20mmNaClatpH7.2and293K. detected at r=1.0 suggest that 3 2 it may result from a specific 2:1 binding mode, such as a sym- equivalents A5/T4’ and G6/C3’, was indicated by similar de- metrical bis-intercalation mode of two individual metal com- greesofbroadeningforthearomaticprotonsofallfourcentral plexesbetweenC3andT4andC3’andT4’. nucleobases.Supportingevidenceforintercalationwasprovid- Similar binding trends could also be observed for the ed by the marked upfield shifts experienced by resonances of [([9]aneS)RhCl(dpq)]Cl/octanucleotide mixture, although in 3 2 the dppz H2/9 and H4/7 protons (see structure of complex 8 this case the degree of line broadening was much less pro- above for the dppz numbering). These signals shifted from nounced than for the dppz complex (see figureS4 in the Sup- 9.75 and 9.43ppm, respectively, to the 9.4–8.6ppm range in portingInformationforone-dimensional1HNMRspectrawithr the complex/octanucleotide mixture at r=2.0 (Figure5). Al- valuesbetween0and3.0).Asillustratedinthetwo-dimension- al NOESY spectrum for the reac- tion mixture at r=3.0 (Figure6), selective broadening was ob- served for the resonances of the four central nucleotides C3–G6. The fact that, with the exception of C3/T4, individual nucleobase H6/H8 resonances could still be clearly assigned at r=3.0 (fig- ureS4 in the Supporting Infor- mation) suggests that the strength of the binding interac- tion with the octanucleotide must be significantly weaker than for the dppz complex. A single intermolecular dpq···nu- cleotide NOE could be observed at r=3.0, but again, not at r= 1.0. On the basis of chemical shift values (2.37/9.27ppm), it was tentatively assigned to a short contact between dpq H2/9 Figure5.The7.1–9.5ppmrangeforaromaticprotonresonancesintheone-dimensional1HNMRspectrumofthe or H4/7 protons and the sugar [([9]aneS)RhCl(dppz)]Cl/d(5’-CGCTAGCG-3’) mixtureatr=2.0inPBScontaining20mmNaClatpH7.2and293K. H2’protonofT4andistherefore 3 2 2 ChemMedChem2010,5,1123–1133 (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 1127 MED W.S.Sheldricketal. site, such as an intercalation site. K values of 5.17(cid:3) b 106 (s=3.4) and 1.26(cid:3)106 (s=1.53), similar to those of 8, have been reported for the dppz complexes [(tpm)Ru(dppz)(py)]2+ and [(h5-CMe)Rh(HmetOMe)- 5 5 2 (dppz)]3+, where tpm=tris(pyrazolyl)methane, py= pyridine, HmetOMe=l-methionyl methyl ester, re- 2 spectively.[31,32] The significantly lower K value for b complex 7 is in accordance with the weaker binding interaction indicated by 1HNMR titration data for 9 with the octanucleotide d(5’-CGCTAGCG-3’). General 2 compliancewiththenearestneighborexclusionprin- ciple for intercalative binding (s(cid:4)2) is possible for the average binding site sizes of 1.9 and 2.2 base pairsofDNA,respectively,for7and8. Viscosity measurements[33,34] generally provide the most convincing evidence for DNA intercalation. In- sertion of planar aromatic ligands, such as dppz, be- tween adjacent nucleobase pairs results in lengthen- ing and shifting of the double helix; such changes arereflectedbyanincreaseinDNAviscosity.Figure8 illustrates the dependence of reduced viscosity func- Figure6.Two-dimensionalNOESYspectrumofthearomaticdpqproton/nucleobaseH6/ H8andsugarH2’/H2’’ + T4-CH 3 regionofthe2:1mixtureof[([9]aneS 3 )RhCl(dpq)]Cl 2 / tion (h/h 0 )1=3 on the [complex]/[DNA] ratio r for the d(5’-CGCTAGCG-3’) 2 atpH7.2and293K. complexes 7–9. As (h/h 0 )1=3 is equal to (L/L 0 ), where L Figure7.Bestleast-squaresfittothemodelofBardandThorp[31,32]forUV/ Figure8.Dependenceof(h/h 0 )1=3onr([complex]/[DNA])forviscositymeas- Vistitrationdataof[([9]aneS)RhCl(dppz)]Cl (8)(20mm)ina10mmphos- urementsofCTDNAwithcomplexes7(&),8(*),9(~)ina10mmphos- 3 2 phatebuffer(pH7.2),withCT-DNAintherange0–350mm(basedonthe phatebuffer.Regressionlinesareincludedtounderlinelineardependence numberofnucleobases). (slopevalues:7,0.34;8,0.50;9,0.18). indicativeofaspecific2:1intercalationbindingmode,possibly is the length of the intercalated DNA and L is the length of 0 similartothatdiscussedaboveforthedppzcomplex. DNAalone,[33]aslopeof1istobeexpectedforidealintercala- AdditionofCTDNAtosolutionsofcomplexes7and8leads tion at lower r values. A slope of 0.97 was determined for to hypochromic shifts in their pp MLCT p–p* absorption ethidiumbromide as a representative strong intercalator using bands.UV/Vistitrationdatacanthereforebeemployedtoesti- ourexperimentalsetup.Infact,theobserved(L(cid:3)L)/(L·r)value 0 0 mate the intrinsic binding constants, K , and average binding of0.50isclosely similartothatof0.53obtained forthestrong b sites,s,fortheinteractionofthesecomplexeswiththeduplex. mono-intercalator [(h6-CMe)RuCl(dppz)](CFSO).[11] Such 6 6 3 3 Absorption data for the MLCT bands at 342 and 366nm, re- valuessuggestthatdppzintercalationbybulkyorganometallic spectively, were used for this purpose and fitted graphically half-sandwich complexes may often be associated with a using the model of Bard and Thorp[29,30] to afford least-squares marked disturbance in BDNA conformation. However, it estimates of K =4.2(cid:3)105 (s=1.9) for 7 and 7.0(cid:3)106 (s=2.2) should be noted that a much higher slope of 0.85 was report- b for 8 (Figure7). The model assumes non-cooperative, nonspe- ed for the analogous [(tpm)RhCl(dppz)]Cl complex.[12] A lower 2 cificbindingwiththeexistenceofonetypeofdiscretebinding slopevalueof0.34isobservedforthedpqcomplex,inaccord- 1128 www.chemmedchem.org (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2010,5,1123–1133 Rhodium(III)CrownThiaetherComplexes ance with the findings of the 1HNMR and UV/Vis titrations, thecomplexes6–8(pp=phen,dpq,dppz)inHEK-293cellsare and indicates a weaker intercalative binding mode for 7. Com- all somewhat higher than for the MCF-7 and HT-29 cancer petition between intercalation and alternative groove-binding cells, the opposite trend is observed for complexes 9 and 10 orcovalent-bindingmodescannotberuledoutonthebasisof of the N ligands bpm and tap. For example, an IC value of 4 50 this evidence. The much lower (L(cid:3)L)/(L·r) value of only 0.18 0.6(cid:2)0.2mmwasdeterminedforthebpmcomplextowardsthe 0 0 for bpm complex 9 confirms that the former binding mode HEK-293 cells in comparison to nearly threefold higher values does not play a significant role in DNA interactions of this of 1.7(cid:2)0.5 and 1.9(cid:2)0.1mm towards MCF-7 and HT-29 cells, highly cytotoxic agent. A similar slope value of 0.20 was ob- respectively. tainedfortheanalogoustapcomplex10. The present invitro cytotoxicity results for the novel com- pounds 1–4 allow two additional structure–activity relation- ships to be postulated: complexes containing a monodentate Cytotoxicityandstructure–activityrelationships kN ligand and two chloride ligands are less active than those In vitro cytotoxicity studies of compounds 1–10 were per- containing a bidentate k2N (imino) ligand and one chloride formed with MCF-7, HT-29 and HEK-293 cell lines, with the re- ligand;andcomplexeswithborderlineorsoftbidentateligand sulting IC values listed in Table1. The antiproliferative effects donor atoms (e.g., k2N(imino) or k2S (thiaether)) are more 50 of the compounds were determined by an established proce- active than those containing hard bidentate ligand donor dure,[35]whichwasadaptedinthisworkforHEK-293cells.Opti- atoms(e.g.,k2N(amino)). Thefirsthypothesisissupportedbyalackofcytotoxicityfor the imidazole complex 3 towards MCF-7 cells, and the low ac- Table1. IC valuesforcomplexesofthetype[([9]aneS)RhCl(LL)]2+ (1–2, tivity of the acetonitrile complex 4 towards all three cell lines. 