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Synthesis, Structural Characterization and Biological Evaluation of Rhenium(I) Tricarbonyl Complexes with β‐Carboline Ligands.

DOI: 10.1002/slct.201701961 Full Papers 1 z 2 Inorganic Chemistry 3 4 Synthesis, Structural Characterization and Biological 5 Evaluation of Rhenium(I) Tricarbonyl Complexes with 6 7 b-Carboline Ligands. 8 9 Iva´n Maisuls,[a,b] Ezequiel Wolcan,[a] Oscar E. Piro,[c] Eduardo E. Castellano,[d] 10 Gabriela Petroselli,[e] Rosa Erra-Balsells,[e] Franco M. Cabrerizo,*[b] and Gustavo T. Ruiz*[a] 11 12 13 14 Two b-carboline rhenium(I) complexes, fac-[Re(CO) with TD-DFT calculations using several hybrid functionals. 3 15 (dppz)(nHo)]OSCF and fac-[Re(CO)(nHo)Cl] where dppz= Cellular uptake and cytotoxic effect of both complexes against 3 3 3 2 16 dipyrido[3,2-a:2’,3’-c] phenazine and nHo=9H-pyrido[3,4-b] human lung carcinoma (A549) cells were evaluated. For 17 indole (norharmane), have been synthesized. These complexes comparative purpose, two related fac-[Re(CO)(L)(nHo)]+ com- 3 18 were fully characterized by structural X-ray diffraction, 1H and plexes (L=bpy and phen) were included in the biological 19 13CNMR, UV-vis absorption and FT-IR spectroscopy and mass studies. The anti-proliferative effect of the compounds was 20 spectrometry. Ground state geometry optimization was carried rationalizedintermsofthedifferentextensionoftheligandsp- 21 out in vacuo with the PBE0 hybrid functional using the system. 22 LanL2DZ basis set. The characterization was complemented 23 24 Introduction in intracellular domains by conventional and/or time-resolved 25 Rhenium(I) complexes comprise a group of compounds emission microscopy. This is appropriate to elucidate their 26 showing quite interesting physicochemical features, i.e., rich cellulardistributionand,potentially,theirmechanismofaction. 27 excited-state behavior,[1] thermal and photochemical stability,[2] Rhenium(I) tri-carbonyl complexes, fac-[Re(CO)(L)(L)] 28 3 1 2 large Stokes shift and long luminescence lifetimes.[3] These (where L and L are different mono or bidentate ligands), 29 1 2 properties make them appealing candidates for diverse representasubgroupofRe(I)complexeshavinga-Re(CO) core 30 3 applications in broad research areas such as photo-induced initsarchitecture.Thiscoremakesthemquiterobustfromthe 31 electron transfer,[4] solar energy conversion,[5] catalysis,[6] oncol- chemical point of view. Moreover, fac-[Re(CO)(L)(L)] com- 32 3 1 2 ogy[7]andnuclearmedicine,[8]amongothers. plexesrepresentversatilestartingmaterialsthatcanbechemi- 33 In particular, due to their luminescent properties, Re(I) callymodifiedatease(thatisin1–3stepsynthesis)introducing 34 complexes are used as biological labelling reagents[9] and also specific ligands to fine-tune their physical or chemical proper- 35 as non-covalent luminescent molecular probes for bio-mole- ties.[2,11] Therefore, rhenium organometallic compounds based 36 cules and cell imaging.[10] As such, they can be easily followed onthe-Re(CO) coredeservetobefurtherinvestigated. 