Synthesis, biological activity, and structure-activity relationships for potent cytotoxic rhodium(III) polypyridyl complexes.

3924 J.Med.Chem.2008,51,3924–3933 Synthesis, Biological Activity, and Structure-Activity Relationships for Potent Cytotoxic Rhodium(III) Polypyridyl Complexes Melanie Harlos,† Ingo Ott,‡ Ronald Gust,‡ Hamed Alborzinia,§ Stefan Wo¨lfl,§ Anna Kromm,† and William S. Sheldrick*,† Lehrstuhlfu¨rAnalytischeChemie,Ruhr-UniVersita¨tBochum,D-44780Bochum,Germany,Institutfu¨rPharmazie,FreieUniVersita¨tBerlin, Ko¨nigin-Luise-Strasse2-4,D-14195Berlin,Germany,andInstitutfu¨rPharmazieundMolekulareBiotechnologie,Ruprecht-Karls-UniVersita¨t Heidelberg,ImNeuenheimerfeld364,D-69120Heidelberg,Germany ReceiVedFebruary19,2008 Thecomplexesmer-[RhCl (DMSO-κS)(pp)]1a-5amaybepreparedbyreactionofmer,cis-[RhCl (DMSO- 3 3 κS) (DMSO-κO)] with the appropriate polypyridyl ligand (pp ) bpy, phen, dpq, dppz, dppn) in CH OH/ 2 3 H Osolutionat75°C.Themerisomersof1a-5aarestableinchloroformsolutionbutthoseof1aand2a 2 isomerizerapidlytoamixtureoffacandmerisomersinDMSO.Thecomplexesarepotentinvitrocytotoxic agentsandexhibitIC valuesthatarestronglydependentonthesizeofthepolypyridylligand.IC values 50 50 of, respectively, 4.0 (0.5) and 1.9 (0.5), 0.40 (0.06) and 0.19 (0.05), and 0.079 (0.012) and 0.069 (0.021) µMareobservedfor1a-3aagainstthehumancelllinesMCF-7(breastcancer)andHT-29(coloncancer). Cellularuptakestudiesshowedarapidandhighaccumulationofthepolypyridylcompounds.Treatmentof HT-29andMCF-7cellswith3aleadstosignificantdecreasesincellularoxygenconsumptionandtherate of extracellular acidification. Introduction Although dirhodium(II, II) carboxylates have received con- siderableattentionasanticanceragents1–4owingtotheirlimited side effects, relatively few reports of cytotoxic rhodium(III) complexes have previously appeared. The trichloridorhod- ium(III) complexes mer,cis-[RhCl (DMSO-κS)(Im) ] (DMSO 3 2 ) (CH ) SO, Im ) imidazole) and mer,cis-[RhCl (DMSO- 3 2 3 κS) (L)] (L ) Im, NH ) were studied by Mestroni et al., who 2 3 establishedaremarkablecytotoxicactivity(IC )1.5(0.4, 50 0.4 ( 0.2, 9 µM) for the latter ammine complex toward the human cell lines A 2780 (ovarian carcinoma), LoVo (colon carcinoma), and Calu (lung carcinoma).5 Replacing ammonia by imidazole leads to an increase in the IC values by about 50 anorderofmagnitudeandmer,cis-[RhCl (DMSO-κS)(Im) ]is 3 2 essentially inactive against A2780 and Calu (IC > 200 µM) Figure1. Structuresofthetrichloridorhodium(III)polypyridylcom- 50 and only moderately active toward the colon carcinoma cell plexesmer-[RhCl 3 (DMSO-κS)(pp)]1a-5a(pp)bpy,phen,dpq,dppz, line LoVo (IC ) 40 ( 15 µM). Significant activity against dppn). 50 mouse P 388 leukemia has, however, been reported for the analogous compound mer,cis-[RhCl (DMSO-κS)(py) ] (py ) oruthenium(II) complexes of the type [(η6-C 6 Me 6 )RuCl(pp)]- pyridine).6 The 2,2′:6′,2′′-terpyridin 3 e (tpy) complex 2 es mer- (CF 3 SO 3 ) are directly correlated to the size of the polypyridyl ligand.9 A similar trend is observed for the cytotoxicities of [RhCl (tpy)]and[Rh(Im)(tpy) ]Cl·3H Oalsoexhibitpotentially 3 2 2 analogous organometallic Rh(III) and Ir(III) complexes [(η5- usefulcytotoxicity,7asdoesthecompoundfac-[RhCl (9-[ane]- 3 C Me )MCl(pp)](CF SO ) (M ) Rh, Ir),10,11 and this finding NS )] (9-[ane]-NS ) 1-aza-4,7-dithiacyclononane).8a 5 5 3 3 2 2 prompted us to prepare and study the biological properties of Thesefindingssuggestthatoctahedraltrichloridorhodium(III) trichloridorhodium(III) polypyridyl complexes. We chose the complexescouldofferconsiderablescopeforthedevelopment ligandsbpy,phen,dpq,dppz,anddppntogenerateaseriesof of anticancer agents. We have recently demonstrated that the mer-complexes (Figure 1) with a steadily increasing aromatic cellular uptake and cytotoxicity toward the human cell lines surface area. Both mer and fac isomers are possible for the MCF-7 (breast cancer) and HT-29 (colon cancer) for organ- complexesofthetype[RhCl (DMSO-κS)(pp)],andtheircyto- 3 toxicitiesmaywellbeexpectedtodependnotonlyonthesize *To whom correspondence should be addressed. Phone: +49-234- of the polypyridyl ligand but also on the rate of ligand 3224192.Fax:+49-234-3214420.E-mail:william.sheldrick@rub.de. substitutioninaqueoussolution.Thelatterparameterwillitself †Ruhr-Universita¨tBochum. depend on both the general kinetic inertness of octahedral ‡FreieUniversita¨tBerlin. complexes of the group 9 metal (k(Rh) > k(Ir)) and on the §Ruprecht-Karls-Universita¨tHeidelberg. aAbbreviations: bpy, 2,2′-bipyridine; phen, 1,10-phenanthroline; dpq, specific trans effect of the constitutive ligands (DMSO-κS > dipyrido[3,2-f:2′,3′-h]quinoxaline; dppz, dipyrido[3,2-a:2′,3′-c]phenazine; Cl > pp-κN > H O). Nonorganometallic complexes of the 2 dppn, benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine; Im, imidazole; DMSO, heavier homologue iridium(III) are generally considered to be dimethylsulfoxide;LSIMS,liquidsecondaryionmassspectrometry;AAS, kinetically too inert to exhibit significant cytotoxicity and an atomicabsorptionspectrometry;ISFETS,ionsensitivefieldeffecttransistors; IDES,interdigitatedelectrodestructures