50 3 5–10) or [([9]aneS 3 )RhCl 2 (L)]+ (3, 4) towards MCF-7, HT-29 and HEK-293 A similar trend has also been reported for half-sandwich RuII celllines. complexes of the type [(h6-arene)RuCl(LL)]+ (LL=k2N(amino) Complex Ligand IC [a][mm] ligand), in comparison to those with two chloride ligands and 50 MCF-7 HT-29 HEK-293 a neutral monodentate ligand (L).[36] Piperazine and 1,4-di- [([9]aneS)RhCl(LL)]2+ thianewerechosenasstructurallyanalogousbidentateligands 3 1 piperazine >100 >100 >100 in complexes 1and 2toallowadirectcomparison of thepos- 2 1,4-dithiane 9.6(2.2) 8.1(3.7) 11.6(2.9) sible influence of hard or soft donor atoms on the activity of 5 bpy 12.8(0.2) 4.4(0.1) >100 the [([9]aneS)Rh]3+ complexes. While the piperazine complex 6 phen 36.3(6.0) 72.2(8.0) >100 3 7 dpq 19.1(0.3) 20.9(2.8) 45.5(1.9) 1 with its hard N(amino) donor atoms is inactive towards all 8 dppz 4.7(0.5) 7.4(2.2) 8.9(1.6) three cell lines, the 1,4-dithiane complex 2 with its soft 9 bpm 1.7(0.5) 1.9(0.1) 0.6(0.2) S(thiaether) donor atoms exhibits promising IC values in the 10 tap 11.5(0.6) 7.5(4.8) 3.3(1.3) 8.1–11.6mmrange. 50 [([9]aneS)RhCl(L)]+ 3 2 3 imidazole >100 n.d. n.d. These findings suggest a possible dependency of the cyto- 4 CHCN 51.1(15.6) 62.6(20) 96.7(2.0) toxicity of rhodium complexes 1–4 on the lability of their Rh(cid:3) 3 Cl bonds. The hard nitrogen atoms of piperazine should be [a]n.d=notdetermined.Valuesinparenthesesarethestandarddeviation (SD)of2–3experiments. much more effective at reducing the high positive charge on the central Rh atom than the soft sulfur atoms of 1,4-di- thiane.[15]Substitutionreactionswith nontarget anionicspecies mum parameters for HEK-293 cell growth were established on in the cell may be favored for 1 in comparison with 2, owing thebasisofagrowthcurveandledtoapreparationprocedure totheresultingmorelabileRh(cid:3)Clbondintheformercomplex. involving an initial 2day incubation period to obtain 2000 The presence of two anionic chloride ligands in complexes 3 cellsmL(cid:3)1 prior to replacement of the initial medium with a and 4 should also lead to more effective charge reduction for medium containing the compounds. Following incubation in the central 4d metal, which will in turn favor ligand exchange 96wellplatesat378Cin5%CO/95%airatmospherefor48h, reactions in comparison to 1, and particularly, 2. The observa- 2 IC values weredeterminedfortheHEK-293cells usingacrys- tion of significantly higher cytotoxicity for the kinetically more 50 tal violet assay. Further details of the procedure are given in stable 1,4-dithiane complex 2 suggests that the rate of chlo- theExperimentalSection. ride exchange may play an important role in timing its activa- The previously reported IC values[13] for the pp complexes tionthroughatargetbiomolecule. 