Reactivity and Transformation of Antimetastatic and Cytotoxic Rhodium(III)-Dimethyl Sulfoxide Complexes in Biological Fluids: An XAS Speciation Study.

Article CiteThis:Inorg.Chem.XXXX,XXX,XXX−XXX pubs.acs.org/IC Reactivity and Transformation of Antimetastatic and Cytotoxic − Rhodium(III) Dimethyl Sulfoxide Complexes in Biological Fluids: An XAS Speciation Study † † † ‡ ‡ ‡ Jun Liang, Aviva Levina, Junteng Jia, Peter Kappen, Chris Glover, Bernt Johannessen, and Peter A. Lay *,† † School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia ‡ Australian Synchrotron, ANSTO, 800 Blackburn Road, Clayton, Victoria 3168, Australia * S Supporting Information ABSTRACT: Rhodium(III)anticancerdrugscanexertpreferentialantimetastaticorcytotoxicactivities,whicharedependent on subtle structural changes. In order to delineate factors affecting the biotransformations and speciation, mer,cis-[RhCl (S- 3 dmso) (O-dmso)](A1)andmer,cis-[RhCl (S-dmso) (2N-indazole)](A2)havebeenstudiedbyX-rayabsorptionspectroscopy 2 3 2 (XAS). Interactions of these complexes with saline buffer, cell culture media, serum proteins (albumin and apo-transferrin), nativeandchemicallydegradedcollagengels,andA549cellshavebeenstudiedusinglinearcombinationfitting(LCF)and3D − scatterplotsofXASdata.FollowinginitialaquationandhydrolysisreactionsinvolvingstepwisedisplacementofCl andS-/O- dmso ligands, the Rh(III) complexes underwent further ligand substitution reactions with biological nucleophiles (e.g., amino acidresiduesofserumproteins).ThereactionofA1withchemicallydegradedcollagengelwaspostulatedtobeakeyreasonfor itsantimetastaticactivity.AnalysesoftheXASofRh-treatedbulkcellswereconsistentwithstructure−reactivityrelationshipsin which the more reactive A1 was predominantly antimetastatic and the less reactive A2 was predominantly cytotoxic, showing relationshipsparalleltotypicalRu(III)anticanceragents,i.e.,NAMI-A([ImH]trans-[RuCl (S-dmso)(N-imidazole) ],ImH= 4 2 imidazolium cation) and KP1019/NKP1339 (KP1019, [IndH] trans-[RuCl (N-indazole) ], IndH = indazolium cation; 4 2 NKP1339, sodium trans-[RuCl (2N-indazole) ]), respectively. 4 2 ■ INTRODUCTION parts.14−16 Notably, the nontoxic mer,cis-[RhCl (S-dmso) (O- 3 2 dmso)] (Chart 1, A1) demonstrated remarkable and selective Although classical Pt-based cytotoxic agents (e.g., cisplatin, carboplatin,oxaliplatin)areeffectiveintreatingawiderangeof activityagainstspontaneouslungmetastasisofMCamammary cancers in combination therapies,1 their broader use has been carcinoma in a CBA mice model, and mer,cis-[RhCl 3 (S- dmso) (3N-imidazole)] (Chart 1, A3) exhibited cytotoxicity hampered by their high systematic toxicities, limited ranges of 2 against human cancer cell lines which was comparable to that activities, the propensity for acquired tumor resistance, and reduced efficacies for metastatic tumors.1,2 Metastasis is of cisplatin.17 Conversely, [ImH]-trans-[RhCl 4 (3N-imida- responsible for >90% of cancer-related deaths;3 hence, it is zole) 2 ], the Rh(III)−imidazole analogue of KP1019, and ofconsiderableinteresttodesigndrugsthattargetmetastases.4 [Na·2DMSO]-trans-[RhCl 4 (S-dmso)(3N-imidazole)], the Rh- Studies on non-Pt anticancer drugs led to the discovery of (III) analogue of NAMI-A, were inactive both in vitro and in many bioactive Ru complexes,2,5−7 and three Ru(III) vivo.17,18 Rh has stable oxidation states and coordination complexes, the antimetastatic NAMI-A and the cytotoxic geometries of Rh(III) (octahedral), Rh(II) (dimeric lantern), KP1019/NKP1339, have entered Phase II clinical trials.8−13 TheanticancerpropertiesofsomeRh(III)complexeshavealso Received: December13,2018 been evaluated alongside those of their Ru(III) counter- ©XXXXAmericanChemicalSociety A DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX .mehC .gronI .ylno esu lanosrep roF .91/20/40 no ETTEYAFAL TA ANAISIUOL FO VINU yb gro.sca.sbup morf dedaolnwoD Inorganic Chemistry Article Chart 1. Structures of Rh(III) Anticancer Drugs: mer,cis- synthesized and fully characterized. The new complex, A2, [RhCl (S-dmso) (O-dmso)] (A1), mer,cis-[RhCl (S- was studied because of high activities of related Ru(III) 3 2 3 dmso) (2N-indazole)] (A2), and mer,cis-[RhCl (S- complexes with indazole ligands: e.g., KP1019/1339.9,49 2 3 dmso) (3N-imidazole)] (A3) Preliminary studies demonstrated that A2 was more stable 2 against aqueous decomposition than both A1 and A3, had greater levels of cellular uptake than A1 and A3 did under identical experimental conditions, and its cytotoxicities on a range of human cancer cell lines were superior to those of A3 and cisplatin (measured with standard MTT assay; exper- imental details are described in the Supporting Information, and IC values of these complexes are given in Table S1).50 50 ThisresearchisaimedatusingsynchrotronXASspeciationto gain a deeper understanding of structure−reactivity−activity relationships for octahedral Rh(III) anticancer complexes − bearing mixed Cl /(O-/S-dmso)/N-heterocycle ligands with Rh(I)(squareplanar),respectively.14,15Anumberofbioactive arangeofantimetastaticandcytotoxicactivitiescomparableto complexes of each oxidation state have been evaluated in ■those observed for NAMI-A and KP1019.17 recent years (e.g., Rh(I) N-heterocyclic carbenes,19−21 photo- active Rh(II)−polypyridyl complexes,22,23 and organometallic EXPERIMENTAL SECTION Rh(III)−Cp* agents (Cp* = pentamethylcyclopentadienyl Model Compounds. A1 was synthesized according to literature ligand)24−26). Notably, dirhodium(II) tetraacetate, methods,51,52 and A2 was prepared by a procedure similar to that reported for A317 using indazole instead of imidazole. Rh(III) [Rh (OAc) ], readily reacted with methionine and cysteine 2 4 complexesusedforfittingtheXASdataweresynthesizedaccordingto under ambient