37 3 Thecombinationoftransitionmetalswithbioactiveligands 38 offers the possibility of designing novel compounds that may 39 [a] I.Maisuls,Prof.E.Wolcan,Prof.G.T.Ruiz INIFTA optimizeorincreasetheintrinsicchemical,photochemicaland/ 40 UNLP(CCTLaPlata-CONICET),Diag.113y64,C.C.16,Suc.4,(B1906ZAA) or pharmacological properties of the free ligands. In this 41 LaPlata,Argentina context, we have undertaken the search of novel complexes 42 E-mail:gruiz@inifta.unlp.edu.ar based on the combination of both -Re(CO) core and a group 43 [b] I.Maisuls,Prof.F.M.Cabrerizo 3 IIB-INTECH–UNSAM-CONICET of alkaloids called b-carbolines (bCs) as ligands. From the 44 I.MarinoKm8,2.CC164,(7130)Chascomffls,BuenosAires,Argentina chemicalpoint ofview,bCsderivefrom9H-pyrido[3,4-b]indole 45 E-mail:fcabrerizo@intech.gov.ar or norharmane (nHo, Scheme1(a)). The interest in these 46 [c] Dr.O.E.Piro alkaloids arises from their biological and biochemical role. In 47 InstitutoIFLP(CCTLaPlata-CONICET)yDepto.deF(cid:3)sica FCE-UNLP,C.C.67 fact, bCs were found to be active compounds in several 48 (1900)LaPlata,Argentina biochemical and pharmacological[12] processes. Morever, bCs 49 [d] Dr.E.E.Castellano have also shown quite unique and interesting photochemi- 50 InstitutodeF(cid:3)sicadeS¼oCarlos cal,[13] photophysical and/or photosensitizing properties.[14] In 51 USP,C.P.369 13560S¼oCarlos,Brazil consequence,theuseofthesealkaloidsindifferentapplications 52 [e] Dr.G.Petroselli,Prof.R.Erra-Balsells in the biomedical fields, such as photodynamic therapy, has 53 CIHIDECAR-CONICET,DepartamentodeQu(cid:3)micaOrg(cid:4)nica,FCEyN been described.[15] Recently, we have synthesized and fully 54 UBA characterizedtwonovel-Re(CO) complexeswithpolypyridines 55 Pabell(cid:5)nII,3erP.,CiudadUniversitaria,(1428)BuenosAires,Argentina 3 and nHo as ligands.[16] Briefly, nHo binds to the metal center 56 Supporting information for this article is available on the WWW under https://doi.org/10.1002/slct.201701961 through the pyridine nitrogen atom. Such a kind of coordina- 57 ChemistrySelect2017,2,8666–8672 8666 (cid:6)2017Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim WileyVCH Freitag,22.09.2017 1 1727/99871 [S.8666/8672] Full Papers HRESI-MSandUV-MALDI-MS 1 2 Mass spectrometry (MS) analysis was performed to determine 3 the structure of rhenium complexes. FigureS1a shows the 4 HRESI mass spectrum in positive ion mode of Re(CO)(nHo)Cl 5 3 2 complex(MW=642.08).Theintactmolecularionwasobserved 6 as sodiated adduct [M+Na]+. Contributions from 187Re and 7 185Re (62.6 and 37.4% natural abundance, respectively) can be 8 observed at m/z values of 665.035 and 663.034. Structure 9 diagnosissignalswereobtainedbyMALDI-MSwhenDCTBand 10 nHo were used as matrices, FiguresS1b and S1c. In negative 11 ionmode,theintactmolecularion[M](cid:2)asanionradicalspecies 12 was detected at m/z=642.53 when LDI-MS was used and at 13 m/z=642.73 when MALDI-MS was used (matrix, nHo), Fig- 14 ureS2. 15 The HRESI mass spectrum in positive ion mode of the 16 cationic organometallic complex [Re(CO)(dppz)(nHo)]+ (MW= 17 3 720.74) is shown in FigureS3a. The intact molecular ion was 18 observed as cation [M]+ at m/z values of 721.098 and 719.095 19 (contributions from 187Re and 185Re, respectively). Additionally, 20 theintactmolecularionforthecomplexwasdetectedascation 21 [M]+ byMALDI-MSwhenDCTBandnHowereusedasmatrices 22 and alsowhen nomatrixwas added (LDI-MS) (FiguresS3band 23 S3c) 24 Scheme1.Schematicstructureof(a)norharmane,(b)dppz,(c)fac-[Re(CO) 3 25 (dppz)(nHo)]+,(d)fac-[Re(CO)(nHo)Cl],(e)fac-[Re(CO)(bpy)(nHo)]+and(f) 3 2 3 26 fac-[Re(CO) 3 (phen)(nHo)]+.CarbonshavebeennumberedfortheNMR StructuralcharacterizationbyX-raydiffraction analysis. 27 Figure1 is an ORTEP[18] drawing of the Re(CO)(nHo)Cl 28 3 2 complex. Crystal data, data collection procedure, structure 29 30 tion arrangement has been referred as a good strategy for the 31 developmentofnovelsystemicdrugsbasedonbCspotentially 32 havingareducedneurotoxiceffects.