50 5–10 towards MCF-7 and HT-29 cells allowed two important structure-activityrelationshipstobeestablished:Thecytotoxic- Apoptosisinductioninlymphomaandhealthyleukocyte ity of the complexes increases significantly with increasing pp cells ligand size in the series pp=phen, dpq, dppz; and complexes containing the nitrogen-rich N ligands bpm and tap are more On the basis of the results shown in Table1, complexes 8 and 4 active than those containing their N counterparts bpy and 9 were selected as the most potent complexes from the 2 phen. rhodium(III) crown thiaether series 1–10 for a comparative Similartrendswereestablishedforthesecomplexes towards apoptosis induction study towards BJAB lymphoma and the immortalized HEK-293 cells. However, while IC values for healthy exvivo leukocyte cells. Additionally, the choice of 50 ChemMedChem2010,5,1123–1133 (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 1129 MED W.S.Sheldricketal. these specific complexes was advantageous due to the possi- Comparativemeasurementswithhealthyleukocytessuggest bility of different modes of interaction with DNA and other ahighdegreeofcellselectivityforthemorepotentbpmcom- biomolecules. Both spectroscopic techniques (CD, NOESY, UV/ plex 9 towards malignant lymphoma cells. As illustrated in Vis) and viscosity titrations clearly confirm that the dppz com- Figure10, 81.0% and 70.5% of theleukocytes are still vital fol- plex intercalates into the DNA duplex. In contrast, no signifi- cantchangesintheCDspectrumofthedoublehelixwereob- servedinthepresenceofthebpmcomplex. StainingwithannexinV–FITC,thencounterstainingwithpro- pidium iodide (PI), provide a means of estimating the number of vital, apoptotic and necrotic cells following incubation with cytotoxicagents.Duringapoptosis,thephospholipidphospha- tidylserine is exposed to the outer leaflet of the plasma mem- brane.[37,38] AnnexinV–FTTC then binds to phosphatidylserine, leading to an increase in fluorescence that is dependent on thetotalpercentageofcellsundergoingapoptosisandwheth- er this is early or late. On the other hand, PI is excluded from cells with intact membranes. PI positivity is, therefore, an indi- cator for cell necrosis, whereas cells that are annexinV–FITC Figure10.PercentagesofviableBJABcellsandhealthyleukocytesfollowing incubationwithcomplexes8and9for48h.Thevaluesarenormalizedwith positiveandPInegativeareclassifiedasapoptotic.[39] respecttothecontrolvalues(100%). Measurements were performed with BJAB cells and exvivo healthy leukocytes, following an incubation period of 48h withcomplexes8and9.Figure9illustratesthecell-statedistri- lowing 48htreatments with10mmand 25mmsolutionsofthe complex, respectively. These values are normalized with re- spect to values measured for the untreated control samples, whicharesetto100%.Althoughthepercentageofvitalleuko- cytes (64.6%) is lower following incubation with a 25mm solu- tionofthedppzcomplex8,thisvalueisstillmuchhigherthan thenormalizedpercentage(23.4%)ofviableBJAB cellsrecord- edundersimilarconditions. Conclusions On the basis of invitro measurements, the rhodium(III) crown thiaether complexes [([9]aneS)RhCl(pp)]Cl 8 (pp=dppz) and 3 2 9(pp=bpm)arepromisingcytostaticagentsforthetreatment of breast and colon carcinoma, as well as lymphoma. Similar Figure9.StateofBJABcellsaftertreatmentwithcomplexes8and9for IC values were determined for immortalized HEK-293 cells in 50 48h.NecroticcellsarePIpositive,whilecellsthatareannexinV–FITCposi- comparison to the similarly adherent MCF-7 and HT-29 cancer tivebutPInegativeareconsideredtobeapoptotic. cells. Both complexes 8 and 9 exhibit significant selectivity for nonadherent lymphoma cells in comparison to healthy leuko- cytes; this distinction is particularly pronounced for the bpm butions for BJAB cells after treatment with dppz complex 8 or complex, suggesting that toxic side effects may be much bpmcomplex 9atconcentrationsof9,10and25mm.Necrosis lowerfor9thantypicallyobservedforrhodiumcomplexesand values ((cid:5)1.7%) can be regarded as negligible for both com- emphasizing its potential as a novel anticancer agent. Com- plexes. While at the lower 9mm concentration, 86.6% of the plexes8and9induceapoptosisviatheintrinsicmitochondrial BJAB cells are still vital after incubation with complex 8 for pathway,[13]andcausenegligiblenecroticdamage. 