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Synthesis of Camphor-Derived Bis(pyrazolylpyridine) Rhodium(III) Complexes: Structure-Reactivity Relationships and Biological Activity.

PMID: 30565467
Article CiteThis:Inorg.Chem.2019,58,307−319 pubs.acs.org/IC Synthesis of Camphor-Derived Bis(pyrazolylpyridine) Rhodium(III) − Complexes: Structure Reactivity Relationships and Biological Activity Angelina Petrovic, ́† Milan M. Milutinovic, ́†,∇ Edward T. Petri, # Marko Z ̌ ivanovic, ́† Nevena Milivojevic, ́† Ralph Puchta, ‡,§,∥ Andreas Scheurer, ‡ Jana Korzekwa, ‡ Olivera R. Klisuric, ́⊥ and Jovana Bogojeski *,† † ́ Faculty of Science, University of Kragujevac, Radoja Domanovica 12, 34000 Kragujevac, Serbia ‡ § Inorganic Chemistry, Department of Chemistry and Pharmacy, Computer Chemistry Center, Department of Chemistry and Pharmacy, and ∥ Zentralinstitut für Scientific Computing, University of Erlangen-Nürnberg, 91058 Erlangen, Germany ⊥ # Faculty of Science, Department of Physics and Faculty of Science, Department of Biology and Ecology, University of Novi Sad, ́ Trg Dositeja Obradovica 4, 21000 Novi Sad, Serbia ∇ Department of Organic Chemistry, University of Paderborn, Warburgerstraße 100, 33098 Paderborn, Germany * S Supporting Information ABSTRACT: Two novel rhodium(III) complexes, namely, [RhIII(X)Cl ] (X = 2 2,6-bis((4S,7R)- 3 7,8,8-trimethyl-4,5,6,7-tetrahydro-1H-4,7-methanoindazol-3-yl)pyridine or 2,6-bis((4S,7R)-1,7,8,8-tet- ramethyl-4,5,6,7-tetrahydro-1H-4,7-methanoindazol-3-yl)pyridine), were synthesized from camphor derivatives ofabis(pyrazolylpyridine),tridentatenitrogen-donorchelatesystem,giving[RhIII(H L*)- 2 Cl ] (1a) and [RhIII(Me L*)Cl ] (1b). A rhodium(III) terpyridine (terpy) ligand complex, 3 2 3 [RhIII(terpy)Cl ] (1c), was also synthesized. By single-crystal X-ray analysis, 1b crystallizes in an 3 orthorhombicP2 2 2 system,withtwomoleculesintheasymmetricunit.Tridentatecoordinationby 1 1 1 theN,N,N-donorlocalizesthecentralnitrogenatomclosetotherhodium(III)center.Compounds1a and 1b were reactive toward L-methionine (L-Met), guanosine-5′-monophosphate (5′-GMP), and glutathione(GSH),withanorderofreactivityof5′-GMP>GSH>L-Met.Theorderofreactivityof theRhIIIcomplexeswas:1b>1a>1c.TheRhIIIcomplexesshowedaffinityforcalfthymusDNAand bovineserumalbuminbyUV−visandemissionspectralstudies.Furthermore,1bshowedsignificantin vitro cytotoxicity against human epithelial colorectal carcinoma cells. Since the RhIII complexes have similar coordination modes, stability differences were evaluated by density functional theory (DFT) calculations (B3LYP(CPCM)/LANL2DZp). With (H L*) and (terpy) as model ligands, DFT calculations suggest that both 2 tridentateligandsystemshavesimilarstability.Inaddition,moleculardockingsuggeststhatalltestcompoundshaveaffinityfor the minor groove of DNA, while 1b and 1c have potential for DNA intercalation. ■ INTRODUCTION Transition-metal complexes have been synthesized with pyridine-containing tridentate triamine ligands, for use in Transition-metal complexes have a range of applications in supramolecular chemistry,1 catalytic chemistry,2 and as catalytic reactions or as potential antitumor agents, etc. medicinal agents.3 In chemical biology, transition-metal Tridentate triamine ligands have advantages, including ready complexes were investigated as inhibitors,4−9 imaging availability, relatively low cost, and low toxicity.28 Various agents,10−12 biological probes,13,14 or catalysts with unique metal complexes with camphor-based pyridine ligands have also been used for asymmetric catalytsis29 and tested for properties. Over the last century, platinum-based complexes havebeenusedasanticancer drugs.15,16 However, sideeffects, biomolecular interactions or antitumor activity.30−33 such as cell-acquired resistance and high toxicity,17 have Previously, we synthesizeda RhIIIcomplex with a tridentate prompted investigation of other metal complexes.18,19 Despite nitrogen-donor pincer-type ligand that displayed promising their variable oxidation states, the anticancer properties of properties and biomolecular reactivity.20 Thus, we sought to rhodium complexes have not been extensively explored.20,21 expand our investigation of RhIII complexes to pincer-type However, kinetically inert transition-metal complexes could ligands with diverse substituent patterns on the pyrazolyl serve as scaffolds for pharmacological agents due to their moiety,creatingdifferencesinspaceconfigurationandelectron inertness, stability, unique geometries, and structural diver- density distribution that could influence biomolecular sity.22 Recently, such metal complexes were shown to have interaction potential or cytotoxicity. affinity both for DNA, their primary target, as well as various proteins,23−27 suggesting potential use in the design of Received: August24, 2018 anticancer agents. Published: December19,2018 ©2018AmericanChemicalSociety 307 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 .913-703:85.9102 .mehC .gronI .ylno esu lanosrep roF .91/41/40 no ETTEYAFAL TA ANAISIUOL FO VINU yb gro.sca.sbup morf dedaolnwoD Inorganic Chemistry Article Therefore, in the present study we designed two new RhIII Synthesis and Characterization of [RhIII(H L*)Cl ] (1a) and complexes ([RhIII(H 2 L*)Cl 3 ] and [RhIII(Me 2 L*)Cl 3 ]), where [RhIII(Me 2 L*)Cl 3 ] (1b). In general, a solution of 2 H 2 L*3 or Me 2 L* H L* and Me L* are camphor derivatives of a previously (0.380 mmol; 1 equiv) in 20 mL of ethanol was slowly added to a 2 2 solution of 100 mg (0.380 mmol) of RhCl·xHO in 60 mL of introduced bis(pyrazolylpyridine) ligand (Figure 1). For 3 2 ethanol. The mixture was stirred and refluxed overnight, affording a yellow precipitate that was filtered and dried under vacuum. The resultingyellowsolidwaspurifiedbyrecrystallization.Crystallization of 1b was induced by slow diffusion of n-hexane into a dichloro- methane solution of the1b complex. Synthesisof[RhIII(HL*)Cl](1a).HL*(162.5mg)yielded1aasa 2 3 2 yellow solid(196 mg; 0.31mmol;81%). 1HNMR(500MHz,CDCN):δ=8.1(t,J =7.8Hz,para-Ar− 3 HH CH,1H),7.82(d,J =7.8Hz,meta-Ar−CH,2H),6.94(brs,NH, HH 2H), 3.35 (d, J = 3.9 Hz, HCCHCH, 2H), 2.41−2.37 (m, HH 2 2 HCCHCH,2H),2.10−2.04(m,HCCHCH,2H),1.65−1.48(m, 2 2 2 2 HCCHCH and HCCHCH, 4H), 1.38 (s, CCH, 6H), 1.08 (s, 2 2 2 2 3 C(CH),6H), 0.89(s,C(CH),6H) ppm. 3 2 3 2 13CNMR(126MHz,CDCN):δ=158.9(ortho-Ar-C,2C),146.0 3 (HN−CCC,2C),139.2(para-Ar−CH,1C),135.4(HN−CCC,2C), 127.0(meta-Ar−CH,2C),121.0(HN−CCC,2C),63.2(CCH,2C), 3 52.1 (C(CH), 2C), 50.5 (HCCHCH, 2C), 36.3 (HCCHCH, 3 2 2 2 2 2 2C), 29.8(HCCHCH, 2C),25.0 (C(CH), 2C),21.6 (C(CH), 2 2 3 2 3 2 2C),11.2(CCH,2C) ppm. 3 Anal. Calcd for (C H ClNRh) C: 50.92; H: 5.22; N: 11.00. 