conditions (aqueous solutions, pH 7.4, room literature procedures (S1,53 S2,54 S3,55 S4,56,57 S5,17 M1,58 M2,59 temperature, and aerobic conditions) and generated various M3,60 M4,61 M5,62 M6,63 M7,64 M8,64 M9,64 O1,65 O266) or monomericRh(III)species.27−29TheRuanticancercomplexes purchased from the commercial suppliers (S6, O3). Structures of have oxidation states of either Ru(III) (e.g., NAMI-A and these Rh(III) model complexes are presented in Chart 2, and their KP1019) or Ru(II) (organometallic Ru(II)−arene com- structural formulas, types of donor atoms, and published synthetic plexes30,31).ReductionofRu(III)toRu(II)hasbeenproposed procedures are summarized in Table S2 in the Supporting tobethekeymechanismofactionfortheanticanceractivities Information. All synthesized compounds were fully characterized by ofRu(III)complexes,butthelatestbiologicalspeciationusing standard analytical techniques; elemental analyses agreed with calculated values. These model complexes had combinations of N, X-ray absorption spectroscopy (XAS) argued strongly against this hypothesis.32,33 Although detailed mechanistic studies on O,Sand/orCl− donorsgroups thatareexpectedtorepresentthose likely to react with A1 and A2 in biologically relevant matrices (cell theseRh(III)complexes were not pursued, theseobservations culture medium and cytoplasm): e.g., amine/imine/imidazole (N- suggested alternative mechanisms of action for the anticancer donors), aqua/carboxylato/enolato (O-donors), thiolato/thioester activitiesoftheRh(III)complexesincomparisontothemuch (S-donors), and chloridoligands, respectively.67 studied Ru and Pt anticancer drugs. Model compounds were classified into three categories according The mechanisms of action of many anticancer Ru(III) and to the types and compositions of donors. Model compounds Rh(III) complexes remain uncertain, partially due to lack of containing exclusively, or predominantly, S and/or Cl donors were speciation data from physiologically relevant conditions.2,34,35 classifiedassoftdonormodels(S1−S6),fortheSdonorsofthiolato/ thiaetherareregardedassoftLewisbasesaccordingtoPearson’sacid The complexity of biological matrices (water, organic and base theory.68 Although the same theory classified Cl as a hard molecules, inorganic salts, and paramagnetic species) makes Lewis base, it was treated as a soft donor for the convenience of speciation using classical spectroscopic methods (e.g., ESI-MS comparison.S4possessesanNdonorinadditiontoSandCl,yetit and NMR, EPR, and electronic absorption spectroscopy) wasclassifiedasasoftdonormodelbecausethekeyparametersofits particularly challenging to perform. AAS and ICP-MS are XAS were similar to those of S1, S2, and S4. Model compounds destructiveandonlyprovidetotalmetaldeterminationwithout containingexclusivelyOdonorswereclassifiedasoxygendonor(O1− informationon metal speciationunless individual components O3) models. The remaining model compounds were classified as are separated.36,37 XAS, including X-ray absorption near-edge mixeddonor(M1−M9)modelsonthebasisofthefollowingreasons: structure (XANES) and extended X-ray absorption fine (1)theirfirstcoordinationshellsareoccupiedbyhard(N,O)and/or soft (S, Cl) donors at various combinations and (2) later studies structure (EXAFS), are superior for having elemental s g p re e a c t i e fi r ci t t o y, ler b a e n i c n e g fo n r on b d io e l s o t g ru ic c a t l i l v y e re t l o eva sp n e t c m im a e tr n i s c , es a .3 n 6 d ,38− h 4 a 0 vi A n l g l a o co n th n d e fi r d rm i g st r e i o d n u c t p t h s e f . ro k In m ey p t p a h a r o r ti a s c e m ul o e a t f r e , t r h s M e o 2 f m − th o M e d i 4 e r l X h c A a o v m S e p w b o e e u r e e n n d s s i p m r c e i l l a p a s r a s r i t e fi o d e e d a to c in h m to o o th t d h e e e r l oftheseadvantagesmakeXASthepreferredtechniqueforthe Rh(III) binding to amine/imine and carboxylato residues of speciationofmetallodrugsinbiologicalsystems.Notably,XAS proteins.67,69 It is worth pointing out that a number of ligands, speciation has been applied successfully to the speciation of such as methyl phenyl sulfide (S3) and DMSO (M6), while not reactionproductsfrombioactivePt(IV)products,41−43Ru(III) present in vivo under normal conditions, are models for methionine anticancer drugs,32,44,45 and Cr(III), V(V)/V(IV), and Mo- andDMSObindingtotheparentcomplexes,respectively.Usingthem (VI) antidiabetics.46,47 XAS investigations on bioactive Rh in subsequent XAS analysis does not presume the presence of these ligands inbiological systems. complexes have been sporadic in comparison to the studies done on other metals, although Rh K-edge XANES48 and Materials and Biological Sample Preparation. Solid samples of A1, A2, and Rh(III) models were prepared via diluting neat EXAFS27,28 investigations have been carried out on a number compounds with boron nitride at a mass ratio of 1:2500. Reaction of dirhodium(II) complexes. productsofA1orA2withbiologicalfluids(bufferedsaline,buffered In the pursuit of Rh(III) complexes with higher activities, protein solutions, serum-free and serum-supplemented cell culture mer,cis-[RhCl (S-dmso) (2N-indazole)] (Chart 1, A2) was media),Rh-treatedcancercells(A549humanlungadenocarcinoma), 3 2 B DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article Chart 2. Structures of Rh(III) Model Compounds S1−S5, M1−M9, and O1−O3 a aS6and O3werepurchased fromSigma-Aldrichand Abcr GmbH, respectively. and A1-treated native or chemically degraded type I collagen gels dry ice, freeze-dried at 223 K under high vacuum (0.8 mbar), and were prepared on the basis of the methods used in the sample storedinadesiccatoratroomtemperaturebeforethedatacollection. preparationforXASspeciationofRu(III)anticancercompounds.32,44 