48h, the cytotoxic influence of the rhodium complex is clearly Our cytotoxicity assays for complexes 1–10 indicate that evidentforthelymphomacellsatthehigher25mmconcentra- complexes of the type [([9]aneS)RhCl(LL)]2+ (LL=pp, 1,4-di- 3 tion, where only 22.3% of the malignant BJAB cells were still thiane)aremuchmoreactivethanthoseoftheform[([9]aneS)- 3 vitaland76.7%wereapoptotic. RhCl(L)]+ (L=Imidazole, CHCN), which have two chloride 2 3 AnnexinV–FITC staining experiments indicated that the ligands. NOESYand UV/Vis binding studies, together with vis- bpmcomplex9isconsiderablymoreactivetowardsBJABcells cositymeasurements,confirmstrongDNAintercalativebinding than its dppz counterpart 8. Only 14.0% of the lymphoma for the dppz complex 8 and indicate a weaker interaction of cells werestillvital after 48hincubation witha10mmsolution the same type for the dpq complex 7. In contrast, no signifi- of 9, and this value decreases marginally to 11.8% at the cant disturbance of the DNA Bconformation is observed in higher 25mm concentration. Respectively, 85.0% and 87.4% of mixturesoftheduplexwiththehighlypotentbpmcomplex9. theBJABcellsunderwentapoptosisfollowingthetreatment. Both these observations and the structure–activity relation- 1130 www.chemmedchem.org (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim ChemMedChem2010,5,1123–1133 Rhodium(III)CrownThiaetherComplexes ships (IC values: bpm<bpy and tap<phen; IC values: 0.4mm sonicated DNA solution in a phosphate buffer (10mm, 50 50 dppz<dpq<phen) suggest that different mechanisms of pH7.2).Complexsolutions(0.2mm)alsocontainedsonicatedDNA actionmaybeinvolvedforthedppzandbpmcomplexes. atthesameconcentrationasintheviscometer(0.4mm)andwere added in 50–800mL increments. Reduced viscosities h (h =re- 0 duced viscosity of the DNA solution in the absence of complex) Experimental Section a w g e a r i e ns c t alc r u ( l r a = te [ d co b m y p th le e x] l / i [ t D er N a A tu ] re w m he e r t e ho [D d, N [33 A ] ] an is d g p i l v o e t n ted in as m (h ( / b h a 0 ) s 1 e =3 pairs). Ethidium bromide was employed as a standard intercalator Materials and instrumentation: UV/Vis spectra were recorded to confirm experimental conditions in a separate determination. withanAnalytikJenaSPECORD200spectrometer.AJascoJ-715in- ConcentrationsofCTDNAinm(basepairs)weredeterminedspec- strument was used to measure CD spectra in the 220–500nm trophotometrically using the molar extinction coefficient e = range for complex/DNA mixtures ([DNA]=M (base pairs)) in a 260 10mm phosphate buffer at pH7.2. Liquid secondary ion mass 13200m(cid:3)1cm(cid:3)1.[47] spectrometry data (LSIMS) were registered with a Fisons VG Auto- UV spectroscopy: Complexes 7 and 8 at 20mm concentration spec employing a cesium ion gun (17kV), with 3-nitrobenzyl alco- weretitratedwithsonicatedCTDNAoverarangeofDNAconcen- hol as the liquid matrix. Elemental analyses were performed on a trations from 0–350mm. All UV spectra were measured after equi- VarioELofElementarAnalysensystemeGmbH.RhCl·3HOwaspur- librium was achieved. Titration curves were constructed from the 3 2 chased from ABCR; piperazine, 1,2-diaminobenzene, imidazole, fractional change in the absorption concentration as a function of 2,2’-bipyridine, 2,2’-bipyridine-4,4’-dicarbonic acid, 1,10-phenan- DNAconcentrationaccordingtothemodelofBardandThorp.