absence of biological activity has indeed been confirmed for 10.1021/jm800173sCCC:$40.75 2008AmericanChemicalSociety PublishedonWeb06/11/2008 PotentCytotoxicRhodium(III)PolypyridylComplexes JournalofMedicinalChemistry,2008,Vol.51,No.13 3925 (60:40) is observed for solutions of 1a and 2a after 15 min in DMSO.Equilibrationismuchslowerfor3a–5a,e.g.3a/3bare present in a 75:25 ratio in DMSO after 24 h. As depicted for the1a/1bmixtureinFigure5,theprotonsH2/H9,H3/H8,H4/ H7 and H5/H6 are magnetically equivalent for the C sym- s metricalfacisomers.Amarkedupfieldshiftfromtwoseparate doublets at 9.78 and 9.83 ppm to a common doublet at 9.47 ppm is observed for the H2 and H9 protons of 1a on isomerization to 1b. The average positions for the remaining bpyprotonsremaineffectivelyunchangedongoingfrom1ato 1b.Weweresuccessfulincrystallizingthelatterfacisomer1b (Figure 6) on slow evaporation of a CH OH/H O solution of 3 2 the original mer isomer 1a. The relative strength of the trans influence of the κS DMSO ligand can be gauged in 1b by comparingthelengthofRh1-Cl1withRh1-Cl2andRh1-Cl3. The former bond [2.366(1) Å] is significantly longer than the latterbonds[2.332(1),2.349(1)Å]intranspositiontothebpy nitrogen atoms N1 and N10. Figure2. Molecularstructureofmer,cis-[RhCl(DMSO-κS)(DMSO- 3 2 κO)]. Rapidisomerizationtoequilibriummixturesofmerandfac isomers is also observed on dissolving 1a and 2a in the polar [ImH][trans-IrCl(DMSO-κS)(Im)]and[(DMSO)H][trans-IrCl- solvents CD OD and D O. This is accompanied by a limited 4 2 4 3 2 (DMSO-κS) ].12 degree of slow DMSO/CH OD exchange in methanol and 2 3 almostcompleterapidDMSOsubstitutioninaqueoussolution. Results and Discussion The observed 1H NMR ratios of coordinated DMSO to free Synthesis.Therhodium(III)complexesmer-[RhCl (DMSO- DMSO are 95:5 and 93:7 for the bpy and phen complexes in 3 κS)(pp)] 1a-5a (pp ) bpy, phen, dpq, dppz, dppn) were pre- methanol after 24 h and 5:95 and 14:86 in water after 5 min. pared by treatment of the precursor mer,cis-[RhCl (DMSO- This indicates that aquation to mer/fac-[RhCl (H O)(pp)] will 3 3 2 κS)(DMSO-κO)]13–15 with an equivalent of the appropriate berapidinbiologicalsystemsandthattheaquacomplexeswill polypyridylligandinCH OH/H Osolution(1/1).Thepresence bethepotentiallybiologicallyrelevantspecies.Registrationof 3 2 ofasingleisomerisconfirmedineachcasebytheobservation satisfactory1HNMRspectraof3a-5ainCD ODorD Owas 3 2 ofjustonestrongνSOband(values1130-1118cm-1)inthe preventedbytheirpoorsolubility.Thecompound[RhCl(DMSO- 3 typical range 1100-1150 cm-1. Although the precursor was κS)(1,4-dithiane-κ2S,S)] 6 containing the cyclic bidentate 1,4- preparedinaccordancewiththeliteratureprocedurebyreaction dithiane ligand (CH CH S) was prepared for comparison 2 2 2 of RhCl 3 ·3H 2 O with DMSO followed by addition of ethanol purposes. Its 1H NMR spectrum in CDCl 3 contains two to achieve precipitation,14 we obtained a novel monoclinic resonancesofapproximatelyequalintegralintensityforDMSO polymorph on crystallization of mer,cis-[RhCl 3 (DMSO-κS) 2 - methylprotonsat3.43and3.55andis,therefore,inaccordance (DMSOκO)] by slow evaporation of a CH 3 OH/H 2 O solution. withthepresenceofa1:1mixtureofthemerandfacisomers BothpolymorphscrystallizeinthespacegroupP2 1 /cbutexhibit in solution. verydifferentunitcellconstantsandpackingarrangements.The InteractionwithDNA.Wehavedemonstratedthatorgano- structure of the new polymorph is depicted in Figure 2. As a metalliccomplexesofthetypes[(η5-C Me )Ir(L)(dppz)](CF - resultofthestrongertransinfluenceofthechlorideligandCl1 5 5 3 SO ) 11,16,17 and [(η6-C Me )Ru(L)(dppz)](CF SO ) 9,18 (L ) in comparison to the O1 atom of the κO DMSO ligand, the 3 2 6 6 3 3 2 Rh1-S3bondlengthof2.305(2)Åissignificantlylongerthan (NH 2 ) 2 CS, methionyl peptides) are strong metallointercalators thatofRh1-S2[2.256(2)Å].AnRh1-O1distanceof2.113(5) forDNAwithbindingconstantsintherange105-107M-1.A significantdegreeofintercalationisalsoobservedforcomplexes Å is observed for the κO DMSO ligand. The1HNMRspectrumforthemercomplex5a(pp)dppn) containingthesmallerdpqligandbutnotforthoseofphen16,11 ordppn,9,11whichareapparentlyrespectivelytooshortortoo inCDCl solutionisdepictedinFigure3asatypicalexample 3 long to facilitate effective side-on intercalation between the fortherhodium(III)polypyridylcomplexes.Asaresultofthe nucleobases of the double helix.16,18 Rapid substitution of the strongertransinfluenceoftheκSDMSOligand,theresonances for the protons H7-H9 are shifted significantly (0.06-0.09 chlorideligandbynucleobaseNatomsleadstostablecovalent ppm) to lower field in comparison to H2-H4 of the pyridine DNA binding for [(η5-C 5 Me 5 )IrCl(dppz)](CF 3 SO 3 ) following ring sited trans to a chloride ligand. Confirmation of the κS initial kinetically favored intercalation.11 It was, therefore, of coordination of the DMSO ligands in 1a-5a is provided by interest to study the DNA interaction of the mer complexes the pronounced downfield shift of their methyl 1H NMR 1a-5a, in which the facial [η5-C 5 Me 5 ]- coligand is replaced resonances to 3.71-3.84 ppm in