27 33 3 5 Found: C: 51.26;H:5.93; N:11.14%. ESI-MS: [M-Cl]+: Calcd: 599.64.09;Found: 599.18. Synthesisof[RhIII(Me L*)Cl](1b).MeL*(173.1mg)yielded1b 2 3 2 asa yellow solid(189.5 mg;0.28mmol, 75%). 1HNMR(500MHz,CDCN):δ=8.14(dd,J =8.5Hz,meta- 3 HH Ar−CH, 2H), 7.82 (t, J = 8.5 Hz, para-Ar−CH, 1H), 4.38 (s, HH NCH,6H),3.33(d,J =3.8Hz,HCCHCH,2H),2.31−2.05(m, 3 HH 2 2 Figure 1.Structuresof the investigatedRhIII complexes. HCCHCH,2H),2.05−1.91(m,HCCHCH,2H),1.52−1.33(m, 2 2 2 2 HCCHCH and HCCHCH, 4H), 1.41 (s, CCH, 6H), 1.08 (s, 2 2 2 2 3 C(CH),6H), 0.78(s,C(CH), 6H)ppm. 3 2 3 2 13C NMR (126 MHz, CDCN): δ = 159.9 (CHN−CCC, 2C), comparison, a RhIII terpyridine (terpy) complex was also 151.9(ortho-Ar-C,2C),143.8 3 (CHN−CCC,2C),1 3 40.1(para-Ar− synthesized and examined. Substitution reactions with CH,1C),128.9(CHN−CCC,2C) 3 ,119.5(meta-Ar−CH,2C),63.6 3 biomolecules were studied, and biomolecular interaction (CCH, 2C), 53.7 (C(CH), 2C), 47.8 (HCCHCH, 2C), 38.5 3 3 2 2 2 potential with calf thymus DNA (CT-DNA) and bovine (NCH,2C),32.6(HCCHCH,2C),26.7(HCCHCH,2C),19.6 3 2 2 2 2 serum albumin (BSA) were measured. New compounds were (C(CH),2C),18.3(C(CH),2C),10.0(CCH, 2C)ppm. 3 2 3 2 3 testedforinvitrocytotoxicityusinga3-(4,5-dimethylthiazol-2- Anal. Calcd for (C H ClNRh) C: 52.39; H: 5.61; N: 10.53. 29 37 3 5 yl)-2,5-diphenyltetrazoliumbromide(MTT)assayandhuman Found: C: 52.26;H:5.53; N:10.01%. epithelial colorectal carcinoma cells (HCT-116). density ESI-MS: [M-Cl]+: Calcd: 628.12; Found:628.14. Instrumentation. NMR spectra were recorded on a 200 MHz functional theory (DFT) calculations were conducted to Varian Gemini-2000 and 500 MHz Bruker Avance spectrometer. explorethesimilaritybetweenourbasicbis(pyrazolylpyridine) NMRsignalswerereferencedtoresidualprotonorcarbonsignalsof ligand system and the well-known terpyridine complex thedeuteratedsolvent(1Hand13CNMR)andarereportedinparts a■nalogue. per million relative to tetramethylsilane (TMS). Elemental analyses (C, H,N) were performed by combustion and gas chromatographic EXPERIMENTAL SECTION analysis with an Elementar Vario MICRO elemental analyzer. pH Chemicals and Solutions. Commercial chemicals were used measurementsweredoneusingaMettlerDelta350digitalpHmeter without purification. L-Methionine (L-Met), glutathione (GSH), with resolution of ±0.01 mV and a combination glass electrode guanosine-5′-monophosphate sodium salt (5′-GMP), 2,2′:6′,2″- calibratedusingstandardbuffersolutions(Sigma)atpH4,7,and9. terpyridine (terpy), RhCl·xHO, N-2-hydroxyethylpiperazine-N′-2- UV−VisandkineticmeasurementswereconductedonaPerkinElmer 3 2 ethanesulfonicacid(HEPES), phosphate-buffered saline (PBS),CT- Lamda25and35double-beamspectrophotometerwiththermostated DNA, ethidium bromide (EB), and BSA were from Sigma-Aldrich. 1.00cmquartzSuprasilcells.Temperaturewascontrolledto±0.1°C. CT-DNAwasdissolvedintriple-distilleddeionizedwaterandstored Fluorescence was measured on an RF-1501 PC spectrofluorometer at 4 °C for less than one week. The UV absorbance ratio (260 / (Shimadzu). Mass spectrometry was measured on a Waters nm 280 )ofCT-DNAsolutionsinPBS(phosphate=0.01M,c(NaCl) QuadrupoleTOF Synapt2Gusing electrosprayionization (ESI). nm =0.137,c(KCl)=0.0027M,pH7.4)was1.8−1.9,indicatingalackof Solubility Measurements. The concentrations of saturated protein contamination.34 Nucleophile stock solutions were freshy solutions of the studied RhIII complexes were determined by UV− prepared before use. Dulbecco’s Modified Eagle Medium (DMEM) vis spectrometry. Therefore, the specific absorptivity of the andPBSwereobtainedfromGIBCO,Invitrogen.Fetalbovineserum compounds in the water was determined first. This was measured (FBS) and trypsin−ethylenediaminetetraacetic acid (EDTA) were using five dilution series (5, 10, 30, 40, 50 mM) of the studied from PAA (The Cell Culture Company). Dimethyl sulfoxide complexes, and then the calibration curve was calculated using (DMSO) and MTT were from SERVA. Doubly distilled deionized Lambert−Beerlaw.Theslopeofthecurvegavespecificabsorptivity. waterwasusedforallexperiments.PreparationsofHL*andMeL* The required quantity of water solution was added to the 5 mL 2 2 ligands were according to published procedures.35 [Rh(terpy)Cl] volumetric flask. The solution was heated to 298 K. A previously 3 (1c) was prepared as published and characterized by standard weighedquantityofRhIIIcomplexwasaddedtothevolumetricflask, analyticalmethods.36 untilthesaturationpointoccurred.Stirringwascontinuedupto7hat 308 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article 298K.Thesamplewasfilteredthrough0.20μmmembranefilter.A observedflowtimeofDNA-containingsolutions(t)correctedforthe measured quantity of filtered sample was transferred into another flow timeof bufferalone(t),η =(t− t)/t. 0 0 0 volumetric flask and made further dilutions. The absorbance was Protein Binding Studies. Protein fluorescence is due to natural measured using UV−vis spectrophotometry. The same process was fluorophores such as tryptophan, tyrosine, and phenylalanine. repeatedtwotimes. Changes in BSA fluorescence were used to monitor interaction with KineticMeasurements.Thehydrolysisofcomplexes1a,1b,and metal complexes. Tryptophan fluorescence quenching experiments 1cwasstudiedbyUV−visspectrometryat298K.Thesamples(0.10 wereconductedusing2.0μMBSAinPBS.Quenchingoftheemission mM)werepreparedinabuffersolution(25mMHEPESbuffer,pH= intensity of BSA tryptophan residues at 363 nm in the presence of 7.2)orPBS.Theworkingwavelengthofeachreactioncorresponded increasingconcentrationsofRhIIIcomplexes1a,1b,and1c(0−10.0 to that of a maximum change in absorption derived from the μM)wasmonitored.Fluorescencespectrawererecordedintherange difference spectra. The absorbance at the selected wavelength was of 300−500 nm with excitation at 295 nm. Excitation and emission recorded at 30 s intervals, and the absorption/time data for each bandwidths wereboth 10nm. complex were fitted to the first-order rate equation, which gave the CellPreparationandCulturing.HCT-116cellswerepurchased k value foreach aquation process. fromtheAmericanTissueCultureCollection.Cellsweremaintained H2O The kinetics of the substitution of coordinated chloride was in controlled physiological conditions and grown in DMEM measured spectrophotometrically by following the change in supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, absorbance at suitable wavelengths as a function of time. Working and 100 μg/mL