[29,30] throline, pyrazino[2,3-f]quinoxaline (tap), ethidium bromide and Equation1 was used to fit the absorption data by least-squares NaN were purchased from Acros Organics. 2,2-Bipyrimidine was refinementofbindingconstants(K)andsitesizes(s): 3 b obtained from Alfa Aesar (Karlsruhe, Germany), and 1,4,7-trithiacy- c S l i o g n m o a n – a A n l e d , ric 1 h ,4 ( - M di u th n i i a c n h e ,G a e n rm d an c y a ) lf .T t h h e ym m u o s de D lp N e A pt ( id C e T sH D - N A A G ) XV fr G o A m - ðe a (cid:3)e f Þ=ðe b (cid:3)e f Þ¼ðb(cid:3)fb2(cid:3)2K b 2C t ½DNA(cid:6)=sg1=2Þ=2K b C t ð1Þ GLIK-OH (X=C, D, H, M) were purchased from PSL (Heidelberg, b¼1þK b C t þK b ½DNA(cid:6)=2s Germany) and the octanucleotide d(5’-CGCTAGCG-3’) from Bio- 2 mers.net GmbH (Ulm, Germany). The ligands dpq[40] and dppz[41] wheree istheextinctioncoefficientobservedatagivenDNAcon- a were prepared in accordance with literature procedures as were centration,e istheextinctioncoefficientofthecomplexintheab- f the complexes [([9]aneS)RhCl(pp)]2+ (pp=bpy, phen, dpq, dppz, senceofDNA,e istheextinctioncoefficientofthecomplexwhen 3 b bpm,tap)5–10.[13] fullybound toDNA,K is theequilibrium bindingconstant inm(cid:3)1, b c isthetotalmetalconcentration,[DNA]istheDNAconcentration Mass spectroscopy: ESI-MS and MS/MS spectra were collected on t in molarity, and s is the size of the binding site. Values of e were a Finnigan LCQ mass spectrometer (Thermo Electron Corp., San b obtained by extrapolation from the y intercept of plots of e/e Jose,USA),whichwasoperatedinthepositiveionmodewithaca- a f versus1/[DNA]. pillarytemperatureof2008Candasprayvoltageof1.8kV.Samples were prepared by incubating the model peptides H-AGXVGAGLIK- [([9]aneS)RhCl(pip)]Cl·2HO (1): RhCl·3HO (100.0mg, 3 2 2 3 2 OH(X=C,D,H,M)withcomplex6ata1:1molarratiofor24hat 0.38mmol) in HO (20mL) was added to a solution of piperazine 2 378C.ThepHofthereactionmixtureswasreducedtopH4.0prior (32.7mg, 0.38mmol) in HO (10mL) then stirred for 2h at 858C. 2 to ESI-MS/MS experiments. Delivery to the mass spectrometer was After addition of [9]aneS (68.4mg, 0.38mmol), the resulting sus- 3 performed at a flow rate of 1.0mLmin(cid:3)1. The relative collision pension was heated at reflux for a further 3h. Following filtration energyforcollision-induceddissociationwassetat30%. ofayellowprecipitateof[([9]aneS)RhCl]andsolventremoval,the 3 3 resulting red residue was washed with HO and dried under NMR spectroscopy: Bruker DPX 200 and DRX 400 spectrometers 2 vacuum (65.5mg, 33%); 1HNMR (200MHz, DO, 258C): d=2.86– were used for the 1HNMR spectroscopic characterization of new 2 3.92 (m, 12H, CH[9]aneS), 3.55 (m, 8H, CH, pip); LIMS: m/z (%): compounds, with chemical shifts reported as d values relative to 2 3 2 439 (17) [M(cid:3)Cl]+. Anal. calcd for C H ClNRhS·2HO (M=511.8): the residual signal of the deuterated solvent. The splitting of 10 22 3 2 3 2 C 23.5%, H 5.1%, N 5.5%, S 18.7%, found C 23.6%, H 5.2%, N protonresonancesaredefinedass=singlet,d=doublet,t=triplet 6.0%,S18.1%. and m=multiplet. The octanucleotide spectra with WATERGATE solvent suppression were recorded at 600.13MHz proton frequen- [([9]aneS)RhCl(1,4-dithiane)]Cl·0.5HO (2): Prepared as for 1 in 3 2 2 cy and at 293K on a Bruker DRX600 spectrometer equipped with HO (30mL) with RhCl·3HO (100.0mg, 0.38mmol) and 1,4-di- 2 3 2 apulsedfieldgradientandatripleresonanceprobehead.