comparison to the signal of bythreekineticallyrelativelyinertchlorideligandsandalabile free DMSO at 2.55 ppm. Whereas mer,cis-[RhCl (DMSO- DMSO,andthe5dtransitionmetalisreplacedbyitslighter4d 3 κS) (DMSO-κO)]exhibitssimilarmarkedshiftsto3.62(trans homologue.ApronounceddecreaseinUV/visabsorbanceand 2 toDMSO-κO)and3.44(transtoCl)foritsκSDMSOligands, bathochromic shifts for the absorption maxima at about 364 onlyamodestlowfieldshiftto2.86ppmisobservedforitsκO and 383 nm on addition of dppz-containing metal complexes DMSO ligand in CDCl .14 to calf thymus DNA are generally indicative of possible dppz 3 The mer complexes 1a-5a are all stable in CDCl solution intercalation. However, only negligible changes are observed 3 overaperiodof24hat25°Cinthepresenceoflight,incontrast fortheabsorbancesofthesemaxima,whentheUV/visspectrum to their solutions in DMSO. Rapid isomerization to mixtures ofa20µMaqueoussolutionof4aisrecordedwithandwithout ofthemerandfacisomers(Figure4)1a/1b(29:71)and2a/2b CT DNA (M(nucleotide) ) 200 µM). The UV/vis spectra for 3926 JournalofMedicinalChemistry,2008,Vol.51,No.13 Harlosetal. Figure3. Aromaticregionofthe1HNMRspectrumofmer-[RhCl(DMSO-κS)(dppn)](5a)inCDCl solution. 3 3 an incubation period of 2 h. The molar elipticities [θ] of the typical negative and positive DNA bands are similar to those observedforDNAaloneandthelackofanegativeCDbandat 300nmisinaccordancewithanabsenceofdppzintercalation asindicatedbytheUV/visstudies.ThecharacteristicDNACD spectrumalsoremainseffectivelyunchangedontreatmentwith the other mer complexes 1a-3a and 5a. InviewoftheapparentabsenceofDNAintercalationfor3a and4a,wealsostudiedthepossibilityofacoordinationofthe rhodium complexes by nucleobase N atoms. To this purpose, thepossiblereactionofa10mMsolutionofcomplex1awith a 2-fold excess of guanosine 5′-monophosphate in a 10 mM Figure 4. Structures of the fac isomers fac-[RhCl(DMSO-κS)(pp)] 1b(pp)bpy)and2b(pp)phen). 3 phosphate buffer (pH ) 7.2) was monitored by 1H NMR spectroscopy.Incontrastto(η5-C Me )Ir(III)polypyridylcom- 5 5 the other complexes 1a-3a and 5a also exhibit no effective plexes[(η5-C 5 Me 5 )IrCl(pp)]+,forwhichtheformationofκN7 changes in absorption on mixing with CT DNA. (guanine)complexesisveryrapidat25°C,14noreactionwith Characteristicchangesinthecirculardichroism(CD)spectra thenucleobasewasobservedfor1aevenaftertreatmentat60 ofDNAinthepresenceofsmallmoleculescanprovideameans °C for 72 h. Our findings therefore suggest that neither inter- of monitoring possible conformational changes for the bio- calation nor covalent binding to DNA may be of significance polymer.19,20 Aromatic molecules often generate CD bands forthecellularactivityofthecomplexes.Theoverallneutrality between300and400nmoninteractionwithDNA,asaresult of the complexes may be assumed to be responsible for the of intercalation, surface or groove binding leading to a rigid absenceofeffectiveintercalationby3aand4a,whosearomatic orientationoftheirdipolemomentswithrespecttothedouble dpqanddppzligandswouldclearlyoffersuitablesurfaceareas helix.Wehavereported,inthiscontext,thatorganoiridium(III) for this DNA binding mode. andorganoruthenium(II)dppzcomplexesexhibitcharacteristic CytotoxicityandCellularUptake.Table1liststheinvitro inducednegativeCDbandsatλ)300nmonintercalationinto cytotoxicity of complexes 1a-5a and 6 toward the human DNA9,11,17andthatadecreaseinthemolarelipticity[θ]ofthe cancer cell lines MCF-7 (breast cancer) and HT-29 (colon positiveDNAbandintherange270-290nmcanbeindicative cancer).Itisapparentforthecomplexes1a-3athattheirIC 50 ofdpqintercalation.9FigureS2oftheSupportingInformation values(bpy>phen>dpq)arestronglycorrelatedtothesurface depictstheCDspectrumforamixtureof4awithCTDNAat areaofthepolypyridylligand.Wehavereportedasimilartrend r ) 0.1 (r ) [4a]/[DNA]) in a 10 mM phosphate buffer after for the organometallic complexes [(η5-C Me )RuCl(pp)](CF - 5 5 3 PotentCytotoxicRhodium(III)PolypyridylComplexes JournalofMedicinalChemistry,2008,Vol.51,No.13 3927 Figure5. 1HNMRspectrumofthemixtureofisomersmer-[RhCl(DMSO-κS)(bpy)]1aandfac-[RhCl(DMSO-κS)(bpy)]1binDMSO-d solution. 3 3 6 Table1. IC50Values(µM)fortheComplexesmer-[RhCl3(DMSO)(pp)] 1a-6andCorrespondingFreePolypyridylLigandsinMCF-7and HT-29Cells;n.d.:NotDetermined MCF-7IC50 HT-29IC50 complex pp complex ligand complex ligand 1a bpy 4.0(0.5) 52.7(7.8) 1.9(0.5) 45.7(4.6) 2a phen 0.40(0.06) 3.5(0.2) 0.19(0.05) 2.7(0.5) 3a dpq 0.079(0.012) 6.7(2.0) 0.069(0.021) 7.0(2.2) 4a dppz 0.095(0.020) 0.8(0.6) 0.073(0.017) 1.8(0.2) 5a dppn 0.051(0.012) 0.15(0.05) 0.070(0.008) n.d. 6 S2(CH2)4 9.0(0.5) n.d. 16.5(6.5) n.d. cisplatin 2.0(0.3) 7.0(2.0) for the relative activity of the complexes toward the different celllines.Whereas1aand2aareabouttwiceasactivetoward HT-29cells,muchsmallerrelativedifferencesareobservedfor 3aand4a,andcomplex5awiththelargestpolypyridylligand is slightly more active toward MCF-7 cells. Replacement of the polypyridyl ligand with dithiane (6) caused significantly higher IC -values. Complexes 3a-5a are extremely potent 50 Figure6. Molecularstructureoffac-[RhCl 3 (DMSO-κS)(bpy)]1b. cytotoxicagentswithIC 50 