streptomycin in a humidified atmosphere with 5% wavelengths were determined by recording spectra of the reaction CO at37°C. 2 mixturefrom220to450nm.Kineticmeasurementswereperformed Viability Effects. A standardized MTT colored reaction under pseudo-first-order conditions, with nucleophile concentrations (Mosmann, 1983)38 was measured on an ELISA microplate reader atleast10-foldinexcess.Reactionswereinitiatedbymixing0.50mL (Rayto-2100C) asdescribed inour previous work.39−41 of a nucleophile complex solution with 2.50 mL of thermally X-ray Diffraction Studies. X-ray diffraction data for equilibratedcomplexsolutioninaUV−viscuvette,andreactionswere [RhIII(MeL*)Cl] (1b) were collected at room temperature on an followed for at least eight half-lives. The observed pseudo-first-order Oxford D 2 iffractio 3 n Gemini S diffractometer. Graphite-monochro- rate constant k represents an average value of three to four mated Mo Kα radiation (λ = 0.7107 Å) was used to measure obs independent kinetic runs for each experimental condition. Some diffraction from suitable single crystals of complex 1b. CrysAlisPro reactionswerestudiedatthreetemperatures(288,298,and310K). and CrysAlisREDsoftwarepackages42 wereusedfordatacollection Experimental data are summarized in the Supporting Information and data integration. Space group determinations were based on (Tables S1−S9). Values for constants and other thermodynamic analysis of the Laue class and systematically absent reflections. parameters were determined using Microsoft Excel 2007 and Collected data were corrected for absorption effects using the OriginPro8. Multiscanmethod,applyinganempiricalabsorptioncorrectionusing UV−Vis DNA Interactions. CT-DNA stock solutions were spherical harmonics as implemented in SCALE3 ABSPACK.42 prepared in PBS, resulting in a UV absorbance ratio A 260 /A 280 of Structuresolutionandrefinementwereperformedwiththeprograms ca. 1.8−1.9, indicating negligible protein contamination. CT-DNA SHELXT and SHELXL-2014/6, respectively.43 MERCURY44 was c c o m n − c 1 e .3 n 7 trations were determined using A 260 with ε = 6600 M−1 p em re p p l a o r y e ed m f a o te r r m ial ol f e o c r ul p a u r b g l r ic a a p t h io ic n s . ,a N n o d n W -hy in d G ro X g 4 e 5 n so at ft o w m a s re w w e a re su r s e e fi d ne t d o Fluorescence spectra were recorded in the range of 550−750 nm anisotropically; C−H hydrogen atoms were included at calculated withexcitationat527nm.Excitation andemissionbandwidthswere positionsridingontheirattachedatomswithfixeddistancesofCH= both 10nm. 0.93Å andCH = 0.96Åwith U (H)= 1.2U (C)formethylene UV−Vis Absorption Studies. To quantitatively compare the andmethynegro 2 ups,andCH =0. i 9 so 7ÅwithU ( e H q )=1.5U (C)for 3 iso eq binding strength of the complexes, the intrinsic binding constant K b methyl groups. [RhIII(Me 2 L*)Cl 3 ] complex had contributions from wasdeterminedbymonitoringchangesinabsorptionatthemetal-to- disordered solvent molecules that were removed by the SQUEEZE ligandchargetransfer(MLCT)bandwithincreasingconcentrationof routineimplementedinPLATON,46andtheoutputfromSQUEEZE CT-DNAusing thefollowing eq1. calculations is attached in the CIF file. Crystal data and refinement parameters are summarizedinTable 1. [DNA]/(ε A −ε f )=[DNA]/(ε b −ε f )+1/[K b (ε b −ε f )] (1) Quantum Chemical Methods. To enable comparison with earlier studies,47 we performed B3LYP/LANL2DZp hybrid DFT K is given by the ratio of the slope to the y intercept in plots of [D b NA]/(ε −ε)versus[DNA],where[DNA]istheconcentration calculations, with pseudopotentials on the heavy elements and the of DNA i A n bas f e pairs, ε = A /[complex], ε is the extinction valence basis set augmented with polarization functions.48,49 During coefficient for the unbou A nd c o o bs m d plex, and ε f is the extinction structure optimization, only symmetry constraints were applied. In coefficient forthe complexinthe fully boundfo b rm. addition, resulting structures were characterized as minima, by computation of vibrational frequencies. Relative energies were EthidiumBromideDisplacementStudies.Therelativebinding corrected for zero-point vibrational energies (ZPE). The influence of complexes to CT-DNA was determined by calculating the ofbulksolventwaterwasevaluatedviasingle-pointcalculationsusing quenching constant (K ) from the slopes of straight lines obtained fromthe Stern−Volmer sv equation(eq 2) CPCM formalism,50 that is, B3LYP(CPCM)/LANL2DZp//B3LYP/ LANL2DZp. The Gaussiansuite ofprograms was used.51 I /I=1+K [Q] (2) MolecularDockingSimulations.Structuralcoordinatesrepresent- 0 sv ing a fragmentof (1) canonical B-DNA (PDB:1BNA) or (2)DNA whereI andIareemissionintensitiesintheabsenceandpresenceof with an intercalation gap (PDB: 1Z3F) were obtained from the 0 quencher (complexes 1a, 1b, and 1c), respectively, [Q] is the total protein data bank (http://www.rcsb.org). Water molecules, ligands, concentration of quencher, and K is the Stern−Volmer quenching and heteroatoms were removed if present. Hydrogen atoms and sv constant,whichwasobtainedfromtheslopeoftheplotofI/Iversus Gasteigerpartialchargeswereadded;nonpolarhydrogenatomswere 0 [Q]. merged in AutoDockTools52 (http://autodock.scripps.edu), and Viscosity Measurements. Changes in DNA viscosity were coordinates were converted to PDBQT format. For ligands (1a, 1b, measured in the presence of increasing amounts of complexes 1a, 1c, or [RhIII(HLtBu)Cl]), nonpolar hydrogen atoms were merged, 2 3 1b, and 1c. Flow time was measured with a digital stopwatch. Each andGasteigerpartialchargeswereaddedinAutoDockTools.52Natual sample was measured in triplicate, and the average flow time was populationanalysis(NPA)partialchargesforRhandClatomswere calculated. Data are presented as (η/η)1/3 against r, where η is the calculated in Gaussian16 with B3LYP/LANL2DZp and merged into 0 DNAviscosity in the presence of complex, and η is the viscosity of final ligand PDBQT files. Grid maps were calculated in AutoGrid4 0 DNA in buffer alone. Viscosity values were calculated from the usingcoordinatesfor1BNAor1Z3F.Gridmapswerecentered,anda 309 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article Table 1. Experimental Details: Crystalographic Data and mass spectrometry (MS). For the complex 1b single crystals Refinement Parameters for [RhIII(Me L*)Cl ] Complex 1b suitable for the X-ray analysis were also obtained. 