XAS Data Collection and Processing. Rh K-edge XAS were collected at the wiggler XAS Beamline ID-1270 of the Australian Typically, samples were prepared via diluting the freshly prepared Synchrotron, ANSTO. The synchrotron has an electron storagering stocksolutionsofA1orA2(100mMinDMSO)inreactionmatrices operated at 3 GeV in top-up mode (200 mA). For photon delivery, andthenincubatingthemixtureat310Kfordifferentperiods(1,4, the beamline had an upstream vertically collimating mirror (Pt- or24h).Asummaryofconditionsforbiologicalsamplepreparationis coated), a liquid-nitrogen-cooled Si(311) double-crystal monochro- giveninTable1.Allaqueoussampleswerecentrifugedat2000gfor3 mator, and a downstream toroidal focusing mirror (Pt-coated). The min to remove solid particles. The supernatant of aqueous samples, monochromatorwasoperatedatthepeakoftherockingcurve(“fully bulk cell samples, and Rh-treated collagen gels were snap-frozen in tuned”), with harmonics rejected by the mirrors. Model compounds C DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article Table 1. Biological Sample Preparation Conditions for the calibration(23222.0eVatRh(0)edge).71Ionchambers(Oken;U= Reaction Products of A1 and A2 2.1 kV; N 2 flow at 0.4 L/min) were used to record incident and transmittedbeamintensities.Dependingonthespectralquality,each incubationtime samplewasmeasuredbetween3and15timesatdifferentspotsofthe designation (h) reactionmatrix pellet. The possibility of photodamage was assessed by comparing A1H1/A2H1 1 HBSa,b both the shape of the edgeregion and the calibrated edge energyin A1H4/A2H4 4 successivescansonasample.72Nosystematicdriftingofedgeenergy A1H24/A2H24 24 or noticeable differences in edge shape can be observed during A1B1/A2B1 1 100μMBSA,a,cHBSb sequential scans on a sample, and the differences in edge energy A1B4/A2B4 4 between two successive measurements were no more than 0.1 eV, A1B24/A2B24 24 indicating the absence ofphotodamage.72 A1T/A2T 4 50μMofapoBTf,a,dHBS,HCO−e The raw spectra were individually evaluated using the Sakura A1M1/A2M1 1 advancedDMEMa,f 3 program,73 and those dominated by monochromator glitches or featured low signal to noise ratios were excluded. Subsequent A1M4/A2M4 4 calibration (23222.0 eV at the first peak of the first derivative of Rh A1M24/ 24 A2M24 foil spectra71), averaging, and normalization of the spectra were A1MS1/ 1 10%v/vFBS,gadvancedDMEMf performedusingtheAthenaprogram.74 Analysesofthespectrawere A2MS1 carriedoutviatwoapproaches:linearcombinationfitting(LCF)and A1MS4/ 4 three-dimensional (3D) scatter plots. LCF was performed using the A2MS4 built-in module of the Athena program, via fitting the spectra of A1MS24/ 24 biological samples with the spectra of model compounds within the A2MS24 same energy range. Since the XAS of all model compounds and A1CN 1 typeIcollagensolution(native)h,i biological samples featured strong postedge EXAFS oscillations, the A1CD 1 typeIcollagengel(degraded)a,i range of fitting was limited to between 23130 and 23600 eV to A1BC/A2BC 4 FBS(10%v/v),advancedDMEM; accommodateboththeXANESregionandthelow-energyendofthe A549cellsj,k EXAFS region of the experimental data. For a fitted spectrum of a a100μMA1/A2.bHEPES-bufferedsaline,containing20mMHEPES biologicalsample,thecalculatedweightedcontributions(%)foreach (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and 140 mM spectrumfromtheindividualmodelcompoundswereconstrainedto NaCl,pH7.4.cBovineserumalbumin.dapoBTf:bovineapotransfer- lie between 0 and 100%, and the sum of the calculated weighted rin. eHEPES-buffered saline with 25 mM NaHCO , pH 7.4. fAdvanced Dulbecco’s modified Eagle medium. gFe 3 tal bovine contributionsofallmodelspectrawasconstrainedtobe100%.Model serum.h100μMA1.iNative:typeIbovinetendoncollagensolution spectrahavingnullorsmallcontributions(<5%)wererejectedduring (5.0 mg mL−1) was neutralized with 0.10 M NaOH and Minimum successiveroundsoffitting,andthegoodnessofafitwasassessedby EssentialMedium(10×),andfurtherdilutedinMilli-Qwaterto1.0 the χ2 value. The best fit was obtained when further exclusion of a mg mL−1. Degraded: native collagen solution was incubated with model spectrum resulted in a sharp increase in the χ2 value. These immobilized tris(2-carboxyethyl)phosphine (TCEP) disulfide reduc- considerations assured that the LCF outcomes were physically inggelat273Kfor1h.Bothsolutionswereincubatedat310Kfor1 meaningful.Alternatively,thekeyspectralparametersweremeasured haftertheadditionofA1causedthegelationofcollagen.j100μMA1 directly from the normalized spectra of model compounds and or5μMA2.kA549cellsat80%confluence(approximately1×107 biologicalsamples(Figure1),includingtheedgeenergy(E 0 ,eV),the cellsfromfive75cm2standardcellcultureflasks)wereincubatedfor normalizedwhitelineheight(H WL ,dimensionless),andtheenergyof 4 h in fully supplemented advanced DMEM containing A1 or A2, thesecondoscillationmaximum(E SOM ,eV).Theseparameterswere respectively. The Rh-containing medium was removed, and the cell represented in scatter plots in three-dimensional Cartesian space, monolayer was washed five times with PBS, detached using trypsin- where the x,y,and z coordinatesof each pointwere represented by EDTA solution, and then pelleted via gentle centrifugation at 1000g the values ofE, H , andE ,respectively. 