[17] 33 Certainly,thesearchofnovel-Re(CO) complexescoordinat- 34 3 ing bCs represents an excellent alternative that deserve to be 35 furtherexplored.Inthiscontext,wereporthereinthesynthesis 36 and full characterization of two novel bC(cid:2)Re(I) complexes, 37 namely fac-[Re(CO)(dppz)(nHo)]+, where dppz=dipyrido[3,2- 38 3 a:2’,3’-c] phenazine, a known DNA intercalating molecule 39 (Scheme1(b)),andthefirstneutralRe(I)complexwithonlynHo 40 as ligands, fac-[Re(CO)(nHo)Cl] (Scheme1(c) and (d), respec- 41 3 2 tively).Moreover,asafirststepinthebiologicalevaluation,the 42 cytotoxicity activity of [Re(CO)(bpy)(nHo)]+ (Scheme1(e)), 43 3 [Re(CO)(phen)(nHo)]+ (Scheme1(f)), [Re(CO)(dppz)(nHo)]+ 44 3 3 andRe(CO)(nHo)Clcomplexesagainsthumancarcinomalung 45 3 2 Figure1.Viewofdimericfac-[Re(CO)(nHo)Cl]complexshowingthedis- cells(A549)aswellastheircellularuptakewasinvestigated. 3 2 46 placementellipsoidsofnon-Hatomsatthe30%probabilitylevel.Metal- 47 ligandbondsareindicatedbyfulllinesandintermolecularN(cid:2)H***Clbonds 48 Results and Discussion b ha y v d e a b sh e e e d n l l i a n b e e s l . le F d o . r S c e la le ri c t t y e , d in b t o h n e d fu le se n d gt h h e s t [ e A˚ ro ] c a y n c d lic an ri g n l g e s s o [8 n ] l a y ro th u e nd N- R a e to (I m ) s 49 ions:Re1;C(1A)-Re(1):1.980(15),C(1B)-Re(1):1.923(12),C(1C)-Re(1):1.873 Two Re(I) complexes with nHo as ligands, fac-[Re(CO) 50 3 (12),N(11A)-Re(1):2.281(14),N(12A)-Re(1):2.247(9),Re(1)-Cl(1):2.483(3).Re2; (dppz)(nHo)]+ and fac-[Re(CO)(nHo)Cl], were obtained and 51 3 2 C(2A)-Re(2):1.900(13),C(2B)-Re(2):1.902(14),C(2C)-Re(2):1.882(19),N(21A)- fully characterized by elemental analysis, FT-IR, NMR and by a Re(2):2.208(10),N(22A)-Re(2):2.164(12),Re(2)-Cl(2):2.495(3). 52 combination of mass spectrometry techniques. Also, the solid 53 state structure of the Re(CO)(nHo)Cl complex was solved by 54 3 2 X-raydiffractionmethods. 55 56 57 ChemistrySelect2017,2,8666–8672 8667 (cid:6)2017Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim WileyVCH Freitag,22.09.2017 1 1727/99871 [S.8667/8672] Full Papers determinationmethodsandrefinementresultsaresummarized best least-squares plane less than 0.024A˚]. The molecular 1 inTable1.Bonddistancesandanglesaroundthemetalionsare planes subtend dihedral angles of 59.8(2)8 (Re1) and 61.7(2)8 2 inTablesS1-S2. (Re2) with each other and the corresponding Re(I) ions lay 3 nearlyontotheirintersections. 4 As shown in Figure1, the pair of complexes is arranged in 5 Table1. CrystaldataandstructurerefinementresultsforRe(CO) 3 (nHo) 2 Cl. the lattice as dimeric units linked through NH***Cl bonds 6 7 Empiricalformula C 25 H 16 ClN 4 O 3 Re [N***Cl distances of 3.29 and 3.26A˚ and (N(cid:2)H***Cl) angles of Formulaweight 642.07 1358]. 8 Temperature 293(2)K 9 Wavelength 0.71073A˚ 10 Crystalsystem Monoclinic Spectroscopiccharacterization Spacegroup Pc 11 Unitcelldimensions a=11.2840(3)A˚ FT-IR absorption spectra of the complexes showed intense 12 b=10.4360(2)A˚ bandsinthe2100–1800cm(cid:2)1region,whichareconsistentwith 13 c=29.8590(8)A˚ both the facial configuration of the carbonyl ligands and with b=91.828(1)8 14 Volume 3514.4(2)A˚3 their Cs symmetry.[19] Absorption bands were attributed to the 15 Z 4 A’1, A’’ and A’2 stretching modes of CO ligands in the 16 Absorptioncoefficient 3.557mm(cid:2)1 complexes according to our previous report on similar 17 F(000) 1240 Crystalsize 0.211x0.068x0.060mm3 complexes with nHo.