Isostructural Re(I)/(99m)Tc(I) tricarbonyl complexes for cancer theranostics.

Organic & Biomolecular Chemistry PAPER Isostructural Re( )/99mTc( ) tricarbonyl complexes I I † for cancer theranostics Citethis:Org.Biomol.Chem.,2015, 13,5182 PatriqueNunes,aGoretiRibeiroMorais,aElisaPalma,aFranciscoSilva,a MariaCristinaOliveira,aVeraF.C.Ferreira,aFilipaMendes,aLurdesGano,a HugoVicenteMiranda,b,cTiagoF.Outeiro,b,c,d,eIsabelSantosaandAntónioPaulo*a Mergingclassicalorganicanticancerdrugswithmetal-basedcompoundsinonesinglemoleculeoffers thepossibilityofexploringnewapproachesforcancertheranostics,i.e.thecombinationofdiagnosticand therapeutic modalities. For this purpose, we have synthesized and biologically evaluated a series of Re(I)/99mTc(I) tricarbonyl complexes (Re1–Re4 and Tc1–Tc4, respectively) stabilized by a cysteamine- based (N,S,O) chelator and containing 2-(4’-aminophenyl)benzothiazole pharmacophores. With the exception of Re1, all the Re complexes have shown a moderate cytotoxicity in MCF7 and PC3 cancer cells(IC valuesinthe15.9–32.1μMrangeafter72hofincubation).ThecytotoxicactivityoftheRecom- 50 plexesiswellcorrelatedwithcellularuptakethatwasquantifiedusingtheisostructural99mTccongeners. ThereisanaugmentedcytotoxiceffectforRe3andRe4(versusRe1andRe2),andthehighestcellular uptakeforTc3andTc4,whichdisplayalongether-containinglinkertocouplethepharmacophoretothe (N,S,O)-chelator framework. Moreover, fluorescence microscopy clearly confirmed the cytosolic accumulation of the most cytotoxic compound (Re3). Biodistribution studies of Tc1–Tc4 in mice confirmedthatthesemoderatelylipophiliccomplexes(logD =1.95–2.32)haveafavorablebioavailabil- o/w Received21stJanuary2015, ity.Tc3andTc4presentedafasterexcretion,astheyundergometabolictransformations,incontrastto Accepted26thMarch2015 complexesTc1andTc2.Insummary,ourresultsshowthatbenzothiazole-containingRe(I)/99mTc(I)tricar- DOI:10.1039/c5ob00124b bonylcomplexesstabilizedbycysteamine-based(N,S,O)-chelatorshavepotentialtobefurtherappliedin www.rsc.org/obc thedesignofnewtoolsforcancertheranostics. Introduction detection of β-amyloid (Aβ) aggregates by positron emission tomography (PET) and single-photon emission computed Manybenzothiazolederivativeshaveshownrelevantbiological tomography (SPECT).3 The lead compound in these studies features for drug development, both for diagnostic and thera- has been the so-called “Pittsburgh compound B” (11C-PIB) peutic purposes. This class of compounds has proven to be (Fig. 1). Studies inAlzheimer’s disease(AD) patientsvalidated particularly useful for the design of radioactive probes for theusefulnessof11C-PIBasaPETimagingprobeforAβdetec- nuclear imaging and as cytotoxic drugs for antitumor therapies.1–3 For nuclear imaging, 2-arylbenzothiazole derivatives have a crucial role in the development of radioprobes for the in vivo aCentrodeCiênciaseTecnologiaseNucleares,IST,UniversidadedeLisboa,Estrada Nacional10,2695-066BobadelaLRS,Portugal.E-mail:apaulo@ctn.ist.utl.pt bInstitutodeMedicinaMolecular,Av.Prof.EgasMoniz,1649-028Lisboa,Portugal cCEDOC,FaculdadedeCiênciasMédicas,UniversidadeNovadeLisboa,Lisboa, Portugal dInstitutodeFisiologia,FaculdadedeMedicinadaUniversidadedeLisboa,Lisboa, Portugal eDepartmentofNeuroDegenerationandRestorativeResearch,UniversityMedical CenterGöttingen,Waldweg33,37073Göttingen,Germany †Electronic supplementary information (ESI) available. See DOI: 10.1039/ Fig.1 Chemical structures of clinically relevant benzothiazole c5ob00124b derivatives. 5182 | Org.Biomol.Chem.,2015,13,5182–5194 Thisjournalis©TheRoyalSocietyofChemistry2015 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online View Journal | View Issue Organic&BiomolecularChemistry Paper tion. However, 11C-PIB is not approved for clinical use.4,5 ated as SPECT- or MRI-imaging compounds for cancer thera- Nevertheless, structurally related 18F-labeled congeners, such nostics,respectively.16–18Thesefirststudiesledtoencouraging as 18F-flutemetamol (Fig. 1), have been recently approved by biological results underlying the relevance of benzothiazole- the US Food and Drug Administration (FDA) and European containing metal complexes in this field of research. In this MedicineAgency(EMA)forthevisualdetectionofAβburdenin