2 3 Elemental analyses on these complexes were in very good crystaldata agreementwithacomplexcompositionof[RhIII(H L*)Cl ]or 2 3 chemicalformula C H ClNRh [RhIII(Me L*)Cl ]. The 1H NMR as well as the 13C NMR 29 37 3 5 2 3 M 664.89 spectra of the 1a and 1b complexes indicated that only this r crystalsystem,spacegroup orthorhombic,P222 distinctspeciesisformed.Theobtainedspectradisplayasetof 1 1 1 a,b,c(Å) 13.4941(3),19.4283(4), signals for the pyrazole moieties, pyridine moiety, and a 28.9799(13) camphor moiety, significantly shifted, compared to the free V(Å3) 7597.6(4) ligand. Further, the complexes are characterized by ESI-MS Z 8 massspectrometry,inthem/zrangeof400−700thatincludes radiationtype MoKα main peaks at m/z = 599.18 (1+) and 628.14 (1+), which No.ofreflectionsforcell 9233 representsfragmentsof the1aand1bcomplexes,whichcame measurement θrange(deg)forcellmeasurement 2.8−28.4 about by losing one chloride. μ(mm−1) 0.68 Crystal Structure Discussion. A perspective view of the molecular structure of 1b with adopted atom-numbering crystalshape prism crystalsize(mm) 0.51×0.42×0.14 scheme is shown in Figure 2. Selected metal−ligand bond datacollection diffractometer Xcalibur,Sapphire3,Gemini absorptioncorrection multiscan Tmin,Tmax 0.882,1.000 No.ofmeasured,independentandobserved 27626,13398,10655 [I>2σ(I)]reflections R 0.052 int θvalues(deg) θ =25.0,θ =2.5 max min (sinθ/λ) (Å−1) 0.595 max rangeofh,k,l h=−16→11,k=−23→15, l=−34→29 refinement R[F2>2σ(F2)],wR(F2),S 0.084,0.228,1.04 Figure 2. MERCURY44 drawing of the molecular structure of complex1bwithlabelednon-Hatoms(onlymoleculeAisshownfor No.ofreflections 13398 clarity).Displacementellipsoidsareshownat30%probability,andH No.ofparameters 701 atoms aredrawn asspheres ofarbitrary radii. No.ofrestraints 13 H-atomtreatment H-atomparametersconstrained ρ ,ρ (eÅ−3) 0.96,−2.41 lengths, bond angles, and torsion angles are listed in Table 2. max min absolutestructureparameter 0.05(3) [RhIII(Me L*)Cl ] complex crystallizes in an orthorhombic 2 3 crystal system and P2 2 2 space group, where each 1 1 1 asymmetric unit consists of two 1b complex molecules maximumgridboxsizewaschosentocovertheentireDNAmolecule. (molecule A and molecule B). From a structural point of For1BNAagridboxof54×56×106wascenteredatx,y,z=14.72, 20.99,8.82.For1ZF3agridboxof62×50×68wascenteredatx,y,z view,thecoordinationoftheRh1AandRh1Batomsinthe1b complex is noteworthy. The rhodium centers in the =2.27,15.76,37.63.Defaultgridspacingof0.375Åwasused.Maps werecalculatedforallligandatomtypesalongwithelectrostaticand [RhIII(Me 2 L*)Cl 3 ] complex are coordinated in a slightly desolvation maps using a dielectric value of −0.1465. Initial ligand distorted octahedral geometry. position, orientation, and dihedral offset were set as random. The Because of the tridentate coordination of the N,N,N-donor, numberoftorsionaldegreesoffreedomforeachligandwasfixedto0 the central nitrogen atom is pushed closer toward the to ensure rigid docking. Docking simulations were conducted in rhodium(III) center, applying a trans influence on Cl2 that AutoDock4 using the Larmarckian genetic algorithm and default explainstheslightlylargervalueoftheRh1Cl2bondlength, parameters: the maximum number of energy evaluations was whichisthesameinbothAandBmolecules(Table2).Bond 2500000, the genetic algorithm (GA) population size was 150, and anglesN3Rh1Cl1(inbothmoleculeAandB)areslightly a total of 10 hybrid genetic algorithm with a local dearch (GA-LS) largerthat90°,whileN3Rh1Cl3angles(inbothmolecule runs were performed. Parameters for Rh atoms were added to AutoDock as follows: R-eqm = 2.93 Å, weighted epsilon = 0.008, Aand B)are smaller than 90°, showing thestericinfluence of At.frag.vol. = 12.000, At.solv.par. = −0.001, Hb R-eqm = 0.000, thebulkyMe L*chelatingligand.ThegroupRh1Cl2N2N3N4 2 weightedHbepsilon=0.000, Hbtype =0, bond index =1. Results is almost perfectly planar, since the largest displacement from w■ereanalyzedinAutoDockTools (http://autodock.scripps.edu).52 the same weighted least-squares plane in both A and B molecules is 0.111(9) Åforatom N3A.Comparing the angles RESULTS AND DISCUSSION between Rh1Cl2N2N3N4 plane and N1N2C3C2C1 and Preparation and Structure of [RhIII(H L*)Cl ] (1a) and N4N5C11C10C9 planes in molecule A (21.0 (7)° and 10.7 2 3 [RhIII(Me L*)Cl ](1b).Complexes[RhIII(H L*)Cl ](1a)and (7)°,respectively)andthesameplanesinmoleculeB(5.2(7)° 2 3 2 3 [RhIII(Me L*)Cl ] (1b) (Figure 1) were synthesized by and17.4(7)°,respectively),onecanobservethatthemolecule 2 3 stirring equimolar amounts of RhCl ·xH O and H L* or A is more disordered from planarity. This leads to the 3 2 2 Me L* ligands in ethanol and by refluxing overnight. The conclusionthatmoleculesAandBof1bcomplexhaveslightly 2 synthesized RhIII complexes 1a and 1b were characterized by different conformations, which is confirmed by comparison of 1H and 13C NMR spectroscopy, elemental analysis, and ESI the values for torsion angles (Table 2). Visually, this result is 310 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article Table 2. Selected Geometric Parameters for solubilityinwater(TableS10).Thesolubilityofthecomplexes [RhIII(Me L*)Cl ] Complex 1b is in line with oxaliplatin and a bit greater of cisplatin. 2 3 Kinetics of Aquation. The kinetics of aquation of bondlength[deg] complexes 1a, 1b, and 1c were quantitatively studied by moleculeA moleculeB UV−vis spectroscopy of 0.1 mM solutions in HEPES buffer Rh1−N3 2.027(9) 1.975(9) and PBS at 298 K. The UV−vis spectra of 1a and 1b Rh1−N4 2.063(14) 2.051(14) complexes show no significant time-dependent changes in the Rh1−N2 2.080(12) 2.051(12) region of 200−800 nm (Figure S2), which indicates that Rh1−Cl1 2.332(4) 2.319(5) complexes 1a and 1b do not undergo hydrolysis in the Rh1−Cl3 2.337(4) 2.341(4) observed time period in either 25 mM HEPES buffer or PBS. Rh1−Cl2 2.351(3) 2.362(3) Complex 1c hydrolyzes with a constant k =3.5 × 10 −5 s −1 H2O bondangles[deg] (t =330min)inPBSandk =1.1×10 −3s −1(t =9.68 1/2 H2O 1/2 N3−Rh1−N4 78.7(4) 78.4(5) min) in 25 mM HEPES buffer (Figures S3). The results N3−Rh1−N2 80.4(4) 79.6(4) obtainedsuggestthatcomplex1cunderwentslowhydrolysisin N4−Rh1−N2 158.3(4) 157.8(4) PBS and significantly hydrolyzed in HEPES buffer, but all N3−Rh1−Cl1 91.8(3) 90.9(3) kineticmeasurementsforthisstudywereperformedinHEPES N4−Rh1−Cl1 89.1(4) 85.4(4) buffer in the presence of 50 mM NaCl to prevent hydrolysis. N2−Rh1−Cl1 86.0(3) 92.1(4) Kinetic Studies. To measure the reactivity of the RhIII N3−Rh1−Cl3 87.0(3) 86.7(3) complex under physiological conditions, substitution reactions N4−Rh1−Cl3 91.2(4) 93.1(4) of RhIII complex (1a, 1b, and 1c) (Figure 1) with selected N2−Rh1−Cl3 93.2(3) 88.5(4) nucleophilesL-Met,GSH,and5′-GMPwereinvestigated.The