valuesintherange0.069-0.079µM, SO ),9 where the IC values for MCF-7 and HT-29 cells thataresome2ordersofmagnitudelowerthanforcisplatin.In 3 50 improvefrom11.1(1.0)and30.3(5.6)forpp)dpqover2.1 this context, it should be noted that we also observed toxic (0.6) and 2.5 (0.6) for pp ) dppz to 0.13 (0.02) and 0.4 (0.1) effects for the free polypyridyl ligands, which increase in the µMforpp)dppn.Incontrast,whereasadramaticimprovement series bpy < phen, dpq < dppz < dppn. However, the cor- isobservedforthesmallerbpy,phenanddpqligandsof1a-3a, responding complexes 1a-5a displayed significantly higher no further significant increase in cytotoxicity is apparent for activity in all the experiments. For the larger free polypyridyl the larger dppz and dppn ligands of 4a and 5a (Figure S3 of ligands (dppz and dppn), limited solubility in the assay media theSupportingInformation).Asizedependenceisalsoapparent was noted, which might limit their bioavailability. 3928 JournalofMedicinalChemistry,2008,Vol.51,No.13 Harlosetal. Figure8. Concentrationdependenceoftheuptakeof4aintoMCF-7 cells. Table2. CellularUptake(ngRh/mgCellProtein)inMCF-7andHT-29 Cellsfor1.0µMoftheComplexesmer-[RhCl3(DMSO)(pp)]1a-6 Complex pp MCF-7 HT-29 1a bpy 41.6(0.5) 24.7(9.2) 2a phen 70.8(12.2) 49.0(0.6) 3a dpq 92.1(1.4) 53.6(2.0) 4a dppz 43.1(1.9) 69.8(18) 5a dppn 74.7(1.7) 39.8(9.7) 6 S2(CH2)4 10.5(1.7) 13.3(2.1) Figure7. Timedependentuptakeof1.0µM4ainto(a)MCF-7cells and(b)HT-29cells. Rhlevelsintheorder6<1a<2a<3a≈4a≈5awastobe expected.Thistrendwasonlyobservedinpart.Ontheonehand, Extending the size of the polypyridyl ligand will confer a lower level of cellular uptake was indeed observed for the lipophiliccharactertothecomplexesandtherebyenhancetheir less toxic compounds 1a and 6, but on the other hand, the passagethroughthecellmembrane.Theneutralityofcomplexes accumulation of 2a was comparable to that of the more toxic 1a-5aand6shouldalsofavorcellularuptake.Aquantitative complexes 3a-5a. With the exception of 6 and 4a, higher ng AASstudy9forthecomplexes[(η6-C Me )RuCl(pp)](CF SO ) Rh/mgproteinlevelswerereachedinMCF-7cellsincompari- 6 6 3 3 has demonstrated that the Ru uptake increases dramatically in sontoHT-29cells.However,ontakingtheindividualcellular theorderdpq<dppz<dppnfrom1.1(1.1)and11.8(8.5)ng parameters into account (e.g., the mean cellular diameter and Ru/mg protein for the dpq complex to 906.7 (1.5) and 1054.7 themeanproteintovolumeratioofHT-2921andMCF-722cells), (94.5)ngRu/mgproteinforthedppncomplex.Ananalysisof itcanbeestimatedthat1.0ngRhpermgproteincorrrespond the IC values listed in Table 1 suggests that an increase in toacellularmolarconcentrationof1.9µMinHT-29cellsbut 50 intracellular concentration on going from 1a to 3a could lead to only 1.1 µM in MCF-7 cells. Thus, 1a-3a and 5a reached totheobserveddramaticincreaseincytotoxicityfortheseries comparablemolarlevelsinbothstudiedcelllines.Forexample, andthatasaturationlevelmaybeachievedforpp)dpq.Further the cellular molar concentration of 5a was 82 µM in MCF-7 increasesinthelipophilicityfor4aand5ahaveapparentlyno cells and 76 µM in HT-29 cells. additionalbeneficialinfluenceonthecytotoxiccellularactivity Theobservedcellularuptakevaluescanbeclassifiedasvery ofthecomplexes.Toevaluatetheuptakecharactaristicsofthe highwhencomparedtothoseofotherestablishedmetallodrugs. targetcompounds1a-5a,wedeterminedtheRhlevelsoftumor In the present study, the highest molar cellular concentration cellsexposedtothecompoundsbyatomicabsorptionspectros- (133µM)wasobservedfor4ainHT-29cells.Withrespectto copy. Initial experiments were performed exemplarily on the the exposure concentration of 1.0 µM, this means that the dppzcompound4a.Onincubationwith1.0µMof4a,cellular compound is accumulated 133 fold in the cancer cells! In Rh levels increased quickly within the first two hours of comparison, similar studies on the cellular uptake of the exposure (Figure 7). Longer incubation periods afforded no clinically used platinum drugs cisplatin, carboplatin, and ox- significantchanges,whichindicatesthatfollowingarapiduptake aliplatinhaveshownthatthesecomplexesareaccumulatedonly processessentiallystablecellularlevelswerereached.Aslight 1.5-6 fold.23 thoughnotsignificanttrendtoloweruptakevaluesisapparent Cellular Metabolism and Morphological Changes. We for the measurements after 4 and 6 h. Exposure to various havestudiedthecellularmetabolismandmorphologicalchanges concentrations of 4a (0.2-5.0 µM) for 6 h led to an almost ofHT-29andMCF-7cellsinresponsetothehighlycytotoxic linear increase (r2 > 0.99) of the cellular rhodium(III) level compounds 3a and 4a with a cell-based sensor chip system, (Figure 8). This demonstrates that the plateau levels in Figure which has the ability to monitor the biological impact of a 7arenottheconsequenceofasaturationeffectforthecellular compoundbymeasuringthreeimportantparametersofcellular uptake process. metabolisminlivingcellcultures.Theseparametersareoxygen On the basis of these results, the tumor cells were exposed consumption, the extracellular acidification rate, and changes to 1.0 µM of 1a-5a and 6 for 6 h for the celluar