context, we have embarked on the synthesis and biological patients suspected of AD.6 Several metal-based compounds evaluation of new benzothiazole-containing 99mTc(I) and Re(I) bearingarylbenzothiazolemoietieswerealsoevaluatedforinvivo tricarbonyl complexes stabilized by a (S,N,O)-donor BFC imaging of Aβ plaques. This included the study of 99mTc com- derivedfromcysteamine.Here,wereportonthesynthesisand plexes for SPECT imaging and Gd complexes as contrast agents characterization of these new complexes, as well as on their formagneticresonanceimaging(MRI).7–9Theresultsformetallic biological evaluation as potential cancer theranostic agents. complexeswerelessencouragingifcomparedwithpurelyorganic FortheRecomplexes,thebiologicalevaluationcomprisedthe molecules, reflecting their poor ability to penetrate the blood– assessment of their cytotoxic activity against human breast brainbarrierandtoreachtheβ-amyloidplaques. cancer – MCF7 – and prostate cancer – PC3 – cell lines using The 2-arylbenzothiazole scaffold is also promising for the the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium development of anticancer drugs. It was shown that such a bromide (MTT) assay, and the visualization of cellular uptake simple scaffold might afford possible drug candidates upon by fluorescence microscopy. Conversely, the 99mTc counter- the introduction of minor functional group changes in its parts were used for the quantification of the uptake in the core. Many benzothiazole derivatives have demonstrated same cell lines, and to ascertain the biodistribution and activityagainstawiderangeofhumantumorcelllines,includ- invivostabilityofthecomplexesinmice. ing ovarian, breast, prostate lung, renal, and colon cancercell lines.10–13 From a large numberof tested molecules, the most explored as an antitumour agent is Phortress (Fig. 1), a 2-(4′- Results and discussion aminophenyl)benzothiazolederivativethatunderwentaPhase 1clinicaltrial.10 Synthesisandcharacterizationoftheligands Merging classical organic anticancer drugs with metal- As mentioned above, we have considered (S,N,O)-donor BFCs basedcompoundsinonesinglemoleculeoffersthepossibility derived from cysteamine to stabilize the fac-[M(CO) ]+ (M = 3 ofexploringnewapproachesforcancertheranostics,basedon Re, 99mTc) core and to be functionalized with the 2-(4′-amino- thecombinationofdiagnosticandtherapeuticmodalities.The phenyl)benzothiazole pharmacophore. In the last few years, metalfragmentsmightconferadvantagesastheranostictools, thiscorehasgainedanincreasingimpactinradiopharmaceu- suchasthepossibilityofexploringalargerdiversityofchemi- tical chemistry following the introduction by Alberto and co- cal structures, which may increase their bioavailability and workers of a convenient and fully aqueous-based kit prepa- resistance to metabolic transformation. For this purpose, a ration of the organometallic precursors fac-[M(OH ) - 23 largevarietyofdandftransitionmetals withnuclearor mag- (CO) ]+.19,20 This so-called tricarbonyl approach has several 3 netic properties suitable for medical imaging is available, for intrinsic advantages, including the high stability and kinetic exampletheaforementionedTcandGd.14,15 inertness inherent to Tc(I) and Re(I) tricarbonyl complexes In the particular case of technetium, non-radioactive Re when appropriate ligands are used.21 Previously, we have complexes are often used as reference compounds to assign shown that the cysteamine-based (S,N,O)-donor ligand 2-(2- thechemicalidentityofthe99mTccongenersduetothesimili- (ethylthio)ethylamino)acetic acid affords low-molecular weight tudeofthechemistryofthesetwogroup7elements.14Hence, and lipophilic 99mTc(I)/Re(I) complexes with high in vitro and whendesigningmetal-basedcancertheranosticagents,theRe in vivo stability.22,23 We have anticipated that the replacement complexes can be incorporated into the cytotoxic entity that oftheS-terminalethylsubstituentofthischelatorbythe2-(4′- will exert a therapeutic effect while the 99mTc congeners are aminophenyl)benzothiazole pharmacophore would not com- partoftheimagingentitythatwillenabletheinvivodetection promise the coordination capability of the (S,N,O)-donor set of tumor