Cl1−Rh1−Cl3 178.73(15) 177.38(17) kinetics of the substitution of coordinated chloride were N3−Rh1−Cl2 176.3(3) 176.5(3) investigated byUV−visspectroscopy,byfollowing thechange N4−Rh1−Cl2 100.9(3) 101.1(4) inabsorbanceasafunctionoftime.NucleophilesL-Met,GSH, N2−Rh1−Cl2 100.3(3) 101.1(3) and 5′-GMP were chosen because of their different Cl1−Rh1−Cl2 91.82(17) 92.51(19) nucleophilicity, steric hindrance, binding properties, and Cl3−Rh1−Cl2 89.33(16) 89.87(17) biological relevance. Since our overal goal is to investigate torsionangles[deg] theanticancerpotentialofRhIIIcomplexes,kineticexperiments Rh1−N2−N1−C1 154.1(11) −178.1(12) weredesignedtomimicphysiologicalconditions:measurments Rh1−N2−N1−C12 −19(2) 6(2) were conducted at pH 7.2 (maintained with 25 mM HEPES Rh1−N2−C3−C4 7.3(15) −3.8(15) buffer)in50mMNaCl(tosuppressthesolvolyticpathway)at Rh1−N3−C4−C5 −175.6(10) −175.0(10) 310 K. Kinetic experiments were performed under pseudo- Rh1−N3−C8−C7 173.5(10) 172.1(11) first-order conditions, with nucleophile concentrations at least Rh1−N3−C8−C9 −1.7(16) −6.1(16) 10-fold in excess. Proposed reaction pathways for all observed Rh1−N4−C9−C10 179.5(10) 166.9(11) substitution processes are shown in Scheme 1. Rh1−N4−C9−C8 11.6(17) −5.5(16) Scheme 1. Schematic Representation of Substitution depicted in Figure 3, which displays an overlay of A and B ReactionsofComplexes1a,1b,and1cwithNucleophiles:L- Met, GSH, and 5′-GMP molecules of 1b complex. Directnucleophilicattackproceedsinareversiblemanneras in Scheme 1. Substitution rate constants were determined under pseudo-first-order conditions by plotting the linear dependenceofk versustotalnucleophileconcentration(see obs eq 3). All kinetic data are summarized in Tables S1−S9 (see Supporting Information). Figure 3. MERCURY44 drawing showing an overlay of two independentmoleculesin[RhIII(Me 2 L*)Cl 3 ]1bcomplex:A(yellow) k obs = k 2 [nucleophile] + k 1 [Cl−] (3) andB (red). Hydrogenatoms wereomittedforclarity. Direct nucleophilic attack is characterized by rate constant k , and the reverse reactions are represented by rate constant 2 k . The second-order rate constant k characterizes product 1 2 The crystal packing of [RhIII(Me L*)Cl ] complex is formation and was evaluated from the slope of a plot k 2 3 obs dominantly arranged by van der Waals forces, since classic versus nucleophile concentration. Experimental results for the hydrogen bonds were not found in intra- or intermolecular displacement of a chloride ion from 1a, 1b, and 1c are shown space. in Table 3. Representative plots are shown in Figure 4. Solubility of the Studied RhIII Complexes. The Ascanbeseen,1a,1b,and1carereactivetowardallofthe prepared RhIII complexes are neutral; UV−vis spectrophoto- testednucleophiles,withorderofreactivity5′-GMP>GSH> metric measurements showed that they have moderate L-Met.RhIIIcomplexesareexpectedtohavethehighestaffinity 311 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article Table 3. Rate Constants for the Substitution Reactions of the RhIII Complex with L-Met, 5′-GMP, and GSH at pH = 7.2 (25 mM HEPES Buffer) in the Presence of 50 mM NaCl [RhIII(HL*)Cl](1a) 2 3 T(K) 1×101k,M−1s−1 1×104k[Cl−],M−1s−1 ΔH⧧,kJmol−1 ΔS⧧,JK−1mol−1 2 1 2 2 L-Met 310 2.4±0.1 0.20±0.01 5′-GMP 288 7.6±0.1 0.27±0.01 40±2 −120±8 298 13±0.1 0.35±0.01 310 27±0.1 0.80±0.02 GSH 288 1.7±0.1 26±1 −183±2 298 2.5±0.1 310 4.0±0.1 [RhIII(MeL*)Cl](1b) 2 3 T,K 1×101k,M−1s−1 1×104k[Cl−],M−1s−1 ΔH⧧,kJmol−1 ΔS⧧,JK−1mol−1 2 1 2 2 L-Met 288 2.1±0.1 0.10±0.01 17±6 −212±22 298 3.3±0.1 0.15±0.01 310 3.8±0.2 0.20±0.01 5′-GMP 288 38±0.1 0.50±0.01 10±2 −212±8 298 43±0.1 2.0±0.1 310 56±0.1 7.8±0.2 GSH 310 19±0.1 0.16±0.01 [RhIII(terpy)Cl](1c) 3 T,K 1×101k,M−1s−1 1×105k[Cl−],M−1s−1 ΔH⧧,kJmol−1 ΔS⧧,JK−1mol−1 2 1 2 2 L-Met 310 0.34±0.01 0.01±0.01 5′-GMP 288 1.3±0.1 35±5 −154±16 298 2.5±0.1 310 4.0±0.1 GSH 310 0.72±0.01 Figure4.Pseudo-first-orderrateconstantsasafunctionofnucleophileconcentration.(right)k vsnucleophileconcentationforreactionof1a, obs 1b,and1ccomplexesand5′-GMP.(left)k obs vsnucleophileconcentationforreactionof1bcomplexand5′-GMP,L-Met,andGSH;pH=7.2and 310K in25mM HEPESand 50mM NaCl. for N-bonding nucleophiles, because in addition to the [RhIII(Me L*)Cl] (1b) > [RhIII(H L*)Cl] (1a) presence of the nitrogen donor atom RhIII ions are borderline 2 3 2 3 hard−softacids.ThelowerreactivityofL-MetoverGSHcould > [RhIII(terpy)Cl 3 ] (1c) be due to the bulky methyl group in L-Met, making access to the RhIII complexes difficult. Furthermore, the N-donor The camphor-derived bis(pyrazolylpyridine) RhIII com- nucleophile 5′-GMP is able to compete with S-donor plexes react ∼10 times faster than [RhIII(terpy)Cl 3 ]. Although nucleophiles L-Met and GSH, and display greater affinity complexes 1a and 1b have similar structure, 1b is more toward the RhIII complexes. These observations are of special reactive. Considering steric effects, 1b is the most bulky RhIII interest,sinceunderbiologicalconditionswithinthecell,these complex, suggesting that electronic effects have a greater S-donor biomolecules are present in relatively high concen- impact on reactivity than steric effects. The obtained rate trations and therefore compete with the DNA; suggesting the constants are in agreement with an earlier studied complex possibility that the investigated complexes could be bound to [RhIII(H LtBu)Cl ].20 2 3 DNA and that DNA could be considered a potential target. The thermodynamic properties and relative stability of the The order of reactivity of the investigated RhIII complexes is RhIII complexes were examined comparing 2,6-di(1H-pyrazol- (Table 3): 3-yl)pyridine (H LH) (representing the smallest possible 2 312 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article Scheme 2. Model Equation to Evaluate (B3LYP/LANL2DZp) the Relative Stability of [Rh(H LH)(Cl) L]+ Versus [Rh(terpy)(Cl) L]+a 2 2 2 aL: Gua, S(CH),HSCH. 3 2 3 ligand without hampering substituents) and terpy. Therefore, complex. In the case of trans-coordinated Gua a hydrogen the following model was calculated as presented in Scheme 2. bond of 1.7 Å between the CO group in Gua and the NH As can be seen in Table 4, in gas phase, regardless of group in H LH is formed (see Figure 5). This moderate 2 whetherthetransorciscomplexesareinvestigated,thegeneral preference for the terpy complexes likely originates from the slightly smaller size of H LH versus terpy, leading to weaker 2 Table 4. Calculated Relative Stabilities for the Model charge stabilization. Incorporating solvent effects in our Equation from Figure 5 a calculations supports this, resulting