uptake incellularadhesionormorphology.Themetabolicsiliconchip measurements reported in Table 2. On taking the cytotoxic includesminiatureClark-typeoxygenelectrodesformonitoring activitiesofthecompoundsintoaccount,anincreaseofcellular thecellularoxygenuptake,24ion-sensitivefieldeffecttransistors PotentCytotoxicRhodium(III)PolypyridylComplexes JournalofMedicinalChemistry,2008,Vol.51,No.13 3929 Figure9. Standardrespirationrates(%)for(a)HT-29cellsand(b) Figure10. Standardextracellularacidificationrates(%)for(a)HT- MCF-7cellstreatedwith2and5µMsolutionsofcompounds3aand 29 cells and (b) MCF-7 cells treated with 2 and 5 µM solutions of 4aovertheperiod0-6h.Theendoftreatmentisindicatedbyavertical compounds3aand4aovertheperiod0-6h.Theendoftreatmentis line.Measurementwascontinuedforanadditional24h(6-30h)after indicated by a vertical line. Measurement was continued for an removalofsubstances. additional24h(6-30h)afterremovalofsubstances. torecordextracellularpHchanges,22andinterdigitatedelectrode rate of MCF-7 cells (Figure 9b) is rapidly affected by 3a structures for measuring the cellular impedance.26 Oxygen during the treatment phase and sinks to about 40% for both consumptionandacidificationrateareimportantparametersfor doses (2 and 5 µM) during the regeneration phase. Only a identifying the contribution of glycolysis and mitochondrial very limited increase is observed at both concentrations of respiration to the energy metabolism of the cells. 3a. MCF-7 cells are more affected by 4a than HT-29 cells Oxygen consumption is generally indicative of enhanced with oxygen consumption falling to about 80% for both 2 or decreased mitochondrial activity (respiration). Other and 5 µM concentrations of the compound. oxygenconsumingprocessesaremuchlessefficientandthus Extracellular acidification is closely linked to the activity unlikelytocontributesignificantlytothissignal.FromFigure of glycolysis. This parameter is chiefly influenced by lactic 9a,itisapparentthattheoxygenconsumptionofHT-29cells acid production, which is the waste product of anaerobic is strongly affected during the first hours of treatment with metabolism. Within the first 10 h (6-16 h) following the 3a. For a 2 µM solution of the complex, a rapid initial treatmentphase,asignificantdosedependentdecreaseinthe decrease to a 60% level is observed, followed by a degree extracellular acidification rate is apparent for HT-29 cells of recovery during the regeneration phase after 12 h. In treated with complex 3a (Figure 10a), and interestingly this contrast, regeneration is no longer visible for the 5 µM startsbeforetreatmentisstopped,whichindicatespermanent solution of 3a, possibly due to permanent damage of the cellular damage associated with the high cytotoxicity of the mitochondria. Both the 2 and 5 µM concentrations have compound. The marked reduction in lactic acid production similareffectsontherespirationratesforHT-29cellstreated isindicativeofamuchlowercellularactivityincomparison with complex 4a. After small positive and negative fluctua- tothenontreatedcells.TreatmentofMCF-7cellswith3aat tionsinoxygenconsumption,thesignalvaluesapproachthe a 5 µM concentration reduces the acidification rate to 40% controllevelsduringtheregenerationphase.Therespiration after 28 h, but the compound has little effect at a lower 2 3930 JournalofMedicinalChemistry,2008,Vol.51,No.13 Harlosetal. theregenerationphase,whichindicatesthepresenceofperma- nent dose dependent cellular damage. Conclusions The complexes mer-[RhCl (DMSO-κS)(pp)] are potent cy- 3 totoxicagentstowardthehumancelllinesMCF-7andHT-29 and exhibit IC values in the range 0.069-0.079 µM for the 50 larger polypyridyl ligands dpq, dppz, and dppn. A strong dependenceonthesurfaceareaofthepolypyridylligandsand therebyonthecompoundlipophilicityand/orthemer/facratio in solution is apparent for the cytotoxicities of 1a-3a, whose IC valuesdecreasefrom4.0(0.5)and1.9(0.5)µMto0.079 50 (0.012)and0.069(0.021)µMongoingfrompp)bpy(1a)to pp)dpq(3a).ThelattervalueforHT-29issome2ordersof magnitude lower than that of 7.0 (2.0) µM determined for cisplatin. Cellular uptake studies revealed high cellular levels of the targetcompounds,whichcorrelatedinparttotheextentofthe antiproliferative effects triggered by the complexes. The ex- pectedincreaseofcellularuptakelevelsongoingtothelarger polypyridylligandsdppzanddppncouldnotbeobserved,which might provide an explanation for the fact that the toxicities of 3a-5adidnotdiffersignificantlyfromoneanother.However, the impact of the pp ligands on the biological activity is demonstrated by results obtained with 6, which was used as a reference substance not containing a pp ligand. In this case, much lower cellular uptake values were obtained and these correlate with elevated IC values of 9.0(0.5) and 16.5(6.5) 50 µM for the compound in the cell growth assay. Our1HNMRstudiesindicatethatDMSOsubstitutionisrapid for1a-5ainaqueoussolutionandthatcomplexesofthetype mer-[RhCl (H O)(pp)]willprobablybethebiologicallyactive 3 2 species.OnthebasisofourUV/visandCDinvestigations,DNA may not be an important cellular target for these aqua complexes. In