[16] Absorption band at 3200–3300cm(cid:2)1 18 #-rangefordatacollection 1.36to25.258. region was assigned to N(cid:2)H peak of nHo ligands which has 19 Indexranges -13(cid:3)h(cid:3)13,0(cid:3)k(cid:3)12,(cid:2)35(cid:3)l(cid:3)35 alsobeenreportedforothermetal-nHocomplexes.[16,20] 20 Reflectionscollected 21612 1H- and 13C-NMR spectra showed the expected signals for 21 Independentreflections 11152[R(int)=0.0827] both nHo and dppz ligands. However, all signals were slightly Observedreflections[I>2s(I)] 9691 22 Completenessto#=25.258 98.0% shifted, with respect to the free ligands, due to their 23 Refinementmethod Full-matrixleast-squaresonF2 coordination with Re atom. It is noteworthy that 13C-NMR 24 Data/restraints/parameters 11152/2/613 spectrumofbothcomplexesshowedasignalathighfields,i.e., 25 Goodness-of-fitonF2 1.058 26 FinalRindicesa[I>2s(I)] R1=0.0441,wR2=0.1230 d ~ 197.5ppm (Re(CO) 3 (nHo) 2 Cl) and d ~ 196.4 ([Re(CO) 3 Rindices(alldata) R1=0.0484,wR2=0.1246 (dppz)(nHo)]+)whichwereassignedtothecarbonylligands. 27 Absolutestructureparameter -0.02(1) The UV-vis absorption spectrum of Re(CO)(nHo)Cl in 28 Largestdiff.peakandhole 0.759and(cid:2)1.321e.A˚(cid:2)3 3 2 MeOH solution consists of three different absorption bands, 29 aR =SjjF j-jFjj/SjF j,wR =[Sw(jF j2-jFj2)2/Sw(jF j2)2]1/2 bluelineinFigure2.Thefirstband,showingthehighestmolar 1 o c o 2 o c o 30 31 32 There are two closely related but independent Re(I) 33 complexes per asymmetric unit. Assuming that the Re(cid:2)Cl 34 bonds define the axis, the molecules can be seen as an 35 incompletefour-bladedpropeller-likestructurelackingthetwo 36 trans blades which are replaced by CO groups. It can be 37 envisagedthatforaright-screwrotationalongtheRe(cid:2)Clbond, 38 Re1 complex has one of its NH group on the front and the 39 other one on the rear edges of the blades, while in Re2 40 complex both NH groups are on the front blades edges. 41 Rhenium(I) ions are in slightly distorted octahedral environ- 42 ments, fac-coordinated to three carbonyl groups [Re(cid:2)C bond 43 distancesintherange from1.87(1)to1.98(2)A˚ (Re1)andfrom 44 1.88(2) to 1.90(1) A˚ (Re2); C(cid:2)Re-C bond angles in the 89.3(5)- 45 92.1(5)8 (Re1) and 89.8(5)-92.2(6)8 (Re2) intervals; C(cid:2)O lengths 46 from 1.08(2) to 1.20(1) A˚ (Re1) and from 1.14(1) to 1.16(2) A˚ 47 (Re2); Re-C(cid:2)O angles are in the 177(1)-179(1)8 and 175(1)-178 48 (1)8rangesforRe1andRe2,respectively]. 49 Twoothercis-positionsareoccupiedbytwonHomolecules 50 acting as mono-dentate ligand through their pyridine N-atoms 51 along their electron lone-pair lobes [Re(cid:2)N bond distances of 52 2.28(1) and 2.247(9) A˚ (Re1) and 2.21(1) and 2.16(1) A˚ (Re2)]. 53 Thesix-foldcoordinationiscompletedbyachlorineion[Re(cid:2)Cl Figure2.AbsorptionspectraofmethanolicsolutionofRe(CO) 3 (nHo) 2 Cl(blue 54 distancesof2.483(3)A˚ forRe1and2.495(3)A˚ forRe2]. solidline),[Re(CO) 3 (dppz)(nHo)]O 3 SCF 3 (redsolidline),nHo(lightbluedotted 55 line),nHoH+(bluedottedline)anddppz(reddottedline). As expected from extended p-bonding delocalization, the 56 nHoligandsareplanar[rmsdeviationofnon-Hatomsfromthe 57 ChemistrySelect2017,2,8666–8672 8668 (cid:6)2017Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim WileyVCH Freitag,22.09.2017 1 1727/99871 [S.8668/8672] Full Papers absorptioncoefficient,e(e~5.7x104M(cid:2)1cm(cid:2)1),iscenteredat Polarizable Continuum Model the differences in SCF energies 1 l =241nm. A less intense band (e ~ 2.8 x 104 M(cid:2)1 cm(cid:2)1) betweenRe1andRe2werelowerthan0.6Kcal/mol. 2 max appears at l =308nm. There are two successive absorption 3 max bands appearing as shoulders of the latter band at l ~ 348 4 max TD-DFTcalculations and365nm(e~1.2x104and8.0x103M(cid:2)1cm(cid:2)1,respectively). 