[42–44]The thiane (33.5mg, 0.38mmol). Following evaporation of HO, 2 one-dimensional 1HNMR spectra were recorded with a time [9]aneS (68.4mg, 0.38mmol) was added to the resulting inter- 3 domain of 32k data points and a spectral width of 12019.23Hz. mediate [RhCl(HO)(1,4-dithiane)] in CHOH/CHCl (1:1; 20mL) to 3 2 3 2 2 Thesweepwidthofthetwo-dimensionalhomonuclearspectrawas give the title complex (130mg, 66%); LSIMS: m/z (%): 473 (100) 12019.23Hz in the direct and in the indirect 1H dimension. The [M(cid:3)Cl]+, 437 (52) [M(cid:3)Cl(cid:3)HCl]+; Anal. calcd for free-inductiondecaywasacquiredfor340.9msandthedwelltime C H ClRhS·0.5HO(M=518.9):C23.2%,H4.1%,S30.9%,found: 10 20 3 5 2 was set to 41.6ms. Homonuclear and NOESY spectra were zero- C23.4%,H4.0%,S30.5%. filledpriortoFouriertransformationandsineapodizationfunctions [([9]aneS)RhCl(Im)]Cl (3): RhCl·3HO (100.0mg, 0.38mmol) in were applied in both dimensions. All spectra were processed with 3 3 2 MeOH (10mL) was added to imidazole (25.8mg, 0.38mmol), and NMR-Pipe and analyzed with NMRView.[45,46] Assignment and data the mixture was stirred at reflux for 2h. After addition of [9]aneS handlingwereperformedusingNMRView.[46] 3 (68.4mg, 0.38mmol) to the colorless solution, the resulting sus- Viscosity measurements: Viscosities for complex/sonicated DNA pension was heated at reflux for 2h. Following filtration of a mixtures were determined for 7–10 using a Cannon-Ubbelhode yellow precipitate of [([9]aneS)RhCl] and solvent removal, the re- 3 3 semi-micro-dilution viscometer (Series No 75, Cannon Instrument sulting residue was washed with MeOH and dried under vacuum Co.), maintained at 258C. The viscometer contained 2mL of to give complex 3 (32.0mg, 18%); 1HNMR (200MHz, DO, 258C): 2 ChemMedChem2010,5,1123–1133 (cid:2)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemmedchem.org 1131 MED W.S.Sheldricketal. d=2.99–3.70(m,12H,CH[9]aneS),7.34(d,1H,Im-b),7.38(s,1H, placedwithmediumcontainingthecompoundsingradedconcen- 2 3 Im-c),8.29(d,1H,Im-a);LSIMS:m/z(%):422(35)[M(cid:3)Cl]+,386(10) trations (six replicates). After further incubation for 96h (MCF-7 [M(cid:3)Cl(cid:3)HCl]+,353(4)[M(cid:3)Cl(cid:3)2HCl]+ Anal.calcdforCH ClNRhS and HEK-293) or 72h (HT-29), the medium was removed and the 9 16 3 2 3 (M=457.2):C23.6%,H3.5%,N6.1%,S21.0%,found:C23.4%,H plates treated with glutaraldehyde and PBS as described above. 3.8%,N6.1%,S21.0%. The cell biomass was determined for HEK-293 and the other cell lines by crystal violet staining. Firstly, the PBS was removed, fol- [([9]aneS)RhCl(CHCN)](CFSO)·1.5HO (4): Ag(CFSO) 3 2 3 3 3 2 3 33 lowed by addition of 100mL of 0.02m crystal violet solution, after (256.9mg, 2mmol) was added to a solution of RhCl·3HO 3 2 whichtheplateswereincubatedfor30min.Thecrystalvioletsolu- (263.3mg, 1mmol) in CHCN (50mL), and the reaction mixture 3 tion was subsequently removed and the cell biomass washed was refluxed for 16h. Following removal of the AgCl precipitate, twice with water. The remaining crystal violet was eliminated by [9]aneS (180.6mg, 1.0mmol) in CHCN (10mL) was added and 3 3 adding more water and leaving the resulting suspension to stand the solution was refluxed for 4h. After volume reduction to 5mL, for 15min. Following the emptying and drying of the plate, the addition of EtO led to precipitation of the product, which was 2 crystal violet was extracted from the adherent cell biomass by washed with CHCN anddried undervacuum to give4(355.7mg, 3 shaking for 3h on a softly rocking rotary shaker with 180mL of 62%); 1HNMR (200MHz, CDOD, 258C): d=2.04 (s, 3H, CHCN), 3 3 70% ethanol.Absorption was thendeterminedat590nmin a Fu- 3.34–3.69 (m, 12H, CH[9]aneS); LSIMS: m/z (%): 506 (8) [M(cid:3)Cl]+, 2 3 siona multiwell-plate reader (Perkin–Elmer) for HEK-293 cells, or a 434 (15) [M(cid:3)Cl(cid:3)HCl(cid:3)CHCN]+, 396 (17) [M(cid:3)CFSO]+; Anal. calcd 3 3 3 Flashscan Analytik Jena instrument for MCF-7 and HT-29 cells. The for CH ClFNORhS·1.5HO: C 18.9%, H 3.2%, N 2.5%, found: C 9 15 2 2 3 4 2 mean absorption of the initial biomass t plate was subtracted 18.3%,H2.8%,N2.5%. 