0 WL SOM for 3 min. No morphological changes were observed during Rh treatments. Cells treated with A2 developed a faint yellow color. General cell culture procedures are given in the Supporting Information. and freeze-dried biological samples were pressed into 1 mm thick pellets that were held by a polymethacrylate sample holder and sandwichedbetweentwo63.5μmthickKaptontapes,givingapellet size of 2 × 4 × 1 mm3, with a window size of 2 × 4 mm2. Samples weresetatanangleof45°totheincidentX-rayandloweredintoan air-free, He-filled closed cycle cryostat (Optistat 4K, Oxford Instruments) that was maintained at 5 K. Spectra were collected in fluorescence detection mode using a 100-element Ge-array detector (Canberra,France)placedperpendiculartotheincidentX-ray.Each measurementwasrecordedoveranenergyscalerangingfrom23020 to24200eVwiththreeconsecutiveregions:pre-edge(23020−23200 eV),9eVstepsizeand2sdwellperstep;near-edge(23200−23270 Figure 1. Representative key XAS parameters measured from the eV),0.4eVstepsizeand2sdwellperstep;EXAFS(23270−24200 spectrum of mer,cis-[RhCl(S-dmso)(O-dmso)] (A1). Highlighted 3 2 eV),0.035Å−1stepsizeandalinearlyscaleddwelltimefrom2supto withdashedlinesarevaluesofedgeenergy(E),measuredatthefirst 0 10 s at the maximal k value. All model compounds and biological maximum of the first-derivative spectrum, normalized white line samplesotherthanbulkcellsweremeasuredoverthekrangefrom0 height (H , dimensionless), measured at the peak of the first WL to16Å−1,andbulkcellsamplesweremeasuredoverthekrangefrom postedge maximum, and energy of the second oscillation maximum 0 to 14 Å−1. A Rh foil (EXAFS Materials, CA, USA) was measured (E ,eV),measuredatthepeakofthesecondpostedgemaximal.μE SOM concurrentlyintransmissionmodeasaninternalstandardforenergy is the normalizedXASabsorbance. D DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article Figure2.Comparisonoflow-energyEXAFSofA1andA2(A)withthoseofselectedRh(III)modelcompounds(B−D)andtypicalbiological s■amples(E−K). Designationsof compoundsand reactionconditionsof biological samples aregiveninChart 2 andTable1,respectively. RESULTS indazole). XAS of the softdonor modelcomplexes had strong EXAFS oscillations beyond 23.3 keV, and their H values ComparisonofXASSpectralFeatures.Onlyasmallset WL of XAS from the model compounds (S1−S4; M3, M6, and were comparable to those of the μE of the SOM. Conversely, M7;O1andO3)contributedtoXASthatthebestfitswereto theXASofthemixeddonor(Figure2B,C)andoxygendonor (Figure2D)complexesfeaturedH valueshigherthanthose thoseobservedfromthereactionproductsofA1andA2under WL at the SOM, and the EXAFS oscillation beyond 23.3 keV the studied conditions. Due to the absence of pre-edge transitions, the postedge XANES and low-energy EXAFS of showedsignificantattenuation.ForthereactionproductsofA1 experimental XAS were compared (Figure 2). The complete andA2inHBS(A1H4/A2H4),inserum-free(A1M4/A2M4) XANESandlow-energyEXAFSspectraaregiveninFiguresS2 and serum-supplemented (A1MS4/A2MS4) advanced and S3 in the Supporting Information. The gross spectral DMEM, with BSA (A1B4/A2B4) and with apoBTf (A1T/ featuresofA1andA2(Figure2A)resembledthoseofthesoft A2T), their white line features were similar to those of the donor model complexes (Figure 2B), an observation that was mixed donor and oxygen donor model complexes, yet their consistent with the nature of their ligands (predominantly Cl low-energy EXAFS showed characters (less attenuation of and S-dmso, minor contributions from O-dmso and 2N- oscillation) that were similarto those of thesoft donor model E DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article Figure3.3DscatterplotsofkeyXASparametersofA1(A)andA2(B)forselectedRh(III)modelcomplexeswithsoftdonors(S1−S4),mixed donors (M3, M6, M7), and oxygen donors (O1, O3) and reaction products in HBS (A1H4/AEH4), with BSA (A1B4/A2B4), with apoBTf (A1T/A2T), in serum-free (A1M4/A2M4) and serum-supplemented (A1MS4/A2MS4) advanced DMEM, in native (A1CN) or chemically degraded(A1CD)typeIcollagengel,andRh-treatedbulkcells(A1BC/A2BC).Thex,y,andzaxesaretheedgeenergy(E,eV),normalized 0 white-lineheight(H ,dimensionless),andtheenergyofthesecondoscillationmaximum(E ,eV)oftheXAS,respectively.Circlesshownin WL SOM red, green, and blue highlight the clustering of the XAS points into the corresponding soft, mixed and oxygen donor clusters in 3D space, respectively. complexes (Figure 2E,F,I,J). Reaction products of A1 in hadadecreasedE value(A1CD,23228.1eV)comparedwith 0 collagen gels (A1CN/A1CD, Figure 2G) and Rh-treated cells that from the reaction with the native collagen gel (A1CN, (Figure 2H,K) gave rise to XAS with small differences from 23229.4 eV). The XAS points from Rh-treated bulk cells also thoseoftheirparentcomplexes,whichwerealsoclosetothose fell within the XAS cluster for soft donor complexes, although features of the XAS of soft donor model complexes. XAS from A2-treated cells had an increased E value in 0 Figure 3 shows the key spectral parameters of experimental comparisonwiththatoftheXASfromA1-treatedcells(A1BC, XAS as single points in 3D plots, including those of both 23228.1 eV; A2BC, 23229.5 eV). modelcomplexes and selected biological samples presented in LCF on Reaction Products in Aqueous Buffers and Figure 2. A full list of XAS parameters for all model Serum Protein Adducts. The LCF results on reaction compounds and their products under different experimental products of A1 and A2 in HBS, with BSA, or with apoBTf at conditions is summarized in Table S3 in the Supporting various time points are summarized in Figure 4. Details of Information, and the complete 3D scatter plots of these XAS weighted contributions from each of the XAS from model are given in Figures S4−S9 in the Supporting Information. In complexes to the XAS fits are given in Tables S4 and S5, and general, soft-donor and mixed-donor model complexes gave experimental, fitted, and expanded residuals of XAS from all XASwithagoodseparationandclusteringalongtheE values, reactionproductsaregiveninFiguresS10−S19.FittedXASof 0 with oxygen donor model complexes having XAS that were the reaction products of A1 in HBS at all reaction times distinct from the others mainly due to higher H and E (A1H1,A1H4,andA1H24)featuredsignificantcontributions WL SOM values.A4hreactionofA1inHBS,withBSA,orwithapoBTf ofXASfrommodelcomplexesS4,M3,andO3;thefittedXAS gave rise to the single XAS points adjacent to those of the of the reaction products of A2 under identical conditions mixed donor complex cluster (A1H4, A1B4, and A1T). The (A2H1, A2H4, and A2H24) were dominated by XAS reactionofA1inserum-freeorserum-supplementedadvanced contributions from models S4 and O3. Minor contributions DMEM (A1M4 and A1MS4) for the same time period gave from the XAS from model complex O1 were present in the risetosingleXASpointswithonlyminorincreasesinE values bestXASfittotheXASfromthereactionproductsafterlonger 0 butsignificantincreasesinH andE values.Singlepoints incubation periods (4 and 24 h, Figure 4A). WL SOM representing the XAS of reaction products of A2 (A2H4, TheLCFresultsfortheXASofthereactionproductsofA1 A2B4, A2T, A2M4, and A2MS4) under reaction conditions and A2 in HBS in the presence of a stoichiometric amount of identical with those of A1 for 4 h gave increased E values in BSA (100 μM) or a 1/2 equiv stoichiometric amount of 0 comparison with that of the parent complex, and the XAS apoBTf (50 μM) are summarized in Figure 4B. The XAS of points were clustered between those of mixed donor and reaction products of A1 after 1 h of incubation (A1B1) was oxygendonorcomplexclusters.PointsrepresentingXASofthe fitted with contributions of XAS from neat A1, S4, M3, and reaction products of A1 in collagen gels fell within the XAS O3. The XAS from the reaction products after longer cluster for soft donor complexes, and the XAS point for the incubation periods (4 and 24 h) had no contribution from reactionproductsofA1withchemicallydegradedcollagengel theXASoftheparentcomplex,A1,tothefittedXAS.Instead, F DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article Figure5.SummaryofLCFresultsofreactionproductsofA1(A)and Figure4.SummaryofLCFresultsofreactionproductsofA1andA2 inHBS(A),orinHBScontaining100μMofBSAorinHBS/HCO− A2 (B) in serum-free and serum-supplemented advanced DMEM. containing 50 μM of apoBTf (B). Designations and reactio 3 n Designations and reactionconditions aresummarized inTable1. conditionsaresummarized inTable1. thatofneatA2aminorcomponentoftheXASfitstoreaction products from 1 or 4 h incubation. the fitted XAS was dominated by contributions from the XAS TheXASofRh-BSAadductsofA1andA2wereincludedas ofS4,O1,andO3(A1B4andA1B24),wherebytheXASfrom additional models for fitting the experimental XAS of reaction O1 and S4 had increased contributions in comparison to the products in serum-supplemented advanced DMEM, on the fits to the reaction product at 1 h. The fitted XAS of reaction basis of the hypothesis that the models of BSA adducts were products of A2 with BSA (A2B1, A2B4, and A2B24) were likely to represent general Rh−protein bonding environments dominated by XAS contributions from model complexes S4, in the presence of serum. In comparison to using the XAS of O1, and O3; a minor contribution of the XAS from M6 was model compounds only (Table S6 and Figure S20 in the present in the XAS of the reaction product after 1 h of Supporting Information), inclusion of Rh−BSA adducts as incubation (A2B1). The fitted XAS of the reaction mixtures XAS models resulted in a substantial improvement in fitting preparedfromA1(A1T)andA2(A2T)werecombinationsof quality, as demonstrated by the significant reduction in χ2 XAS from model complexes S4, O1, and O3. Although A1T, values(TablesS5andS6intheSupportingInformation).The A2T,A1B24,andA2B24werepreparedfromdifferentparent fitted XAS of the reaction products of A1 in serum- complexes and serum proteins, their fitted XAS had the same supplemented advanced DMEM featured contributions from combinationofXASfrommodelcomplexes(S1,O1,andO3) XAS of neat A1/M6 (A1MS1, A1MS4) and S2 (A1MS24). and numerically similar weighted contributions. TheLCFresultsalsoidentifiedA1B4,theexperimentalXASof LCFonReactionProductsinSerum-FreeandSerum- the reaction product of A1 with BSA obtained after 4 h of SupplementedCellCultureMedia.TheLCFresultsonthe incubation,asthedominatingcontributortothefittedXASof XAS of reaction products of A1 and A2 in serum-free and A1 in serum-supplemented advanced DMEM. For the fitted serum-supplemented advanced DMEM are summarized in XAS of A2 under identical reaction conditions, minor XAS Figure5.ThefittedXASofA1inserum-freeadvancedDMEM contributions were made from those of the neat A2 and O1, atalltimepoints(A1M1,A1M4,andA1M24)werefittedwith andthemajorcontributorsweretheXASofA2B1andA2B24, contributions predominantly from XAS of models S4 and O1, each of which corresponded to the reaction products of A2 withminorcontributionsfromtheXASofneatA1after1hof withBSAobtainedafter1and24hofincubation,respectively. incubation. Similarly, predominant contributors to the fitted LCF on Reaction Products in Collagen Gels and Rh- XAS of A2 under identical conditions (A2M1, A2M4, and Treated Bulk Cells. The LCF results on the XAS of the A2M24)wereXASofmodelcomplexesS4,O1,andO3,with reaction products of A1 with native and chemically degraded G DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article typeIcollagengelsaresummarizedinFigure6.Thefitstothe increasesinfreethiols,thefitstotheXASwereconsistentwith XAS from these samples (A1CN and A1CD) had significant the reaction products from A1CD having more Rh(III)−S bonds in comparison to the reaction products from A1CN under the same conditions. TheXASofA1-treatedA549lungcancercells(A1BC)were fitted with a combination of XAS from model complexes S4 andM6,whiletheXASofA2-treatedcells(A2BC)werefitted with a substantial contribution of XAS from the neat A2, as well as contributions of XAS from model complexes S1 and M7. In