5 When comparing the absorption spectra of the complex with TD-DFT calculations were performed on the optimized struc- 6 those recorded for free neutral (nHo) and protonated (nHoH+) turesofRe1andRe2conformers.Atthebeginning,therelative 7 norharmane (light blue and blue dotted lines, respectively in performance of several hybrid functionals (see experimental 8 Figure2),[16,21] it is evident that, despite other electronic section) was assayed by their ability to reproduce the 9 transitions,bothnHo-likeandnHoH+-likebC’sringscontribute, experimental absorption spectrum in MeOH of both Re1 and 10 atleastinpart,totheoverallabsorptionofRe(CO)(nHo)Cl. Re2. The results of the simulations are shown in FiguresS4a-g 11 3 2 This is in agreement with TD-DFT calculations discussed incomparisonwiththeexperimentalabsorptions.Itisobserved 12 below. In addition, absorbance observed at l > 300nm also that the M06 functional gives the best agreement with the 13 suggeststhepresenceofnewchargetransfertransitionsbands experimentalabsorptionspectrumofRe(CO)(nHo)ClinMeOH, 14 3 2 due to the coordination of nHo with the metal center (vide Figure3. 15 infra). 16 The UV-vis absorption spectrum of [Re(CO)(dppz)(nHo)] 17 3 OSCF in MeOH solutions, red line in Figure2, consist in one 18 3 3 intensebandcenteredinl =280nm(e~6.4x104M(cid:2)1cm(cid:2)1) 19 max andseveraloverlappingbandsin325–425nmregion(e~1.5x 20 104 M(cid:2)1 cm(cid:2)1), i.e. corresponding to typical vibronic compo- 21 nentsofdppz(reddottedlineinFigure2)andnHoligands. 22 23 24 TheoreticalcalculationsonRe(CO) (nHo) Cl 3 2 25 DFTand TD-DFTcalculations have shed light on the nature of 26 the absorption bands of fac-[Re(CO)(L)(L)] complexes. They 27 3 1 2 are comprised of a set of IL, MLCT and LMCT electronic 28 transitions.[11c,22] In particular, these methods have been em- 29 ployed recently to unravel the character of the electronic 30 transitions in [Re(CO)(bpy)(nHo)]+.[16] Since the replacement of 31 3 bpy by nHo could deeply modify the electronic structure not 32 only of complex but also of each nHo ligand, TD-DFT 33 calculations on Re(CO)(nHo)Cl complex were performed in 34 3 2 order to get a deeper understanding of its experimental 35 absorptionspectrum.Further,sincetheX-raystructurerevealed Figure3.ComparisonoftheobservedUV-visabsorptionspectrumofRe(CO) 3 36 (nHo)ClinMeOH(blackcurve)withTD-DFTcalculatedelectronictransitions that there are two un-equivalent complexes per asymmetric 2 37 (verticallines)andsimulatedspectraforRe1(dashedbluecurve)andRe2 unit (vide supra) ground state geometry optimization was (dashedredcurve)undertheM06/6-311G/6-311G*/LanL2TZ(f)leveloftheory. 38 previously performed on two conformers of Re(CO)(nHo)Cl 39 3 2 (namedasRe1andRe2inFigure1). 40 41 Electronic transitions resultscalculated at thesamelevel of 42 Groundstategeometryoptimization theory for Re1 and Re2 in MeOH are summarized in TablesS3 43 Additional structural information was obtained by DFT calcu- andS4. 