in all RhIII−H LH 2 complexes being favored: the cis-[Rh(H LH)(Cl) Gua]+ by [kcalmol−1] cis trans 2 2 more than 5 kcal/mol. Generally, the complexes are best ligand(L) B3LYP B3LYP(CPCM) B3LYP B3LYP(CPCM) addressed as equally stable. HS−CH 3 1.51 −0.72 2.78 −1.40 Inallinvestigatedterpycomplexes,thedistancebetweenthe CH 3 −S−CH 3 −0.98 −0.98 2.40 −2.11 central pyridine moiety nitrogen atom and the RhIII center is TU 1.37 −0.41 1.88 −0.95 ∼1.5%smallerthaninH LHsystems.TheN−Rhbondsinthe 2 Gua 1.23 −2.40 −3.94 −5.53 equatorialplanecistothecentralpyridineringisinthecaseof Imi 0.96 −0.09 3.70 −0.13 theterpy-ligand∼2%longer,whereastheRh−Ldistancedoes aB3LYP: RB3LYP/LANL2DZp + ZPE(B3LYP/LANL2DZp) not seem affected by the type of tridentate ligand (see Tables B3LYP(CPCM): RB3LYP(CPCM)/LANL2DZp // RB3LYP/ S11 and S12). Rh−L coordination is much more affected by LANL2DZp+ ZPE(B3LYP/LANL2DZp). the trans influence of pyridine or Cl − depending on the investigated isomer, indicating that average Rh−N coordina- trend favors the terpy complexes, with two exceptions, L: tion in all test complexes is similar and the H LH ligand is a 2 dimethyl sulfide in the cis complex and Gua in the trans good alternative to terpy (see Figure S1). Figure 5.Calculated (B3LYP/LANL2DZp)structureof cis-and trans-[Rh(terpy)(Cl)Gua]+andof cis-and trans-[Rh(HLH)(Cl)Gua]+. 3 2 2 313 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article Figure6.UV−Vistitrationspectraof1a,1b,and1ccomplexes(8μM)inPBSpH7.4withincreasingconcentrationofCT-DNA(0−40μM).The arrowshows hyperchromism inthe spectralband. (inset) Plots of[DNA]/(ε − ε)] vs[DNA]. a f Table 5. Interaction Constants for RhIII Complexes with CT-DNA and BSA CT-DNA BSA K,M−1 K ,M−1 K ,M−1 b sv sv [RhIII(HL*)Cl](1a) (5.0±0.1)×104 (5.0±0.1)×104 (3.5±0.1)×104 2 3 [RhIII(Me2L*)Cl3](1b) (8.3±0.1)×104 (5.5±0.1)×104 (3.9±0.1)×104 [RhIII(terpy)Cl](1c) (7.0±0.1)×104 (3.8±0.1)×104 (3.4±0.1)×103 3 [RhIII(HLtBu)Cl]20 (9.7±0.1)×104 (1.9±0.1)×104 (3.0±0.1)×104 2 3 While tridentate complexes allow only one ligand to be Absorptionspectraof1a,1b,and1ccomplexesintheabsence substituted due to the chelate effects in square-planar and presence of CT-DNA at varying concentrations are given complexes, octahedral complexes offer three possibilities: in in Figure 6. ourcase,twoequivalentcistothecentralpyridinemoietyand Results show that the RhIII complexes have a strong onetrans.Toexaminetheoverallthermodynamicequilibrium, absorption at ca. 258 nm. The absorption spectra of all three wecomparedthestabilityofcisandtransisomersofcalculated complexes showed hyperchromism and red shift, ∼2 or 3 nm, model complexes [Rh(H LH)(Cl) L]n+ and [Rh(terpy)- at the maximum peak with increasing CT-DNA concen- 2 2 (Cl) L]n+, where in all cases the cis isomer was considered trations. This observed hyperchromism and shift at the 2 to be the 0 value (Table S13). maximum peak could indicate an intercalative binding mode, As shown in Figure 5, application of the CPCM solvent possibly involving stacking interactions between the planar modelreducesthecalculatedenergygapequalizingthecisand aromatic chromophore of the complex and nucleotide base trans isomer. However, the overall trend in the gas phase and pairs in CT-DNA.54 However, the exact mode of binding especially after application of a solvent model favors the trans cannot be proposed based on UV−vis spectroscopic isomers. This is likely because, as shown in Figure 5, L has methods.55−57 Intrinsic equilibrium binding constants (K b ) more free space in front of the chelating ligand trans to the of the RhIIIcomplexes withCT-DNA were evaluated usingeq central pyridine ring compared to the cis position, where L is 1. The intrinsic binding constant K b (Table 5) obtained for above or below the chelating ring. these complexes with CT-DNA follows the order: 1b > 1c > ■ 1a, with 1b displaying the strongest interaction with DNA. However, both camphor-based ligand and terpiridine ligand DNA BINDING STUDIES complexes have nearly the same binding affinity; the intrinsic Electronic Absorption Method. Electronic absorption binding constants, K , do not differ considerably among the b spectrometry was used to investigate the interactions of metal studied complexes. Kinetic data show that 5′-GMP reacts ion complexes and CT-DNA molecules. In the present study, much more slowly with the RhIII terpy complex than with the metal complex absorption titration studies were conducted at camphor-based RhIII complexes. Considering that DNA is a room temperature using fixed concentration of complexes (8 relatively “crowded” molecule, it is possible that steric effects μM) in PBS and varying amounts of CT-DNA (0−40 μM).53 play a significant role in binding RhIII complexes, and steric 314 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article effectsarethusmorepronouncedinthecaseofcamphor-based inlengtheningandstiffeningoftheDNAdoublehelix,leading RhIII complexes. to increased DNA viscosity.59,60 Fluorescence Spectroscopic Methods. Fluorescence Addition of increasing amounts (up to r = 1.0) of RhIII spectroscopic studies were used to confirm DNA interactions complexes to a CT-DNA solution (0.01 mM) resulted in an withrhodiumcomplexes.BecauseourRhIIIcomplexesinteract increase in the relative viscosity of CT-DNA (Figure S6), with DNA by UV−vis, competitive binding experiments were which was most pronounced upon addition of 1b, in also conducted by adding ethidium bromide (EB)as a known accordance with UV−vis and fluorescence measurements. In DNA intercalator. While EB itself fluorescences weakly,54 in thecaseofclassicintercalation,DNAbasepairsmustseparate the presence of CT-DNA, EB intercalates its planar to host the bound compound, resulting in increased DNA phenanthridinium ring between adjacent base pairs of the viscosityasafunctionofinteractionstrength.Thus,ourresults DNAdoublehelix,resultinginstrongfluorescenceemissionat are in agreement with an increase in overall DNA length, ∼600nm.58Additionofcomplexes1a,1b,and1ctoEB-DNA possiblycausedbycompoundintercalationbetweenDNAbase resulted in fluorescence quenching due to displacement of EB pairs via aromatic chromophores such as pyridine or pyrazole from the DNA. Quenching parameters for RhIII complexes l■igands in the complexes. were calculated using the Stern−Volmer equation (eq 2). EB PROTEIN BINDING STUDIES displacement studies were performed by changing the concentrationofmetalcomplexesandmonitoringtheemission Fluorescence Spectroscopy of BSA. Metal ion com- intensityoftheEB-DNAcomplex.53Increasingconcentrations plexes with antitumor activity often interact with specific of complexes 1a, 1b, and 1c (0−10 μM) resulted in a proteins, both as part of their mechanism of action as well as significant decrease in fluorescence intensity with a noticeable transport and metabolism. In the present study we examined redshift(Figure7;seealsoSupportingInformationFiguresS4 the affinity of 1a, 1b, and 1c for BSA, using tryptophan and S5). fluorescence quenching experiments. Fluorescence spectrosco- py can monitor changes in protein structure, dynamics, and folding.61−63ThechangeinBSAfluorescenceuponadditionof increasing concentrations of 1a, 1b, and 1c (0−30 μM) over the range of 300−500 nm (λ , = 295 nm) is presented in ex Figures S7−S9, Suporting Information. As shown, a decrease influorescenceintensityat363nmwasobserved.Fluorescence quenching data were analyzed using the Stern−Volmer equation(eq2),andaquenchingconstant(K )wascalculated sv from I /Iversus[Q], FiguresS7−S9, SupportingInformation. 