comparison with A1, A2 is significantly more inert against aquation and hydrolysis and is more readily accumulated in intracellular space. The A2-treated A549 cells developed a faint yellow color at the end of treatment due to thisaccumulation.Therefore,theseobservationssuggestedthat the majority of intracellular A2 was intact. Also worth mentioning for the A1BC and A2BC treatments were the Figure6.SummaryofLCFresultsofreactionproductsofA1andA2 zero contributions of XAS from model complexes containing in native type I collagen gel (A1CN), in chemically degraded type I Cl − and O donor ligands. The XAS contributions to the fits collagengel(A1CD),andbulkA549cellstreatedwitheither100μM from amodelcomplexwithSdonorligands,S4, indicatedthe of A1 (A1BC) or 5 μM of A2 (A2BC), in fully supplemented − likelysubstitutionofCl ligandsfromtheparentcomplexesby advanced DMEM. Designations and reaction conditions are intracellular thiolato ligands. It is unclear, however, whether summarizedinTable 1. the indazole ligand of A2 was dissociated or remained coordinated to the Rh(III) metal center after it had been contributions from the XAS of neat A1, an observation that internalized by A549 cells. Nevertheless, the substantial XAS was hypothesized to be due to the reduced reaction rates, molar contribution from XAS of M7, a model that features resulting in the incomplete substitution of A1 in these highly exclusivelyRh−Ncoordination,suggestedthatthedissociation viscous solutions. Other XAS from model complexes that of indazole was less likely. contributed to the fit to XAS were that from S4, which ■ accounted for 30% of overall contributions to A1CN, and DISCUSSION those from S1 and S3, which accounted for 22% and 28% of overall contributions to the fits to the XAS of A1CD, XAS speciation of the reaction products of antimetastatic respectively. The immobilized TCEP used in the preparation mer,cis-[RhCl (S-dmso) (O-dmso)] (A1) and the cytotoxic 3 2 of A1CD is capable of reducing the disulfide bonds (cysteine mer,cis-[RhCl (S-dmso) (2N-indazole)] (A2), in various bio- knots) of collagen molecules,75−77 which increases the logical media 3 , in nativ 2 e and chemically degraded type I abundance of free thiols in A1CD. As expected, due to collagen gel, and in bulk A549 lung cancer cells revealed a Scheme 1. Proposed Generalized Mechanisms of Action of A1 and A2 aLdenotesthe tightlybound ligands(e.g., heterocycles), andX isthe leavinggroup(Cl− and S-/O-dmso ligands). H DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article extensive biotransformation of the parent agents (Scheme 1). toward transferrin under identical experimental condi- Different biotransformations of A1/A2 were likely to have tions,86−88 the Rh−transferrin adducts of A1/A2 were less occurred in the extracellular fluids, extracellular matrix, cell likely to be implicated in the overall speciation of parent membrane, cytoplasm, and nucleus. complexes. Rhodium Speciation in Extracellular Fluids. Immedi- Rhodium Speciation in Extracellular Matrix. For a atelyafterdissolutioninextracellularfluids(e.g.,bloodplasma, parent complex bearing labile ligands (e.g., A1), species 2 and cell culture media), both A1 and A2 (simplified as L Rh-X, 3 were capable of undergoing further reactions with n where L denotes the tightly bound ligands (heterocycles) and extracellular targets, such as the collagen fibers in the tumor − Xistheleavinggroup(Cl andS-/O-dmsoligands))undergo ECMand/orcellsurfaceproteins(Scheme1,4and5),which aquation and hydrolysis to various extents. XAS speciation on would disrupt key molecular machineries that are responsible their reaction products in HBS featured primarily Rh−O for cell motility and eventually contribute to antimetastatic coordination contributions, which confirmed the formation of activities of A1 in vivo.17 Native collagen fibers have a triple- aqua/hydroxido species as the predominant reaction products helical structural motif, which is maintained by the formation for A1 and A2 under extracellular-like conditions (aqueous of disulfide bonds (cysteine knots) between the monomeric media, 310 K, [Cl − ] > 100 mM, pH 7.4) (Figures 3 and 4A precollagen proteins.75−77 The decomposition of the extrac- and Figure S5 in the Supporting Information). These ellular collagen network is catalyzed by matrix metal- intermediates (Scheme 1, 1) are reactive toward substitution loproteinases (MMPs), a family of zinc-dependent endopepti- reactions with a wide variety of biomolecules in extracellular dasesthatareupregulatedtofacilitateECMdegradationwithin fluids,intheextracellularmatrix,andoncellmembranes.Such the metastatic tumors.89 Type I collagen had been selected to ligand exchange reactions between 1 and biological nucleo- investigatethepossibleinteractionsbetweenA1andtheECM philes (e.g., amino acids, peptides and protein residues) of cancerous tissue, because of its high abundance in resultedintheformationofRh(III)specieshavingmixedRh− connective tissues of the human body.90 In order to mimic N,Rh−O,andRh−Sbonds.LCFresultsontheXASfromthe theECMlesionsthatmayoccurinsolid tumors,asolutionof reaction products with serum albumin and apotransferrin native type I collagen was subjected to chemical degradation (Figures 3 and 4B and Figure S6 in the Supporting viatreatmentwithimmobilizedTCEPdisulfidereducingresin, Information) suggested the formation of both covalent an agent that breaks the disulfide bonds present in native (Scheme 1, 2) and noncovalent (Scheme 1, 3) adducts. collagen. The preparation of chemically degraded type I Formation of covalent Rh−protein adducts proceeded via the collagen gel was driven by the hypothesis that the chemical stepwise substitution of Cl − and S-/O-dmso ligands of the equilibrium that originally