tissues. In addition, there are two beta-emitting towards fac-[M(CO) ]+ (M = Re, 99mTc) and the biological pro- 3 radioisotopesofrhenium(186Reand188Re)thatpresentphysi- pertiesofthepharmacophore. cal properties suitable for radionuclide therapy.14 Therefore, Four new BFCs, L1–L4, were synthesized by linking 2-aryl- Tc and Re can be seen as a unique match-pairof d-transition benzothiazole pharmacophores to the 3-((2-mercaptoethyl)- elements that enables the design of radiopharmaceuticals for amino)propanoicacidcoordinatingunitbyusingtwodifferent diagnostic (99mTc) and therapeutic applications (186/188Re), aliphatic spacers, displaying or not an ether function offering further possibilities for cancer theranostics by allow- (Schemes 1 and 2). The synthesis of L1–L4 has been achieved ing the combination of SPECT imaging with chemo- and/or in a convergent way, using compounds 3 and 4 as common radiotherapeuticmodalities. intermediates. As depicted in Scheme 1, the 2-aryl benzothia- Recently, the 2-(4′-aminophenyl)benzothiazole pharmaco- zole rings of 3 and 4 were formed through cyclocondensation phore was linked to acyclic or macrocyclic bifunctional chela- of the o-aminothiophenols 1 and 2, by reaction with the ade- tors (BFCs), which were used to obtain 99mTc(I) tricarbonyl quateN-substitutedbenzaldehydeunderbasicconditions.For complexesandGd(III)complexes.Thesecomplexeswereevalu- L1 and L2, which do not contain any ether function in their Thisjournalis©TheRoyalSocietyofChemistry2015 Org.Biomol.Chem.,2015,13,5182–5194 | 5183 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Paper Organic&BiomolecularChemistry Scheme1 SyntheticrouteforthepreparationofligandsL1andL2.Conditions:(i)N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde,pyridine, reflux;(ii)CH Cl ,CBr ,PPh ,RT;(iii)NaOH,EtOH,2-aminoethanethiolhydrochloride,RT;(iv)CH CN,KI,K CO ,ethyl2-bromoacetate,reflux;(v) 2 2 4 3 3 2 3 THF,water,NaOH,reflux. Scheme2 Syntheticroutefor thepreparationofL3and L4. Conditions:(i)THF,NaH,1,4-dibromobutane; (ii)NaOH,EtOH,2-aminoethanethiol hydrochloride,RT;(iii)CH CN,KI,K CO ,ethyl2-bromoacetate,reflux;(iv)THF,water,NaOH,reflux. 3 2 3 structure, the synthesis involved the brominated derivatives 5 S-alkylation reactions of 2-aminoethanethiol, followed by the and6asintermediates.Compounds 5and6wereobtainedin N-alkylation of the resulting compounds (7, 8, 13, and 14, moderate yield (η = 56–64%) from the benzothiazoles 3 and 4 respectively) with ethyl bromoacetate. Finally, basic hydrolysis using the Appel reaction (Scheme 1). The synthesis of L3 and of the obtained benzothiazole ester derivatives (9, 10, 15, and L4,havinganetherfunctioninthelinkerbetweenthepharma- 16, respectively) gave the ligands L1–L4. All the compounds cophore and the S,N,O-coordinating unit, was also achieved were characterized using common spectroscopy techniques using brominated intermediates (compounds 11 and 12); 11 (1H and 13C NMR) and by ESI-MS, which confirmed the pro- and 12 were obtained in good yield (η = 40–45%) by the posedformulations. O-alkylation of 3 and 4 with an excess of 1,4-dibromobutane SynthesisandcharacterizationoftheorganometallicReand (Scheme 2). For all the ligands, the incorporation of the ben- 99mTccomplexes zothiazole moieties into the (S,N,O)-donor coordination unit wasaccomplishedinasequentialmanner,inwhichthebromi- The synthesis of the Re(I) tricarbonyl complexes Re1–Re4 nated derivatives 5, 6, 11, and 12 acted as electrophiles in was performed by ligand exchange reactions of fac- 5184 | Org.Biomol.Chem.,2015,13,5182–5194 Thisjournalis©TheRoyalSocietyofChemistry2015 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Organic&BiomolecularChemistry Paper Fig.2 1H NMR spectra of complex Re1 at various temperatures in DMF-d. 7 Scheme3 Synthesis of the Re(I) and 99mTc(I) tricarbonyl complexes. Conditions: (i) [Re(H O) (CO) ]Br, methanol, reflux, 16 h; (ii) [99mTc 2 3 3 (H 2 O) 3 (CO) 3 ],H 2 O,100°C,30min. probably contain ligands coordinated in a N,S,O-tridentate fashion, a coordination mode that was confirmed by X-ray diffractionanalysisinthecaseofRe(CO) (NSO).23 3 [Re(H O) (CO) )]Br with L1–L4 in refluxing methanol In aqueous solution and at 100 °C, L1–L4 readily reacted (Schem 2 e 3 3). A 3 fter adequate purification, complexes Re1–Re4 with fac-[99mTc(CO) 3 (OH 2 ) 3 ]+ affording complexes Tc1–Tc4, respectively (Scheme 3). The reactions are almost quantitative wererecoveredasyellowmicrocrystallinesolidsinmoderateto high yield (57–81%). Their characterization was performed (>95%) for final concentrations of L1–L4 as low as 10 −4 M, using common spectroscopy techniques (IR, 1H and 13C complexes Tc1–Tc4 being the unique species formed. Their chemicalidentitywasconfirmedbycomparisonoftheirHPLC NMR),ESI-MS,HPLCandelementalanalysis. ThepositiveESImassspectraobtainedforRe1–Re4showed profiles with those of the corresponding rhenium complexes, asexemplifiedforTc1/Re1inFig.3. thepresenceofprominentpeakscorrespondingtotherespect- ive[M+H]+molecularions;unlikeRe1andRe2,intensepeaks The characterization of Tc1–Tc4 comprised also the evalu- thatwereassignedto[M+Na]+werealsoobservedinthemass ationoftheirlipophilicity,whichwasassessedbythemeasure- spectra of Re3 and Re4. This difference certainly reflects the ment of the respective logD o/w values (n-octanol/0.1 M, phosphate buffered saline (PBS), pH 7.4) using the “shake- presence of an ether function in the aliphatic spacers of L3 flask” method. The logD values and the HPLC retention andL4,whichmightenhancetheinteractionwiththesodium o/w times of Tc1–Tc4 are shown in Table 1. All the complexes are cation. The IR spectroscopic data obtained for all the com- plexes corroborated the presence of the fac-[Re(CO) ]+ core, moderatelylipophilicwithlogD o/w valuesspanninginarather 3 detecting two strong bands assignable to ν(CvO) in the narrow range, from 1.98 to 2.32, suggesting that the presence 1893–2028 cm −1 range. Moreover, the HPLC analysis of Re1– of the ether linkage balances the increase of lipophilicity Re4 showed that the complexes were of high purity, as only singlepeaksweredetectedintherespectivechromatograms. The chemical characterization of Re1–Re4 by NMR spec- troscopy was more demanding. At room temperature, the 1H NMR spectra of these complexes in DMF-d displayed a set of 7 relatively broad and ill-defined resonances in the area of the methylenic and methyl protons, ranging from 1.71 to 3.96 ppm. The aromatic protons from the 2-(4′-aminophenyl)- benzothiazole pharmacophore gave rise to better defined and well resolved signals that appear between 6.90 and 8.10 ppm. The broadness of the resonances from the aliphatic protons suggestedthatcomplexesRe1–Re4undergodynamicprocesses in solution, as confirmed for Re1 by variable temperature 1H NMRstudies(Fig.2).ThedynamicbehaviorobservedforRe1– Re4ismostprobablyduetotheoccurrenceofpyramidalinver- sion at the coordinated sulfur atom, as we have previously observed for the congener Re(I) tricarbonyl complex with 2-(2- (ethylthio)ethylamino)acetic acid (herein designated as Re- Fig.3 HPLCchromatogramsofcomplexesRe1(UVdetection)andTc1 (CO) 3 (NSO)).22 These findings indicate that Re1–Re4 most (radiometricdetection). Thisjournalis©TheRoyalSocietyofChemistry2015 Org.Biomol.Chem.,2015,13,5182–5194 | 5185 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Paper Organic&BiomolecularChemistry Table1 HPLCretentiontimesandlogD ofcomplexesTc1–Tc4 Re3, was assessed by fluorescence microscopy in MCF7 and o/w PC3 human cancer cells. For comparative purposes, the Complex ra,b(min) logD t o/w studieswerealsoperformedfortherespectiveligandL3.Com- Tc1 24.4(23.8) 1.98±0.02 poundsL3andRe3weredetectedbytheemissionofbluefluo- Tc2 24.9(24.1) 2.01±0.05 rescence and the nucleus stained with propidium iodide (red Tc3 24.0(23.3) 1.95±0.01 emission). Tc4 26.0(25.9) 2.32±0.04 AsshowninFig.4,bothcomplexesRe3andL3(toalesser aUsing a gradient of aqueous 0.1% CF 3 COOH and methanol as the extent) were detected in the cytoplasm of both cell lines. No solvent.bThevaluesinparenthesesaretheretentiontimesfortheRe detectable fluorescence in the blue channel was observed for congeners. theDMSOcontrol,confirmingthatthedetectedblueemission is from the compounds L3 and Re3, and not due to the un- specificsignal. caused by the larger number of methylenic groups in the The cell entrance/uptake seems to be reduced for L3 when linker. compared with