0 from the mean absorption of each experiment and control. IC 50 values were determined as those concentrations causing 50% in- hibitionofcellproliferation.Resultswerecalculatedfrom2–3inde- Biologicalinvestigations pendentexperiments. Cell cultures: MCF-7 breast cancer and HT-29 colon carcinoma AnnexinV–propidiumiodidebindingassay:Forthisassay,1(cid:3)105 weremaintainedin10%(v/v)fetalcalfserum(FCS)containingcell cellswere washedtwicewithcoldPBSandthenresuspendedina culture medium (minimum essential Eagle’s supplement with binding buffer (10mm N-(2-hydroxyethyl) piperazin-N0–3(propan- NaHCO (2.2g),sodium pyruvate (110mgL(cid:3)1)and gentamycinsul- sulfonic acid) (HEPES)/NaOH (pH7.4), 140mm NaCl, 2.5mm CaCl) 3 2 fate (50mgL(cid:3)1) adjusted to pH7.4) at 378C under 5% CO and at a concentration of 1(cid:3)106 cellsmL(cid:3)1. Next, 5mL of AnnexinV– 2 passagedtwiceweeklyaccordingtostandardprocedures.ForHEK- FITC (BD Pharmingen, Heidelberg, Germany) and 10mL of 293cells,Dulbecco’smodifiedEaglemedium(Invitrogen,Karlsruhe, 50mgmL(cid:3)1 PI (Sigma–Aldrich, Taufkirchen, Germany) were added Germany), supplemented with 10% fetal bovine serum and 100 to the cells. Analyses were performed on a FACScan (Becton Dick- unitsmL(cid:3)1 each of penicillin and streptomycin was employed at inson,Heidelberg,Germany)usingtheCellQuestanalysissoftware. 378C/5% CO. The cells were split and aliquots were seeded in 2 35mm culture dishes three times a week. Burkitt-like lymphoma (BJAB)cellsweremaintainedat378CinRPMI1640(GIBCO,Invitro- Acknowledgements gen) supplemented with 10% heat-inactivated FCS, penicillin (100 000UL(cid:3)1),streptomycin(0.1gL(cid:3)1)andl-glutamine(0.56gL(cid:3)1).The cells were sub-cultured every 3–4 days by diluting the cells to a Financial support for this work in Berlin and Bochum from the concentration of 1(cid:3)105 cellsmL(cid:3)1. 24h before the start of the Deutsche Forschungsgemeinschaft (DFG), within the research assay,cellswereculturedataconcentrationof3(cid:3)105cellsmL(cid:3)1to group FOR 630 “Biological function of organometallic com- ascertainstandardizedgrowthconditions.Fortheapoptosisassays, pounds,” is gratefully acknowledged. R. Bieda wishes to thank cells were diluted to a concentration of 1(cid:3)105 cellsmL(cid:3)1 immedi- both the Ruhr University Research School and the WASAG Foun- atelybeforeadditionofthecomplexes. dation, and M. Dobroschke thanks the Kind-Philipp Foundation Cytotoxicity measurements: Antiproliferative effects of the com- of the Stifterverband f(cid:2)r die Deutsche Wissenschaft for scholar- poundsweredeterminedbyestablishedproceduresfortheMCF-7 ships. We also thank Prof. Dr. R. Stoll and Dr. J.-A. Bangert for and HT-29 cells.[35] A modification of this procedure was used in helpful discussions, and C. Frias and H. Scheffler for excellent this work for the HEK-293 cells. Initial determination of a growth technicalsupport. curve provided the HEK-293 specific parameters of cell concentra- tion and incubation periods. Cells were suspended in cell culture medium (MCF-7: 10000 cellsmL(cid:3)1; HT-29: 2850 cellsmL(cid:3)1; HEK- Keywords: apoptosis · cell selectivity · cytotoxicity · 293: 2000 cellsmL(cid:3)1) and 100mL aliquots of each were plated in polypyridylligands·rhodium 96-well plates and incubated at 378C/5% CO for 72h (MCF-7), or 2 48h (HT-29 or HEK-293). 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