0 On the basis of these results, 1a and 1b have reasonable affinity for BSA, while 1c displayed slightly lower affinity (Table 5). Quenching constants (K ) for camphor-derived sv RhIII complex interactions with CT-DNA and BSA are approximately the same (Table 5 and Figure 8), although Figure 7. Fluorescence titration spectra of EB-DNA and of EB (10 μM)boundtoDNA(10μM)inthepresenceofvaryingamountsof complexes 1a. [Arrow shows changes in fluorescence intensity upon increasing concentration of 1a (0−10 μM)]. (inset) Stern−Volmer plots forEB-DNAfluorescencetitration with 1a. These results suggest that EB is released from the EB-DNA complex due to displacement by the RhIII complexes, indicating that complexes 1a, 1b, and 1c have moderate DNA intercalative ability, with 1b somewhat stronger than 1a and 1c. As seen in Table 5, the studied RhIII complexes interact moderately with CT-DNA. UV−Vis and fluorescence spectro- scopic studies both show that the 1b complex interacts more Figure8.ObtainedK valuesforcomplexes1a,1b,and1cwithBSA strongly than 1a or 1c. A previously studied complex, sv or CT-DNA. [RhIII(H LtBu)Cl ], was shown to have very similar affinity 2 3 toward CT-DNA as the 1a, 1b, and 1c complexes.20 All three complexes show somewhat higher affinity for CT-DNA than somewhat higher for 1b, whereas the terpiridine−RhIII BSA, in agreement with obtained result from kinetic studies, complex1cappearstointeractstrongerwithCT-DNA.Values where all complexes reacted faster with 5′-GMP than with for these constants for 1a, 1b, and 1c are in agreement with sulfur-donor molecules. those obtained for a previously investigated complex Viscosity. Viscosity measurements were conducted to [RhIII(H LtBu)Cl ], Table 5. 2 3 further confirm interactions between 1a, 1b, and 1c with UV−VisSpectroscopyofBSA.UVspectroscopywasused CT-DNA. In classical intercalation, complex formation results to investigate the mode of binding between RhIII complexes 315 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article Figure9.(left)Dose−responsecurvesfortheeffectof1bonHCT-116cellgrowthafter24and72hofexposure.Theantiproliferativeeffectwas measured by MTT assay. All values are mean ± standard error, n = 3, *P < 0.05 as compared with controls. (right) IC values for studied 50 complexes1a,1b,and 1cafter incubation withHCT-116cancer celllinesfor24and 72h. and BSA, which can be considered to be either dynamic quenching or static quenching. In dynamic quenching, upon addition of a quencher, only the excited-state fluorescence molecule is affected, resulting in no change in the UV absorptionspectraforBSA,whereasinstaticquenching,anew compound is formed between BSA and the quencher, considerably affecting the UV−vis spectra. UV spectra for BSAwererecordedfollowingadditionofequalconcentrations of 1a, 1b, or 1c. Figure S10 shows a peak at ∼280 nm due to aromatic residues (Trp, Tyr, and Phe).64 As shown, the absorption spectra of a mixture of BSA + RhIII complexes descreasesinintesity,indicatingthat themicroenvironmentof these aromatic side chains is changed, possibly due to interaction with one of the RhIII complexes and consistent with a static quenching mechanism for 1a, 1b, and 1c. CellViability.Transition-metalcompoundshavebeenused toinhibitcancerproliferation,65andwehavepreviouslyshown that RhIII complexes display cytotoxicity against HCT-116 cells.40 Thus, in the present study we investigated the Figure 10. Molecular docking suggests that compounds 1b, 1c, and antiproliferative potential of 1a, 1b, and 1c as well as [RhIII(HLtBu)Cl]havepotentialforDNAintercalation,whileallof 2 3 corresponding ligands on human colorectal cancer HCT-116 the tested compounds have affinity for the minor groove of DNA. cells using an MTT assay. Compound 1b showed the most Minorgroove bindingby 1ato B-DNA isshown(upperleft). significant effects with an IC of 80.01 and 7.26 μM after 24 ■ 50 and72htreatment,respectively;seeFigure9,FigureS11,and CONCLUSIONS Table S14. This antiproliferative effect by 1b was concentration- and We used a camphor-derived bis(pyrazolylpyridine), tridentate nitrogen-donor chelate system for the synthesis of two new time-dependent. In contrast, 1a and 1c were not cytotoxic RhIII complexes. The crystal structure of 1b complex was against HCT-116 cells under our laboratory conditions. determined. Kinetic experiments were performed with small Molecular Docking. Molecular docking simulations were biomolecules (L-methionine, L-histidine, and glutathione) used to test if DNA intercalation or minor groove binding is under pseudo-first-order conditions as a function of complex possible between the studied compounds and DNA. Using concentration and temperature by UV−vis spectroscopy. Autodock4, ligands (1a, 1b, 1c, or [RhIII(H L tBu)Cl ]) were These results show that the synthesized complexes have 2 3 docked into DNA fragments representing either (1) canonical affinityforthestudiedligands,withorderofreactivity5′-GMP B-DNA (PDB 1BNA) or (2) DNA with an intercalation gap > GSH > L-Met. The interaction between the synthesized (PDB1Z3F).1BNAisthecrystalstructureofasyntheticDNA complexes and CT-DNA and BSA were also examined by dodecamer,while1Z3Fisthecrystalstructureofa6bpDNA absorption (UV−vis) and emission spectral studies (EB displacement studies). Overall, results show that our RhIII fragment in complex with an intercalating anticancer drug, complexes have good affinity to interact with CT-DNA and ellipticine. All test compounds were predicted to interact with BSA, with somewhat higher affinity toward CT-DNA, with 1b the minor groove of the B-DNA