44 lations on two Re1 and Re2 conformers. X-ray crystallographic The most relevant MOs which are responsible for the 45 data was usedas referencein order to control the precision of electronic transitions in the absorption spectrum of Re(CO) 46 3 theoretical values obtained. TablesS1-S2 show the calculations (nHo)Cl complex in the 230–400nm wavelength range are: 47 2 results of selected bond distances and angles for Re1 and Re2. HOMO, LUMO, and a set of MOs H-10 through H-1 and L+1 48 Bond lengths and angles differed only by 0.1-0.2A˚ and 38 or throughL+6. 49 less showing a good agreement between data obtained by By using the AOMIX program, five contributions to those 50 crystal structures and those obtained by computational MOs were considered from a Mulliken population analysis: (i) 51 chemistry. Self consistent field (SFC) energies in vacuo of both Re atom, (ii) the three carbonyls, (iii) nHo1 molecule, (iv) nHo2 52 Re1andRe2conformersdifferedbyonly0.16Kcal/mol.Though moleculeand(v)Clatom.Thecalculated%compositionsofall 53 a systematic study varying the angles between the two nHo fragments at each MO for Re1 and Re2 in MeOH are shown in 54 ligands in both Re(CO)(nHo)Cl conformers was not carried TablesS5 and S6. In the 300–400nm region, the most relevant 55 3 2 out,sincekT~0.6Kcal/molat298K,afreerotationofthenHo electronictransitionsareH!L,H-1!L+1,H!L+1,H-3!L,H- 56 coordinated ligands in solution is highly probable. Under the 4!L, H-3!L+1 and H-4!L+1. Figures4 and S5 show the 57 ChemistrySelect2017,2,8666–8672 8669 (cid:6)2017Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim WileyVCH Freitag,22.09.2017 1 1727/99871 [S.8669/8672] Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure4.SpatialplotsofmostrepresentativeMOsinRe1conformerofthe 17 Re(CO)(nHo)ClcomplexinMeOH(isovalue=0.02). 3 2 Figure5.CellviabilityofA549cellswithincreasingconcentrationsofthe 18 fourinvestigatedcomplexesandnHoascontrol. 19 20 spatial plots of H-1, H, L and L+1 MOs for Re1 and Re2 in 21 MeOH,whichgiveinsighttotheelectronictransitions. Table2. IC 50 oftheReI-nHocomplexesandnHoinA549cells. 22 For Re1, Figure4, it is observed that in MeOH H is a MO Complex IC value(mM) 23 50 mostly centered on the Re atom (~ 43%) with smaller 24 Re(CO)(nHo)Cl 85(cid:4)1 contributions from the three CO and nHo2 (~ 20–23%) and 3 2 25 [Re(CO) 3 (bpy)(nHo)]+ 88(cid:4)1 from nHo1 and Cl (~ 6–8%). H-1 is a MO mostly centered on [Re(CO)(phen)(nHo)]+ 65(cid:4)1 26 3 nHo1 (~ 58%) with smaller contribution from the Re atom (~ [Re(CO)(dppz)(nHo)]+ 10(cid:4)1 3 27 21%), the three CO and Cl (each ~ 9%) and nHo2 (~ 3%). H-3 nHo >250 28 (not shown) is a MO which is widespread between the Re 29 atom,nHo1andnHo2withlessercontributionsfromthethree 30 COandCl.H-4(notshown)isaMOmostlycenteredontheRe ligand. IC values show that the cytotoxic activity of the 31 50 atom(~49%)withsmallercontributionsfromthethreeCOand investigated ReI-nHo complexes increases with the size of the 32 nHo1 (~ 22 and 17%, respectively) and from Cl (~ 9%). On the ancillary or extension of the p-system of the ligands: IC dppz < 33 50 other hand, L and L+1 are MOs centered on nHo2 and nHo1, IC phen < IC bpy < IC nHo. This accounts for the impact the 34 50 50 50 respectively.Therefore,alltheelectronictransitionsinMeOHin chemicalstructureoftheligandshasontheoverallcytotoxicity 35 the300–400nmregionareMLLCT andMLLCT of the investigated rhenium complexes, as it was shown for 36 (Re(CO)3!nHo1) (Re(CO)3! . ruthenium[23]andplatinum[24]complexes. 