favors the formation of disulfide parent complexes or aqua ligands of the intermediates by bondsmightbedrivenintheoppositedirectionbythehypoxic aminoacidresidues.Thisisconsistentwithsimilarmixturesof and acidic microenvironment that is typical of solid S, N, and O donors obtained from LCF analysis of XAS from tumors.91−94 differentRh−proteinadducts(Figure4,A1B4/A2B4,A1B24/ The results of LCF analysis of the XAS (Figure 6, A1CN/ A2B24, A1T/A2T). These results also underlined that the A1CD) and 3D XAS scatter plot (Figure 3 and Figure S9 in binding of parent A1/A2 to apoBTf proceeded more readily the Supporting Information) illustrated an increase in soft via ligand-substitution reactions with surface residues than at donor character of the ligand donors in the reaction products the Fe-binding domains, since the substantial molar con- from A1 treatment of chemically degraded collagen gel in tributionsfromS4tothefittedXASof A1TandA2T(Figure comparisonwiththosefromnativecollagengel.Sincechemical 4, A1T/A2T) was inconsistent with the expected donor degradation produces more free thiols in comparison to its composition at the domain (two tyrosyl phenolates, a histidyl native counterpart, the increased soft donor character of the imidazole, a aspartate carboxyl, and a synergistic carbonate, XAS of the reaction product of A1 in the former matrix overall5Oand1N).78 TheminorXAScontributionsfromthe indicated the formation of Rh−S coordination bonds via neat A1/A2 in the fitted XAS of reaction products in serum- ligand-exchange reactions between free thiols and the original supplemented cell culture media (Figure 5) represented the ligands of A1 (Cl and S-/O-dmso). These reactions likely formationofnoncovalentRh−proteinadductsviahydrophobic resulted in the cross-linking of the otherwise loose collagen interactions, similar to those observed from the albumin network within and around the periphery of solid tumors, adducts of NAMI-A, KP1019, and their analogues.79,80 More which increased the difficulty of cancer cell migration. The importantly, the presence of XAS of Rh−BSA adducts in the cross-linkingofthecollagennetworkbyA1wasthusproposed fitted XAS of reaction products from the same matrices to be one of the key factors that was responsible for its stronglyarguedfortheformationofcovalentRh−BSAadducts antimetastatic activities on animal models evaluated in earlier in reaction mixtures, since albumin is the most abundant studies.17 Similarly, transmission electron microscopy exami- protein in serum.81 nation of the tissues of a tumor-bearing animal model treated The biological activities of these Rh−protein adducts have with NAMI-A confirmed the selectively accumulation of Ru yettobeunderstood.Nevertheless,albuminadductsofA1and species in collagen fibers and the basement membrane A2arelikelytoactastheirreservoirsandcarriersthatfacilitate surrounding the solid tumors,95 and a recent XANES analysis the accumulation of bioactive Rh species in solid tumors, due on the biotransformation of NAMI-A in collagen matrix also to the enhanced permeability and retention effect.82,83 Similar identified substantial S-donor character in its binding to BSA reaction products have been deduced in similar XAS Ru(III) species.32 experiments with various metal-based anticancer agents, Rhodium Speciation in the Cytoplasm. Parent com- including NAMI-A44 and a series of Ru(II)−halido−thiaether plexes that are highly kinetically inert toward aquation and complexes,84,85 as well as the latest organometallic hydrolysis (e.g., A2) are capable of diffusing across the cell [RhIII(Cp*)Cl(cur)] complex.24 Owing to the higher reac- membrane and accumulating in the cytoplasm without tivities of metal-based anticancer agents toward albumin than undergoingextensiveligand-exchangereactionsinextracellular I DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article fluids (Scheme 1, 6), thus acting as the intracellular drug dissociate from the metal core after cellular uptake and reservoir.DuetothesignificantlydecreasedCl − concentration metabolism.101 Although Rh−imidazole coordination can be inthecytoplasm,6undergoesmoreextensiveligand-exchange modeledbymodelcompoundsS5andM1,theirXAShadzero reactions with various intracellular nucleophiles. Species contributions to the best LCF fit to the XAS from A2BC. generated subsequently via these reactions were likely the Rh Nevertheless, Figure S9 shows that the point representing adducts with cytosolic thiols (e.g., GSH) and S-rich enzymes A2BCwasincloseproximitytothatofS5,indicatingthatheir (Scheme 1, 7 and 8). This hypothesis is supported by the spectralfeaturesweresimilartoacertainextent.Thismightbe degreeofRhspeciationinRh-treatedbulkcells(Figures3and due to the imidazole model compounds used in LCF analysis 6 and Figure S9 in the Supporting Information), which not objectively modeling the spectral features of Rh−N featured predominant soft donor coordination environments. coordination species in A2BC: e.g., any Rh−imidazole species In the case of A2-treated cells, binding of free GSH with Rh that resulted from the ligand-exchange reactions between A2 species was capable of causing the depletion of this key andcytosolicproteins/peptides.PursuingamodelXASlibrary intracellular antioxidant.96,97 Since many types of cancer cells that conclusively addressed all possible ligand combinations have increased levels of reactive oxygen species (ROS) in and stereochemistries was not practical. The minor molar comparison to their normal counterparts,98 depletion of GSH contribution of Rh−S coordination bonds, together with no could eventually result in an increase in intracellular ROS and contributions from Cl and O donor model complexes to the may trigger apoptosis by elevated oxidative