Re3, as less fluorescence was detected from cellsincubatedwithL3ifcomparedwiththoseincubatedwith BiologicalevaluationoftheRecomplexes:cytotoxicityassays Re3. It is important to mention that in Fig. 4 all the images andmicroscopystudies were collected using the same conditions, in particular the acquisition time. A second set of images was collected with a The cytotoxic activity of the ligands and respective Re com- plexeswastestedonhumanbreastcancer–MCF7–andpros- longeracquisition time to furtherevaluate the cellular uptake tate cancer – PC3 – cell lines. These cancer cells were treated and localization of L3 (Fig. 5), showing intracellular cyto- withdecreasingconcentrations(200–0.002μM)ofthedifferent plasmicaccumulation. In addition, the fluorescence spectra obtained for L3 and compounds and incubated for 72 h at 37 °C. All the tested Re3 (at the same concentration) have shown no detectable compounds were firstsolubilized in DMSO anddiluted inthe differences in the maximum wavelength of excitation and cell media. The percentage of DMSO never exceeded 1%, a emission (Fig. 5). Moreover, these experiments have shown non-toxicconcentration.Afterthetreatment,thecellularviabi- that L3 is a slightly more efficient fluorophore than Re3. lity was assessed by the MTT assay. The inhibition of growth Hence, the more intense fluorescence detected in the cells (%) was calculated by correlation with vehicle-treated cells. TheIC values,expressedinμMconcentrations,areaverage± incubated with Re3 is not due to its intrinsic fluorescence 50 standarddeviationsof3independentexperimentswith4repli- cateseach,andtheresultsarepresentedinTable2. In general, the complexes Re2–Re4 exhibited higher cytotoxicity than the respective ligands L2–L4 in the MCF7 breast and PC3 prostate cell lines. In contrast, in these cell linesL1ismorecytotoxicthanRe1.Fromallthestudiedcom- pounds, the complexes Re3 and Re4 were shown to be the most potent, with IC values between 15.9 ± 1.1 and 32.1 ± 50 1.2μMinbothcelllines. Taking advantage of the fluorescence emission character- istics of the benzothiazole unit of the newlysynthesized com- pounds, the cellular uptake of the most cytotoxic Re complex, Table2 Cytotoxicityof L1–L4 and Re1–Re4 in MCF7 breast and PC3 prostatecancercells IC a(µM) 50 Compound MCF7 PC3 L1 25.6±1.4 32.5±1.1 Re1 90±1.2 94±1.3 L2 241±1.8 89±1.1 Re2 30.7±1.0 30.6±1.0 L3 52.2±1.1 95±1.2 Re3 15.9±1.1 28.8±1.1 L4 39.3±1.1 71.3±1.1 Re4 16.3±1.2 32.1±1.2 Fig.4 Fluorescence microscopy evaluation of the uptake of L3 and Re3(blue)inhumanMCF7andPC3cells.Nuclei(red)werestainedwith aIC :theconcentrationthatcauses50%reductionofthecellgrowth. propidiumiodide.Scalebar,10μm. 50 5186 | Org.Biomol.Chem.,2015,13,5182–5194 Thisjournalis©TheRoyalSocietyofChemistry2015 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Organic&BiomolecularChemistry Paper properties. Altogether, these data indicate that Re3 is able to enterintocancercelllinesmoreeasilythanthecorresponding ligand L3. Eventually, this might explain the highest cyto- toxicity that has been found for Re3 in the MCF7 and PC3 cancercelllines,incomparisonwiththerespectiveligand. Biologicalevaluationof99mTccomplexes:cellularuptakeand biodistributionstudies The microscopy studies performed for Re3 have confirmed thatthiscomplexcanreachthecytoplasmofMCF7breastand PC3 prostate human cancer cells. The isostructural 99mTc complex, Tc3, was used to quantify the cellular uptake in the samecelllines.Thesestudieswereextendedtotheotherradio- active complexes (Tc1, Tc2 and Tc4) aiming to correlate their cellular uptake with the cytotoxicity exhibited by the corres- ponding Re complexes. The breast and prostate cancer cell lines were treated with Tc1–Tc4 for different times (0 to 18 h) and the results show that complexes Tc1–Tc4 display a high cellularuptakeinbothcelllines,inatime-dependentmanner, with values at 18 h in the range of 14–29% and 23–48% for MCF7 and PC3 cells, respectively (Fig. 6). The highest uptake was observed in the PC3 cells, in particular for the complexes Tc3andTc4withvaluesof38.6%and31.5%at5handvalues of47.1%and47.9%at18h,respectively.Theseresultsdemon- strate that the complexes Tc3 and Tc4 display the highest