37 nHo2) Thehighenergypartofthespectrum,i.e.,below300nm,is Thehighestcytotoxicity of[Re(CO)(dppz)(nHo)]+ mightbe 38 3 more complex and the main electronic transitions does not associated to the intrinsic intercalative property of the dppz 39 seem to be “pure” excitations but a mixture of nearly all of ligand with DNA (nuclear and/or mitochondrial)[25] and/or also 40 them. However, they appear to be a composition of IL , with the differential cell penetration, which can enhance the 41 (nHo) LLCT and MLLCT transitions. This is a distinct toxicity. In fact, this complex shows a quite similar cytotoxic 42 (Cl!nHo) (Re(CO)3!nHo) feature when compared to the [Re(CO)(bpy)(nHo)]+ complex, effect (IC =10mM) to the one described for cisplatin (IC = 43 3 50 50 where neat p!p* electronic transitions centered in bpy and 8.0mM) against the same cell line.[20] Therefore, is important to 44 nHo fragments, respectively, were obtained by TD-DFT calcu- emphasize that [Re(CO)(dppz)(nHo)]+ might represent an 45 3 lations.[16] attractivealternativeasapotentialantitumordrug. 46 ReI-nHo complexes with bpy, phen and nHo as ligands, 47 show moderated effect on A549 cells when comparing their 48 CytotoxicityandcellularuptakeofReI-nHocomplexes cytotoxicity with related metal complexes based on nHo as 49 Cytotoxicity of four ReI-nHo complexes (Scheme1) against ligands, i.e., AgI-nHo and polypyridyl RuII-nHo complexes.[20,23a] 50 A549 cells was evaluated, Figure5. Data provided herein show Therefore,itwouldbeinterestingtostudythepotentialuseof 51 that the coordination of the -Re(CO) core to nHo ligand thesecomplexesasbiologicallabelsofintracellulardomains. 52 3 increases the toxicity of the bC moiety to A549 cells. All the Re(I)-complexescanbetaken upbydifferenttypesofcells. 53 investigated complexes are more toxic than free nHo (Table2). The efficiency of this process is closely related to their type of 54 Moreover, the intrinsic cytotoxicity of the complexes ligandsandnetcharge.[10c]Thus,tofurtherevaluatetheuptake 55 depends on the chemical nature of the accompanying ligand of this family of ReI-nHo complexes, A549 cells were co- 56 rather than on the net charge of the molecules or the nHo incubated in independent experiments and then visualized by 57 ChemistrySelect2017,2,8666–8672 8670 (cid:6)2017Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim WileyVCH Freitag,22.09.2017 1 1727/99871 [S.8670/8672] Full Papers fluorescence microscopy by making use of the intrinsic possible to estimate this parameter due to the high hydro- 1 luminescentproperties(FigureS6)ofthesecomplexes.Figure6 phobicityofthecomplexes.Thisresultleadsustoassumethat 2 theymightlocalizeintoanyhydrophobicintracellularcompart- 3 ments such as mitochondria, lysosomes or the Golgi body, as 4 seen in other related compounds.[26] In particular, the neutral 5 Re(CO)(nHo)Cl complex represents an excellent alternative to 6 3 2 Re(I) complexes reported in the literature for cellular imaging 7 orbiologicalprobes,sincemostofthemarepositively(cationic) 8 or negatively (anionic) charged.[10c,d] Nevertheless, further 9 studiesareneededtobringlightonthis. 10 11 12 Conclusions 13 TwoRe(I)complexeswithnHohavebeen obtained. Solidstate 14 Structural X-ray diffraction analysis of Re(CO)(nHo)Cl complex 15 3 2 revealed that there are two closely related but independent 16 Re(I) complexes per asymmetric unit in the crystal lattice. The 17 molecules of each complex differed in the orientation of both 18 NH groups of the nHo ligands. The pair of complexes is 19 arranged inthelatticeasdimericunitslinked throughNH***Cl 20 bonds.GroundstategeometryoptimizationbyDFTcalculations 21 Figure6.Phasecontrast(left),fluorescence(center)andmergedimages showed thatin solutionthe nHo ligands may rotate freely.TD- 22 (right)of(a)nHo(control)(b)Re(CO) 3 (nHo) 2 Cland(c)[Re(CO) 3 (dppz)(nHo)]+ DFTcalculationsestablishedthatthemostimportantelectronic ofsuspendedA549cells. 