stress. The high XAS fits, implied the complete displacement of chlorido affinityofRhtowardSdonorsmayalsocausetheinhibitionof ligandsfromtheparentcomplexbyintracellularSdonors,such cytosolic enzymes having thiol and thiaether residues at the astheGSH,S-richpeptidesandproteins.Thismodeofaction activesite,amodeofactionthatissimilartothosediscovered has been established for a number of organometallic Ru(II)− in earlier studies on dinuclear Rh(II)−tetracarboxylato arene anticancer complexes, where their cytotoxicity occurred complexes.14,99,100 Both factors are believed to be the key viacatalyzingtheoxidationofintracellularGSH,whichcaused contributions to the remarkable cytotoxicities of A2 toward apoptosis of cancer cells via elevated oxidative stress.102 cancer cell lines. Rhodium Speciation in the Nucleus. Similar to the UnlikethatobservedfromA2-treatedcells,thefittedXASof established mode of actions for cisplatin, nuclear DNA might the A1-treated bulk cells had XAS contributions from Rh−S, be a potential target of A1/A2.103,104 Synchrotron X-ray Rh−N, and Rh−O coordination bonds, while an XAS fluorescencemicroprobe(XFM)mappingonsingleA549cells contribution from the parent A1 was absent in the fit. This treated with A1 or A2 revealed the presence of Rh species in can be explained on the basis of a common mode of actions the nucleus.50 However, these findings did not necessarily proposed for both A1 and A2. In general, the contrasting showthatthePt-basedandRh-basedcomplexeswereforming anticancer efficacies exhibited by these structurally similar similarnuclearDNAadducts.Earlierinvestigationshadshown analogues (A1, antimetastatic; A2, cytotoxic)50 were the that A1 and A3 predominantly formed monofunctional outcomes of two competing processes: extracellular aqua- adducts with the plasmid DNA, with greater preference to tion/hydrolysis and subsequent substitution with the cell coordinate with pyrimidines.17 These properties were in stark surface and ECM, or intracellular accumulation. The contrast to those of cisplatin, which interacts with DNA via biotransformation of A1 featured extensive ligand-substitution formation of the covalent adducts with adjacent guanines.105 reactions with extracellular biomolecules, such as the serum KP1019 induces nuclear DNA lesions that were different in proteins and collagen networks of tumor ECM, as well as the quantity and nature from those caused by cisplatin,106,107 and cell surface proteins, as was demonstrated by the LCF XAS its pronounced cytotoxicity has been attributed to the results of reactionproducts in HBS, in cell culture media, and induction of apoptosis via an intrinsic mitochondrial path- on A1-treated cells. Given its low levels of cellular uptake, the way108 (also observed in recent mitochondrial respiration proposed biotransformation mode of A1 is believed to impair assays)84andthepromotionofintracellularROSformation.106 the mobility and invasiveness of cancer cells, which eventually Similar to that observed for KP1019, the pronounced gives rise to its antimetastatic activities in vivo.17 The bulky mitochondrial toxicity of A2 has also been confirmed via and hydrophobic indazole ligand resulted in A2 being more mitochondrialrespirationassays.50Thisevidencesuggestedthe inert to aquation/hydrolysis and ligand substitution than was redox modulation, but not the direct nuclear DNA damage, A1. The hydrophobicity of the indazole ligand would also was most likely to be the mode of action for the anticancer facilitatethepassivediffusionofA2acrossthecellmembrane. activities of A2. Targeting the defective redox balance of ThiswouldleadtorapidaccumulationofA2initsnativeform cancerous tissue is becoming an effective approach for the in substantial quantities (Figure 6, 40% contribution to the developmentofmetal-basedanticanceragents,49andanumber overall fitting) in the cytoplasm of the treated cancer cells. ofprecious-metal-basedorganometalliccomplexesdesignedon Therefore, A2 acted mainly as an intracellular cytotoxin, and the basis of this principle have produced promising outcomes the substantial XAS contribution to the fitted XAS of A2- i■n preclinical studies.30,109−111 treatedcellsfromtheXASofparentA2wasdirectevidenceof its inertness. Additional LCF contributions from Rh−N CONCLUSIONS coordination bonds (S4 and M7) to the fitted XAS of A2BC Overall,theworkpresentedaboveconsolidatedthestructure− werelikelymadebytheindazoleligandsoftheparentA2and reactivity−bioactivity relationship reported in previous studies the imidazole/amine/imine residues of proteins and/or onothermetalcomplexes.TheXASspeciationresultsreported peptides.32,40 An earlier X-ray fluorescence microprobe hereinlaythefoundationforamoredetailedunderstandingof (XFM) mapping study on A549 cells treated with 5- the cellular biochemistry for cancer cells treated with A1 and iodoindazole analogues of NAMI-A and KP1019 showed A2. Also demonstrated was the feasibility and robustness of identical intracellular distributions of Ru and I, which synchrotron XAS speciation in preclinical studies on metal- highlighted that N-heterocyclic ligands were less likely to based anticancer compounds, especially its capability of J DOI:10.1021/acs.inorgchem.8b03477 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article providing valuable biochemical information on the physio- yl)-2,5-diphenyl-2H-tetrazolium bromide; TCEP, tris(2- logical transformation and speciation of metallodrugs that carboxyethyl)phosphine; μE, normalized X-ray absorbance; would be otherwise difficult to acquire by conventional SOM, second oscillation maximum ■ s■pectroscopic methods. REFERENCES ASSOCIATED CONTENT * (1) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. 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