view of the rapid aquation of the original complexes 1a-5a, it is possible that the hard carboxylate O atoms of aspartate or glutamate side chains or the hydroxy functionsofserineorthreonineresiduescouldofferthecentral Figure11. Standardcellimpedance(%)for(a)HT-29cellsand(b) Rh(III)atomattractivecoordinationsitesinintracellularproteins. MCF-7cellstreatedwith2µMand5µMsolutionsofcompounds3a Studiesonthecellularmetabolismandmorphologicalproper- and4aovertheperiod0-6h.Theendoftreatmentisindicatedbya tieswereexemplarilyperformedon3aand4aandshowedthat verticalline.Measurementwascontinuedforanadditional24h(6-30 allmeasuredparameters(cellularoxygenuptake,extracellular h)afterremovalofsubstances. pH changes, and cellular impedance) were influenced by the presenceofthecomplexes.However,theeffectswereingeneral µM concentration (Figure 10b). The impact of complex 4a more marked for 3a. In particular, the strong effects of 3a on is much weaker for both cell lines, in particular for MCF-7, cellularoxygenconsumptionareofspecialinterestastheymight wheretheacidificationrateiscloseto100%after28h.This indicate an antimitochondrial mode of action. This is in line indicates that MCF-7 cells depend more on glycolysis for withtheresultsofarecentlypublishedstudyshowingthatRh energy production in response to treatment with the rhod- intercalators can lead to (oxidative) damage of mitochondrial DNA.27 ium(III) compounds. The impedance measurements indicate that a modification IDESmeasurementsofcellularimpedancereflecttheinsulat- of the cellular adhesion properties most probably contributes ing properties of the cell membrane. Morphological changes tothebiologicalactivityofthecompounds.Onthebasisofthe andthestatusofcellularadhesionproperties,includingcell-cell high cellular uptake rates noted for the complexes it appears andcell-matrixcontactsarealsomonitoredbycellimpedance. reasonable that a significant quantity of the agents is ac- TheimpedancesignalforHT-29andMCF-7cellswitha5µM cumulatedinthelipophilicmembraneenvironmentandcauses solutionof3adecreasesrapidlyafteraperiodofapproximately morphological changes. 3-4 h (Figure 11a,b) from commencement of treatment, Experimental Section whereas the impact of the 2 µM solution is somewhat less pronounced and delayed by a further 2-4 h in comparison. MaterialsandInstrumentation.UV/visspectrawererecorded with an Analytik Jena SPECORD 200 spectrometer and FTIR Complex4aalsocausesasignificantthoughsomewhatsmaller spectra with a Perkin-Elmer 1760X as KBr discs. A Jasco J-715 decreaseincellularimpedanceforHT-29andMCF-7cells,but instrument was employed to measure CD spectra in the range theonsetoftheeffectisdelayedforabout6haftertheendof 220-500 nm for 1:10 complex/[DNA] mixtures [complex ) 20 treatment.Thedeclineincellimpedancenowtakesplaceduring µM,DNAconcentrationinM(nucleotide))200µM]ina10mM PotentCytotoxicRhodium(III)PolypyridylComplexes JournalofMedicinalChemistry,2008,Vol.51,No.13 3931 phosphatebufferatpH7.2.Then1%DMSOwasaddedtoensure 533(19)[M-Cl]+,458(9)[M-Cl-DMSO]+,420(65)[M- solubilityof1a-5a.LSIMSspectrawereregisteredforthemass 2Cl-DMSO]+,385(100).1HNMR(CDCl)δ:3.75(s,6H,CH) 3 3 rangem/z<3000withaFisonsVGautospecemployingacesium 8.00 (dd, 1H), 8.02 (dd, 1H), 8.11 (s, 1H), 8.13 (s, 1H) 8.38 (d, ion gun (voltage 17 kV) and 3-nitrobenzyl alcohol as the liquid 1H),8.40(d,1H),9.76(d,1H),9.82(d,1H),10.22(d,1H),10.28 matrix. A Bruker DRX 400 was employed for the registration of (d,1H)ppm.IR:ν˜ )1130s(νSO)cm-1. 1Hand13CNMRspectrawithchemicalshiftsreportedasδvalues mer-[RhCl(DMSO)(dppn)]5a.Synthesisasfor1awithben- 3 relative to the signal of tetramethylsilane. Atomic absorption zo[i]dipyrido[3,2-a:2′,3′-c]phenazine(149.6mg,0.45mmol).Yield: spectrometricmeasurementswereperformedonaVario6(Analytik 68%.Anal.(C H ClNORhS)C,H,N.LSIMS:m/z(%)583(38) 24 18 3 4 Jena)andelementalanalysesonaVarioEL(ElementarAnalysen- [M-Cl]+,548(11)[M-2Cl]+435(41)[M-3Cl-DMSO]+. systeme).RhCl ·3HOwaspurchasedfromChempur,phenfrom 1HNMR(CDCl)δ:3.80(s,6H,CH)7.68,7.70(2d,2H),8.07 3 2 3 3 Acros, and bpy and DMSO from J. T. Baker. The polypyridyl (dd, 1H), 8.16 (dd, 1H), 8.25, 8.27 (2d, 2H) 9.06, 9.08 (2s, 2H), ligandsdpq,28dppz,29anddppn30werepreparedinaccordancewith 9.80,9.86(2d,2H),10.25,10.31(2d,2H).IR:ν˜ )1118s(νSO) literatureproceduresaswasthestartingcompoundmer,cis-[RhCl- cm-1. 3 (DMSO-κS)(DMSO-κO)].14 [RhCl(DMSO){{(CH)S}}] 6. Synthesis as for 1a with 1,4 2 3 22 2 X-rayStructuralAnalyses.Intensitydataformer,cis-[RhCl- dithiane(54.2mg,0.45mmol).Yield:40%.Anal.(CH ClORhS) 3 6 14 3 3 (DMSO-κS)(DMSO-κO)]andfac-[RhCl(DMSO-κS)(bpy)]·HO C,H,S.LSIMS:m/z(%)371(100)[M-Cl]+,1HNMR(CDCl) 2 3 2 3 1b were collected using ω scans on a Siemens P4 diffractometer δ:3.43(s,3H,CH),3.44,3.50(m,8H,CH),3.55(s,3H,CH).IR: 3 2 3 equipped with graphite-monochromated Mo KR radiation (λ ) ν˜ )1122s(νSO)cm-1. 