cel- lularuptakeintheMCF7andPC3cells,accumulatinginboth celllinesovertime.Thistrendseemstobeinagreementwith thehighestcytotoxicityoftheRecongeners(Re3andRe4),par- ticularlyintheMCF7breastcancercellline. As discussed before, all complexes are lipophilic with logD valueswithinthe relatively narrowrangeof1.98–2.32. o/w Therefore, the lipophilicity is not expected to be the major cause of the large differences observed between the cellular uptakeofTc1–Tc4.Thisisclearlycorroboratedbythefactthat Tc1 (logP = 1.98 ± 0.02) and Tc3 (logP = 1.95 ± 0.02), having Fig.5 Emission and excitation spectra of L3 (a) and Re3 (b). Fluo- almostequallogPvalues,presentalargedifferenceinthecel- rescence microscopy evaluation of human MCF7 and PC3 cancer cell lular uptake values, which for Tc3 is as high as 30.9% of the uptakeofL3(blue)(c).Nuclei(red)werestainedwithpropidiumiodide. Note: images were collected with higher acquisition times than those appliedactivity/million cellsin thePC-3celllineversus13.3% presentedinFig.4.Scalebar,10μm. forTc1at5h. Fig.6 CellularuptakeofTc1–Tc4inhumanMCF7breastandPC3prostatecells,expressedasapercentageoftotalradioactivitypermillioncells. Thisjournalis©TheRoyalSocietyofChemistry2015 Org.Biomol.Chem.,2015,13,5182–5194 | 5187 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Paper Organic&BiomolecularChemistry Table3 Biodistributiondata(%IDg−1)ofTc1–Tc4at2and60minpost-injection(p.i.)inCD-1mice %IDg−1±SD Tc1 Tc2 Tc3 Tc4 Organ 2min 60min 2min 60min 2min 60min 2min 60min Blood 6.40±1.10 0.58±0.11 7.00±0.50 1.00±0.09 8.00±2.40 0.70±0.20 5.20±0.90 0.84±0.05 Liver 43.3±1.90 37.5±1.60 19.9±2.60 15.2±2.40 35.8±1.60 11.3±1.20 35.8±4.40 29.5±0.60 Intestine 3.10±0.40 19.5±1.70 1.40±0.40 11.5±3.20 3.90±0.50 10.3±1.20 2.40±0.30 17.2±0.50 Spleen 3.90±0.20 0.50±0.20 3.70±0.03 0.78±0.01 2.90±0.60 0.26±0.03 5.20±0.90 0.70±0.10 Heart 5.40±0.60 0.90±0.30 3.80±0.70 0.90±0.10 3.70±0.80 0.42±0.08 4.40±0.20 1.10±0.20 Lungs 7.00±1.00 1.10±0.20 13.9±0.70 3.00±0.80 3.50±1.00 0.70±0.20 9.00±1.00 3.70±0.60 Kidney 21.6±2.90 2.70±0.20 11.8±4.20 1.90±0.80 14.9±2.20 2.00±0.50 13.2±0.70 2.10±0.40 Muscle 1.20±0.20 0.60±0.10 0.90±0.30 0.55±0.08 0.80±0.30 0.29±0.07 1.40±0.10 0.67±0.06 Skeletal 1.50±0.20 0.46±0.09 1.30±0.30 0.45±0.06 1.40±0.20 0.19±0.03 1.96±0.07 0.54±0.04 Stomach 1.20±0.40 2.60±0.70 0.69±0.10 1.30±0.30 0.90±0.60 1.50±0.50 1.80±0.50 3.60±0.70 Brain 0.30±0.08 0.03±0.01 0.19±0.04 0.04±0.00 0.20±0.04 0.02±0.01 0.40±0.10 0.05±0.01 Excretion(%IA) 2.70±0.50 3.60±2.40 18.4±1.70 12.4±3.40 The most striking structural differences in complexes Tc1– I.A. At 1 h p.i.,most of the activity isretained in theliverand Tc4 are related to the linkers used to attach the 2-arylben- intestine due to the predominantly hepatobiliary excretion of zothiazole pharmacophore to the 3-((2-mercaptoethyl)amino)- these moderately lipophilic compounds. Despite their neu- propanoic acid coordinating unit. A shorter ethylenic linker trality and lipophilicity, Tc1–Tc4 showed a negligible brain hasbeenusedincomplexesTc1andTc2,whichhaveshowna uptake (<0.40% ID g −1) confirming their inability to crossthe much lower cellular uptake than Tc3 and Tc4, containing a blood–brainbarrierinmice. longer 2-n-butoxyethyl linker. The use of a longer linker is TheinvivostabilityofTc1–Tc4hasbeenassessedbyHPLC expected to minimize the interference of the organometallic analysis of the urine and plasma of mice injected with these fragment in molecular interactions involving the pharmaco- complexes. At 2 min p.i., most of the radioactivity detected in phore and biologically relevant targets. It is worthwhile men- the plasma corresponds to the intact complexes (Fig. 7). tioning that many benzothiazole derivatives like the parent However, the complexes with an ether-containing longer compound 2-(4′-aminophenyl)-benzothiazole and its deriva- linker, Tc3 and Tc4, have ahigher tendencyto undergo meta- tives bearing 3′ substituents undergo a selective uptake and bolic transformations, as indicated by the HPLC chromato- biotransformation only in sensitive cancer cell lines, such as grams obtained for the plasma at 1 h p.i. As exemplified for MCF-7 breast cancer cells. In these sensitive cell lines, the Tc4 in Fig. 7, a significant