fragment, with estimated having the highest affinity toward CT-DNA and BSA. A RhIII affinities in the micromolar range. In addition 1b, 1c, and complex with terpiridine ligand, 1c, was also synthesized, and [RhIII(H L tBu)Cl ] showed similar potential for DNA 2 3 the same type of interactions were examined. In all studied intercalation in the gap created by ellipticine, with estimated interactions the RhIII−terpy complex reacted slower than the binding affinities also in the micromolar range (Figure 10). camphor-derived bis(pyrazolylpyridine) complexes, indicating These results are in agreement with our experimental UV−vis that introduction of a camphor-derived spectator ligand can and fluorescence studies and further support the anticancer improvethereactivityofrhodium(III)complexesoveruseofa potential of these RhIII complexes. terpy spectator ligand. In vitro viability effects against human 316 DOI:10.1021/acs.inorgchem.8b02390 Inorg.Chem.2019,58,307−319 Inorganic Chemistry Article epithelial colorectal carcinoma HCT-116 show that 1b has Serendipity and Rational Design. Angew. Chem. 2008, 120, 8924− significant cytotoxic activity, while 1a and 1c showed no 8956; Angew. Chem.,Int. Ed. 2008,47,8794−8824. cytotoxic effects. Molecular docking suggests that all test (2) Beller, M.; Bolm, C. Transition Metals for Organic Synthesis: compounds have affinity for the minor groove of DNA, while BuildingBlocksandFineChemicals;Wiley-VCH:Weinheim,Germany, 2004. ■1b and 1c have potential for DNA intercalation. (3) Alessio, E. Bioinorganic Medicinal Chemistry; Wiley-VCH: Weinheim, Germany, 2011. ASSOCIATED CONTENT (4)Kastl,A.;Wilbuer,A.;Merkel,A.L.;Feng,L.;Meggers,E.;etal. * S Supporting Information Dual anticancer activity in a single compound: visible-light-induced The Supporting Information is available free of charge on the apoptosis by an antiangiogenic iridium complex. Chem. Commun. ACS Publications website at DOI: 10.1021/acs.inorg- 2012,48, 1863−1865. chem.8b02390. (5) Peña, B.; David, A.; Pavani, C.; Baptista, M. S.; Pellois, J.-P.; Turro, C.; Dunbar, K. R. Cytotoxicity Studies of Cyclometallated Illustrated superposition of calculated structures, UV− Ruthenium(II)Compounds:NewApplicationsforRutheniumDyes. vis absorption spectra, plot of absorbance versus time, Organometallics 2014, 33,1100−1103. fluorescencetitrationspectra,plotofrelativeviscosityof (6) Noor, F.; Wüstholz, A.; Kinscherf, R.; Metzler-Nolte, N. A CT-DNA in PBS with varying amounts of complexes, Cobaltocenium−Peptide Bioconjugate Shows Enhanced Cellular emission spectra of BSA in presence of increasing Uptake and Directed Nuclear Delivery. Angew. Chem., Int. Ed. 2005, amounts of 1a, 1b, and 1c, dose-response curves, 44, 2429−2432. tabulated observed pseudo-first-order rate constants, (7) Pongratz, M.; Schluga, P.; Jakupec, M. A.; Arion, V. B.; water solubility of RhIII complexes at 298 K, tabulated Hartinger,C.G.;Allmaier,G.;Keppler,B.K.Transferrinbindingand selected calculated distances, tabulated calculated transferrin-mediated cellular uptake of the ruthenium coordination relative stabilities, tabulated IC values (PDF) compound KP1019, studied by means of AAS, ESI-MS and CD 50 spectroscopy. J. Anal.At.Spectrom. 2004,19,46−51. Accession Codes (8)Ernst,R.J.;Komor,A.C.;Barton,J.K.SelectiveCytotoxicityof CCDC 1847938 contains the supplementary crystallographic Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells. data for this paper. These data can be obtained free of charge Biochemistry 2011, 50,10919−10928. via www.ccdc.cam.ac.uk/data_request/cif, or by emailing (9) Harris, A. L.; Yang, X.; Hegmans, A.; Povirk, L.; Ryan, J. J.; Kelland, L.; Farrell, N. P. Synthesis, Characterization, and data_request@ccdc.cam.ac.uk, or by contacting The Cam- Cytotoxicity of a Novel Highly Charged Trinuclear Platinum bridge Crystallographic Data Centre, 12 Union Road, Compound. Enhancement of Cellular Uptake with Charge. Inorg. ■Cambridge CB2 1EZ, UK; fax: +44 1223 336033. Chem. 2005,44, 9598−9600. (10) Lauffer, R. B. Paramagnetic metal complexes as water proton AUTHOR INFORMATION relaxation agents for NMR imaging: theory and design. Chem. Rev. Corresponding Author 1987,87, 901−927. *Phone: +381(0)34336223. Fax: +381(0)34335040. E-mail: (11) Della Rocca, J.; Liu, D.; Lin, W. Nanoscale Metal−Organic jrosic@kg.ac.rs. Frameworks for Biomedical Imaging and Drug Delivery. Acc. Chem. Res. 2011, 44,957−968. ORCID ́ (12) Anderson, C. J.; Welch, M. J. Radiometal-Labeled Agents Milan M. Milutinovic: 0000-0003-4838-3998 (Non-Technetium) for Diagnostic Imaging. Chem. Rev. 1999, 99, Andreas Scheurer: 0000-0002-2858-9406 2219−2234. Jovana Bogojeski: 0000-0002-3433-7774 (13)Domaille,D.W.;Que,E.L.;Chang,C.J.Syntheticfluorescent sensorsforstudyingthecellbiologyofmetals.Nat.Chem.Biol.2008, Author Contributions 4,168−175. The manuscript was written with contributions from all (14)Minus,M.B.;Kang,M.K.;Knudsen,S.E.;Liu,W.;Krüger,M. authors. J.; Smith, M. L.; Redell, M. S.; Ball, Z. T. Assessing the intracellular Notes fate of rhodium(II) complexes. Chem. Commun. 2016, 52, 11685− T■he authors declare no competing financial interest. 11688. (15) Sliwinska, U.; Pruchnik, F. P.; Pelinska, I.; Ułaszewski, S.; ACKNOWLEDGMENTS Wilczok, A.; Zajdel, A. Synthesis, structure and antitumor activity of Theauthorsgratefullyacknowledgefinancialsupportfromthe [ 2 R 00 h 8 C , l3 1 ( 0 N 2, − 1 N 9 ) 4 ( 7 D − M 19 S 5 O 1. )] polypyridyl complexes. J. Inorg. Biochem. Ministry of Education, Science and Technological Develop- (16) Cwikowska, M.; Pruchnik, F. P.; Starosta, R.; Chojnacki, H.; ment Serbia, Project Nos. 172011, 172057, and III41010. We Wilczok, A.; Ułaszewski, S. Dinuclear Rh(II) complexes with one alsothankProf.T.ClarkforhostingthisworkattheComputer polypyridylligand,structure,propertiesandantitumoractivity.Inorg. Chemistry Center and the Regionales Rechenzentrum Chim. Acta2010,363,2401−2408. ■Erlangen for a generous allotment of computer time. (17)Frade,R.F.M.;Candeias,N.R.;Duarte,C.M.M.;Andre,V.; Duarte, M. T.; Gois, P. M. P.; Afonso, C. A. M. New dirhodium DEDICATION complex with activity towards colorectal cancer. Bioorg. Med. Chem. Lett. 2010, 20,3413−3415. This article is dedicated to the memory of Professor Dr. Z ̌ ivadin D. Bugarc ̌ ic ́ (1954−2017), a great mentor and (18)Morrison,D.E.;Aitken,J.B.;deJonge,M.D.;Issa,F.;Harris, H.H.;Rendina,L.M.SynthesisandBiologicalEvaluationofaClass ■excellent chemist. ofMitochondrially-TargetedGadolinium(III)Agents.Chem. -Eur. J. 2014,20, 16602−16612. 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