0.71073Å,4°e2θe50°).Thedatawerecorrectedsemiempiri- Cytotoxicity Measurements. MCF-7 breast cancer and HT- callyforabsorption(Ψscans),andthestructuresweresolvedby 29humancoloncarcinomacellsweremaintainedin10%(v/v) directmethodsandrefinedbyfull-matrixleast-squaresagainstF2 fetal calf serum containing cell culture medium (minimum 0 usingSHELX97.31Anisotropictemperaturefactorswereemployed essential eagle supplemented with 2.2 g NaHCO , 110 mg/L 3 for the non-hydrogen atoms with the exception of the disordered sodium pyruvate and 50 mg/L gentamicin sulfate adjusted to wateroxygenatomof1andprotonswereincludedatgeometrically pH7.4)at37°C/5%CO andpassagedtwiceaweekaccording 2 calculated positions as riding atoms. The final R factors were to standard procedures. The antiproliferative effects of 1a-6 respectivelyR )0.047and0.033forI>2σ(I)withwR )0.119 were determined by an established procedure.32 Cells were 1 2 and 0.083 for all independent reflections. CCDC 677679 and suspendedincellculturemedium(MCF-7:10000cells/mL;HT- 677680containthesupplementarycrystallographicdataformer,cis- 29:2850cells/mL),and100µLaliquotsthereofwereplatedin [RhCl(DMSO-κS)(DMSO-κO)]and1bandmaybeobtainedfree 96 well plates and incubated at 37 °C/5% CO for 72 h (MCF- 3 2 2 ofchargeatwww.ccdc.cam.ac.uk/conts/retrieving.htmlorfromthe 7)or48h(HT-29).StocksolutionsofthecompoundsinDMSO Cambridge Crystallographic Data Centre, 12 Union Road, Cam- were freshly prepared and diluted with cell culture medium to bridge CB2 1EZ, U.K. (fax: int. code +44(0)1223/336-033, thedesiredconcentrations(finalDMSOconcentration:0.1%v/v). E-mail:deposit@chemcrys.cam.ac.uk). The medium in the plates was replaced with the medium Synthesis.Complexes1a-5awerepreparedbydisplacingcis- containing the compounds in graded concentrations (six repli- sited DMSO and chloride ligands in mer,cis-[RhCl(DMSO- cates).Afterfurtherincubationfor96h(MCF-7)or72h(HT- 3 κS)(DMSO-κO)]withtheappropriatepolypyridylligandinCHOH/ 29), the cell biomass was determined by crystal violet staining 2 3 HOsolutionat75°C.Thegeneralprocedureisdescribedbelow and the IC values were established as those concentrations 2 50 for1a. causing 50% inhibition of cell proliferation. Results were mer-[RhCl(DMSO)(bpy)]1a.mer,cis-[RhCl(DMSO-κO)(DM- calculated from 2-3 independent experiments. 3 3 SO-κS)] (200 mg, 0.45 mmol) was dissolved in 10 mL of a 1:1 CellularUptakeStudies.For cellular uptake studies, HT-29 2 mixture of methanol and water. After addition of 2,2′-bipyridine and MCF-7 cells were grown until at least 70% confluency in (70.3mg,0.45mmol),thereactionmixturewasstirredfor2hat 175cm2cellcultureflasks.Stocksolutionsofcomplexes1a-6 75°Candthenlefttostandat4°Cforafurther24h.Theresulting in DMSO were freshly prepared and diluted with cell culture yellowprecipitatewasfilteredoff,treatedwith5mLofmethanol, medium to the desired concentrations (final DMSO concentra- andreprecipitatedbyadditionofdiethylether.Thesolidwasfiltered tions:0.1%v/v;finalcomplexconcentration:0.2-5.0µM).The off,washedanddriedinvacuo.Yield:76%.Anal.(C H ClN- cellculturemediumofthecellcultureflaskswasreplacedwith 12 14 3 2 ORhS) C, H, N. LSIMS: m/z (%) 407(100) [M - Cl]+, 372(37) 10 mL of the cell culture medium solutions containing 1a-6 [M - 2Cl]+. 1H NMR (CDCl) δ: 3.71 (s, 6H, CH), 7.63 (dd, andtheflaskswereincubatedfor0-6hat37°C/5%CO .Then 3 3 2 1H),7.71(dd,1H),8.04(dd,1H),8.11(d,1H)8.14(dd,1H),8.16 theculturemediumwasremoved,thecelllayerwashedwith10 (d, 1H), 10.01 (d, 2H). IR: ν˜ ) 1126 s (νSO) cm-1. Crystals of mL PBS (phosphate buffered saline pH 7.4), treated with 2-3 fac-[RhCl(DMSO-κS)(bpy)]·HO 1b suitable for X-ray analysis mLtrypsinsolution(0.05%trypsin,0.02%EDTAinPBS),and 3 2 were grown over a period of 7 days by slow evaporation of a incubatedfor2minat37°C/5%CO afterremovalofthetrypsin 2 solutionof1ainwater/methanol. solution. Cells were resuspended in 10 mL of PBS, and cell mer-[RhCl(DMSO)(phen)]·HO2a.Synthesisasfor1awith pelletswereisolatedbycentrifugation(RT,2000g,5min)The 3 2 1,10-phenanthroline (81.1 mg, 0.45 mmol). Yield: 74%. Anal. isolated cell pellets were resuspended in 1-5 mL of twice- (C H ClNORhS) C, H, N. LSIMS: m/z (%) 431(100) [M - distilled water, lysed by using a sonotrode, and approximately 14 16 3 2 2 Cl]+,391(38)[M-2Cl]+.1HNMR(CDCl)δ:3.78(s,6H,CH), diluted using twice distilled water. The rhodium content of the 3 3 7.93(dd,1H),8.03(dd,1H),8.04(dd,1H),8.05(d,1H)8.50(dd, samples was determined by atomic absorption spectroscopy 1H), 8.58 (d, 1H), 10.17 (d, 2H), 10.24 (d,1H). IR: ν˜ ) 1118 s (AAS, see below) and the protein content of separate aliquots (νSO)cm-1. by the Bradford method. To correct for matrix effects in AAS, mer-[RhCl(DMSO)(dpq)] 3a. Synthesis as for 1a with dipy- measurementssamplesandstandardswereadjustedtothesame 3 rido[3,2-f:2′,3′-h]quinoxaline(104.7mg,0.45mmol).Yield:76%. protein concentration by dilution with twice-distilled water Anal.(C H ClNORhS)C,H,N.LSIMS:m/z(%)483(28)[M (matrix matched calibration). Prior to AAS analysis, 20 µL of 16 14 3 4 - Cl]+, 405(9) [M - Cl - DMSO]+, 370(100) [M - 2Cl - Triton X-100 (1%) and 20 µL of nitric acid (13%) were added DMSO]+, 335(83) [M - 3Cl - DMSO]+. 