part of the circulating activity still compounds bind to the aryl hydrocarbon receptor (AhR) with corresponds to the intact complex but two metabolites were AhRtranslocationtothenucleusandsubsequentinductionof detected at retention times of 17.2 and 18.7 min, respectively. the expression and activation of the cytochrome P450 isoform These metabolites are also the major radiochemical species CYP1A1. In contrast, insensitive cells like the prostate carci- that are present in the HPLC chromatograms of the urine of noma PC3 cells do not show AhR translocation and CYP1A1 mice injected with Tc4, at 1 h p.i. (data not shown). We did activationupontreatmentwiththesamebenzothiazolederiva- not identify these metabolites but their formation might be tives.12,13,24 We did not fully investigate the mechanisms related to transformations involving the ether function from involvedinthecellularuptakeofTc1–Tc4andinthecytotoxic the aliphatic linker used to couple the benzothiazole pharma- activity of the congener Re1–Re4, but the higher uptake cophorewiththe(N,S,O)chelatorframeworkincomplexesTc3 observed for the 99mTc complexes in the PC3 cell line in and Tc4. For instance, the ether function can be involved in comparison with the MCF7 cell line and similar cytotoxic O-dealkylationprocesses,followedbyoxidationoftheresulting activityoftheRecomplexesinbothcelllinesindicatethatthe alcoholfunctions,aswehaverecentlyshownfor99mTc(I)tricar- interactionwithAhRismostlikelynotresponsibleforthebio- bonyl complexes with ether-containing pyrazolyl derivatives.25 logicalactivityexhibited. Theformationofthesemetabolitesinvivocancertainlyjustify Finally, biodistribution studies of complexes Tc1–Tc4 were thatcomplexesTc3andTc4displayafasterexcretionratethan performed in CD-1 mice to assess their pharmacokinetic Tc1andTc2. profile and in vivo stability. The biodistribution data obtained forTc1–Tc4at2minand1hpostintravenousinjection(p.i.), expressed in percent of the injected dose per gram of tissue Conclusions (%IDg −1),arepresentedinTable3. All complexes show a relatively fast blood clearance but a The functionalizationof the 3-((2-mercaptoethyl)amino)propa- ratherlowrateofexcretionthatvariedbetween2.70and18.4% noicacidcoordinatingunitwiththe2-(4′-aminophenyl)benzo- 5188 | Org.Biomol.Chem.,2015,13,5182–5194 Thisjournalis©TheRoyalSocietyofChemistry2015 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Organic&BiomolecularChemistry Paper Experimental section Generalinformation All chemicals and solvents were of reagent grade and were used without purification unless stated otherwise. 2-Amino-5- methylbenzenethiol(2)waspreparedasdescribedinthelitera- ture.26 fac-[Re(H O) (CO) ]Br was synthesized according to the 2 3 3 literature.27 Na[99mTcO ] was eluted from a commercial 4 99Mo/99mTc generator, using a 0.9% saline solution. The fac- [99mTc(H O) (CO) ]+ precursor was obtained by labelling of a 2 3 3 Isolink®-kit with Na[99mTcO ], following the procedure 4 described elsewhere.20 The NMR spectra were recorded on a VarianUnity300NMRspectrometeratfrequenciesof300MHz Fig.7 InvivostabilitystudiesofcomplexesTc2andTc4.HPLCchro- (1H) and 75 MHz (13C). Chemical shifts are reported in parts matogramsof:(a)injectedpreparation;(b)plasmacollectedat2minp.i.; per million. 1H and 13C NMR chemical shifts were referenced (c)plasmacollectedat1hp.i. with the residual solvent resonances relative to tetramethyl- silane. FTIR spectra were recorded using KBr pellets on a Bruker, Tensor 27 spectrometer. Electrospray ionisation mass thiazole pharmacophore, using two different aliphatic spacers spectrometry(ESI-MS) was performed on aQITMS instrument containing or not an ether function, led to a new family of inpositiveandnegativeionizationmode.Thin-layerchromato- benzothiazole-containing chelators: L1–L4. These chelators graphy(TLC)wasperformedonprecoatedsilicaplates60F 254 were successfully applied in the synthesis of M(I) (M = Re, (Merck). Visualization of the plates was carried out using UV 99mTc) tricarbonyl complexes (Re1–Re4 and Tc1–Tc4), which light (254 and 365 nm) and/or an iodine chamber. Gravity were biologically evaluated as isostructural tools for cancer column chromatography was carried out on silica gel (Merck, theranostics. 