23 transitions present in the spectrum of the Re(CO)(nHo)Cl 24 3 2 complexareMLLCT alongwithamixtureofIL 25 (Re(CO)3!nHo1,nHo2) (nHo) shows, as a representative example, images of A549 cells andLLCT transitions. 26 (Cl!nHo) stained with Re(CO)(nHo)Cl and [Re(CO)(dppz)(nHo)]+. Cells Data reported herein show that this group of complexes 27 3 2 3 are clearly stained by these types of rhenium(I) tri-carbonyl allowsforagreatdealofstructureandchemicalvariability,and 28 norharmane complexes. Visual inspection of the A549 cells canbefine-tunedtomeettherequirementsofawiderangeof 29 indicatedhealthycellmorphologyinalltestedsamples. biological applications (fluorescence markers and/or antitumor 30 As semi-quantitative test of cell viability, cells were co- drugs) that deserve to be further investigated. Moreover, their 31 incubated with the rhenium complexes and propidium iodide, relatively high hydrophobicity makes these complexes suitable 32 which is only taken up by dead or dying cells. Cell counting formicelle-baseddrugdelivery.[27] 33 showed no evidence of damage on treated cells with respect 34 to untreated cells as control, FigureS7. This evidences that 35 SupplementaryInformationAvailable there is no cellular death during the time (1h) of the staining 36 protocol(seeExperimentalSection). Additional computational details. General experimental proce- 37 Cellswerealsoco-incubatedwithSYBRgreeninanattempt dures. Mass spectra (ESI, LDI and MALDI) of the complexes 38 to elucidate the intracellular localization of Re(CO)(nHo)Cl, (Figs. S1, S2 and S3). Comparison of the experimental UV-vis 39 3 2 Figure7. The absence of co-localization between both dyes absorption spectrum with TD-DFT calculated electronic tran- 40 suggeststhatthecomplexdonotaccumulateintothenucleus. sitions under different levels of theory (Figs. S4a-g). Spatial 41 plotsofMOsinRe2conformer(Fig.S5).Emissionspectraofthe 42 rhenium complexes (Fig. S6). Cells stained with Propidium 43 Iodide (Fig. S7). FTIR spectra of Re(CO)(nHo)Cl and [Re(CO) 44 3 2 3 (dppz)(nHo)]OSCF (Figs. S8a-b). 1HNMR and 13CNMR spectra 45 3 3 of Re(CO)(nHo)Cl and [Re(CO)(dppz)(nHo)]OSCF (Figs. S9 46 3 2 3 3 3 and S10). Bond lengths and angles (TablesS1 and S2). TD-DFT 47 calculations in MeOH (TablesS3 and S4). HOMOs and LUMOs 48 compositions (TablesS5 and S6). Full bond lengths and angles 49 Figure7.AttachedA549cellsstainedwithRe(CO)(nHo)Cl(Blue)andSYBR- 3 2 (TableS7). Atomic coordinates and isotropic displacement of 50 Green. the non-H atoms (TableS8). Atomic anisotropic displacement 51 parameters (TableS9). Hydrogen coordinates and isotropic 52 displacementparameters(TableS10). 53 The cellular uptake characteristics of these complexes can 54 be estimated by their lipophilicities, which are commonly 55 referred to as the n-octan-1-ol/water partition coefficients, 56 expressed as log P .[23d] In the present study, it was not 57 o/w ChemistrySelect2017,2,8666–8672 8671 (cid:6)2017Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim WileyVCH Freitag,22.09.2017 1 1727/99871 [S.8671/8672] Full Papers Acknowledgements Piro,M.P.Juliarena,G.T.Ruiz,E.Wolcan,M.R.F(cid:9)liz,DaltonTrans.2002, 1 2194–2202. 2 [12] a)M.L. Alomar, F.A.O. Rasse-Souriani, A. Gamuza, V.M. C(cid:5)ceres, F.M. 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