1H NMR (CDCl) δ: toeach200µLsampleofthecellsuspensions.Cellularuptake 3 3.84(s,6H,CH),8.13(dd,1H),8.23(dd,1H),9.19(s,2H),9.70 was expressed as ng rhodium per mg cell protein for data 3 (d,1H)9.78(d,1H),10.33(d,1H),10.39(d,1H).IR:ν˜ )1118s obtainedfrom2-3independentexperiments.Conversionofthe (νSO)cm-1. ng rhodium/mg protein value to the micromolar cellular con- mer-[RhCl(DMSO)(dppz)]·1.5HO 4a. Synthesis as for 1a centration was performed as described previously.21,22 3 2 withdipyrido[3,2-a:2′,3′-c]phenazine(127.3mg,0.45mmol).Yield: AtomicAbsorptionSpectroscopy.AVario6graphitefurnace 71%. Anal. (C H ClNO RhS) C, H, N. LSIMS: m/z (%) atomic absorption spectrometer (Analytik Jena) was employed 20 19 3 4 2.5 3932 JournalofMedicinalChemistry,2008,Vol.51,No.13 Harlosetal. for the Rh quantification using a wavelength of 343.5 nm with (4) Qu,P.;He,H.;Liu,X.Antitumoractivityandmechanismofrhodium a bandpass of 0.5 nm. A deuterium lamp was used for complexes.Progr.Chem.2006,18,1646–1651. background correction. Matrix containing standards were ob- (5) Mestroni,G.;Alessio,E.;SessantioSanti,A.;Geremia,S.;Bergamo, A.;Sava,G.;Boccarelli,A.;Schettino,A.;Coluccia,M.Rhodium(III) tainedbyadditionofarhodiumstocksolution(1mg/mLRhin analogues of antitumor-ative ruthenium(II) compounds: the crystal 5%HCl)tountreatedcellsuspensions.Probeswereinjectedat structureof[ImH][trans-RhCl4(Im)2](Im)imidazole).Inorg.Chim. a volume of 20 µL into standard graphite tubes. Drying, Acta1998,273,62–71. pyrolysis,andatomizationinthegraphitefurnacewasperformed (6) Colamarino,P.;Orioli,P.Crystalandmolecularstructureoftrichlo- accordingtotheconditionslistedinTableS5oftheSupporting ro(dimethylsulphoxide)bis-pyridinerhodium(III).J.Chem.Soc.,Dalton Information.Duringthetemperatureprogramthegraphitetube Trans.1976,845–848. (7) Pruchnik,F.P.;Jakimowicz,P.;Ciunik,Z.;Zakrzewska-Czerwin´ska, was purged with a constant argon gas flow, which was only J.;Opolski,A.;Wietrzyk,J.;Wojdat,E.Rhodium(III)complexeswith halted during the zeroing and atomization steps. Pyrolysis and polypyridylsandpyrazoleandtheirantitumoractivity.Inorg.Chim. atomizationtemperatureswereoptimizedpriortotheexperiments Acta2002,334,59–66. and the recovery rates of the metallodrugs using the above- (8) Medvetz,D.A.;Stakleff,K.D.;Schreiber,T.;Custer,P.D.;Hindi, mentioned conditions were determined (data not shown). The K.;Panzner,M.D.;Blanco,D.D.;Taschner,M.J.;Tessier,C.A.; mean recovery rate of the metallodrugs (74 ( 12%) was used Youngs,W.J.Ovariancanceractivityofcyclicaminesandthiaether metalcomplexes.J.Med.Chem.2007,50,1703–1706. for calculation of the final values. The mean integrated absor- (9) Scha¨fer,S.;Ott,I.;Gust,R.;Sheldrick,W.S.Influenceofthepoly- bances of duplicate injections were used throughout the study. pyridyl(pp)ligandsizeontheDNAbindingproperties,cytotoxicity Thecharacteristicalconcentrationforthedescribedmethodwas andcellularuptakeoforganoruthenium(II)complexesofthetype[(η6- 0.85 ( 0.05 (µg Rh L-1)/1% A. C6Me6)Ru(L)(pp)]n+[L)Cl,n)1;L)(NH2)2CS,n)2].Eur. Cellular Metabolism. Changes in cellular metabolism and J.Inorg.Chem.2007,3034–3046. morphology were analyzed using a Bionas 2500 sensor chip (10) Scharwitz,M.;Ott,I.;Geldmacher,Y.;Gust,R.;Sheldrick,W.S., Cytotoxichalf-sandwichrhodium(III)complexes:polypyridylligand system(Bionas,Rostock,Germany).Thesensorchipallowsto influence on their DNA binding properties and cellular uptake. J. continuousmeasurementofthreeimportantparametersofcellular Organomet.Chem.2008,DOI10.1016/j.jorganchem.2008.04.002. metabolism: oxygen consumption using oxygen sensitive elec- (11) Scha¨fer, S.; Sheldrick, W. S. Coligand tuning of the DNA binding trodes,changeinthepHofthemediumusingion-sensitivefield propertiesofhalf-sandwichorganometallicintercalators:influenceof effecttransistorsandtheimpedancebetweentwointerdigitated polypyridyl(pp) and monodentate ligands (L ) Cl, (NH2)2CS, electrodestructurestoregistertheimpedanceunderandacross (NMe2)2CS)ontheintercalationof(η5-pentamethylcyclopentadienyl) iridium(III)-dipyridoquinoxalineand-dipyridophenazinecomplexes. thecelllayeronthechipsurface.24–26Beforemeasurement,cells J.Organomet.Chem.2007,692,1300–1309. wereseededonthesensorchipinDMEM(PAA,E15-883)with (12) Messori,L.;Marcon,G.;Orioli,P.;Fontani,M.;Zanello,P.;Bergamo, penicillin/streptomycinand10%(v/v)FCS(PAA)andincubated A.; Sava, G.; Mura, P. Molecular structure, solution chemistry and in a standard tissue culture incubator at 37 °C, 5% CO 2 , and biologicalpropertiesofthenovel[ImH][trans-IrCl4(DMSO)],(I)and 95%humidityfor24huntil90%confluencywasreached.Sensor oftheorangeformof[(DMSO)2H][trans-IrCl4(DMSO)2],(II),com- plexes.J.Inorg.Biochem.2003,95,37–46. chipswithcellswerethentransferredtotheBionas2500analyzer (13) Sokol,V.I.;Porai-Koshits,M.A.Coordinationofdimethylsulfoxide in which medium is continously exchanged in 8 min cycles (4 in rhodium(III) complexes. Crystal and molecular structure of tris- minexchangeofmediumand4minwithoutflow)duringwhich (dimethylsulfoxide)trichlororhodium.SoV.J.Coord.Chem.1975,1, theparametersweremeasured.Therunningmediumusedduring 577–583. analysiswasDMEMwithoutcarbonatebufferandonlyweakly (14) James, B. R.; Morris, R. H. Sulfur-bonded sulfoxide complexes of buffered with 1 mM Hepes and reduced FCS (0.1%). 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