70–230 mesh). HPLC analysis of the Re and 99mTc complexes TheRecomplexeshaveshownamoderatecytotoxicactivity was performed on a PerkinElmer LC pump coupled to an LC againstPC3andMCF7humantumorcells(IC 50 values<50μM tuneable UV/Vis detector and to a Berthold LB-507A radio- after 72 h of incubation), with the exception of Re1 that metric detector, using an analytical C18 reversed-phase showedrelativelyhighIC 50 values(>50μM).Ingeneral,theRe column(Nucleosil100-10,250×3mm)withaflowrateof1ml complexesare more cytotoxicthan thecorrespondingligands, min −1. UV detection, 254 nm. Eluents: A – aqueous 0.1% which is most probably due to the better ability of the com- CF COOH solution, B – MeOH. The HPLC analysis was per- 3 plexes to enter into the cells, as confirmed by fluorescence formed with gradient elution, using the following method: microscopystudiesinthecaseofRe3/L3. 0–8min,100%A;8–8.1min,100%–75%A;8.1–14min,75%A; Thereisaclearinfluenceofthelinkeronthecytotoxicityof 14–14.1min,75%–66%A;14.1–25min,66%–0%A;25–30min, the complexes, those which display the ether-containing 0%A;30–30.1min,0–100%A;30.1–35min,100%A. linker,i.e.Re3andRe4,beingthemostactive.Thecytotoxicity activityoftheRecomplexesiswellcorrelatedwiththecellular Synthesisoftheligands uptake,whichhasbeenquantifiedusingthe99mTccongeners, 2-[N-Methyl-N-(2′-hydroxyethyl)-4′-aminophenyl]-benzothia- Tc1–Tc4. Interestingly, the nature of the linker also strongly zole(3). Asolutionofo-aminothiophenol(557mg,4.5mmol) influences the in vivo behavior of the complexes, as Tc3 and and N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde Tc4 are more prone to metabolic transformations and display (660 mg, 3.7 mmol) in pyridine (6 mL) was refluxed for 3 h. afasterexcretionrateinmice. After cooling at room temperature the intense yellow suspen- In summary, the moderate cytotoxicity exhibited by the Re sion was neutralized with a 3 M HCl solution and the solvent complexes, together with the remarkably high cellular uptake was concentrated. The reaction crude was taken in water andthereasonablygoodbioavailabilitypresentedbythe99mTc (50 mL) and extracted with CH Cl (3 × 30 ml). The organic 2 2 congeners, show that the benzothiazole-containing Re(I)/ phase was dried over Na 2 SO 4 , filtered and evaporated. The 99mTc(I)tricarbonylcomplexesdescribedhereholdpromiseto residuewassubjectedtocolumnchromatographyonsilica-gel befurtherappliedinthedesignofnewtoolsforcancerthera- (CH Cl –MeOH, 98:2) to give 3 (510 mg, 49%); R = 0.25 2 2 f nostics. The marked influence of the linker on the biological (n-hexane–AcOEt 1:1); 1H NMR (CDCl , 300 MHz): δ 1.78 (bs, 3 fate of the complexes and its easy structural modification, as OH, 1H), 3.07 (s, 3H), 3.58 (t, 2H, J = 5.7 Hz), 3.86 (t, 2H, J = well as the different possibilities available to attach the 2-(4′- 5.7Hz),6.79(d,2H,3J=8.7Hz),7.29(dd,1H,3J=8.1Hz,3J= aminophenyl)benzothiazole pharmacophore (e.g. N-alkylation 8.1Hz),7.42(dd,1H,3J=8.1Hz,3J=8.1Hz),7.82(d,2H,3J= vs. amide-forming reactions) to the cysteamine based (N,S,O)- 8.1 Hz),7.94(d, 2H,3J=8.7 Hz),7.98 (d,2H,3J= 8.1 Hz);13C chelatorare expectedto help inthesearchfor betterperform- NMR (CDCl , 75 MHz): δ 39.05, 54.67, 60.17, 111.98 (2C), 3 ing compounds and on the elucidation of structure–activity 121.37 (2C), 122.15, 124.38, 126.14, 129.07 (2C); ESI/MS relationships(SAR). C H N OS(284)m/z284.9[M+H]+(100%). 16 16 2 Thisjournalis©TheRoyalSocietyofChemistry2015 Org.Biomol.Chem.,2015,13,5182–5194 | 5189 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Paper Organic&BiomolecularChemistry 6-Methyl-2-[N-methyl-N-(2′-hydroxyethyl)-4′-aminophenyl]- 3J=7.8Hz);13CNMR(CDCl ,75MHz):δ28.5,36.4,38.6,41.1, 3 benzothiazole(4). Compound4wassynthesizedandpurified 52.4, 111.4 (2C), 121.3, 121.5, 122.2, 124.2, 125.9, 128.9 (2C), as described above forcompound 3, starting from 4-methyl-o- 134.4,150.3,154.2,168.5. aminothiophenol (320 mg, 2.30 mmol). Yield: 60% (329 mg, 6-Methyl-2-[N-methyl-N-(2′-(2″-aminoethylthio)ethyl)-4′-ami- 1.38 mmol); R = 0.43 (petroleum ether–AcOEt 1:1); 1H NMR nophenyl]-benzothiazole (8). Compound 8 was synthesized f (CDCl , 300 MHz): δ 2.45 (s, 3H), 3.06 (s, 3H), 3.52 (t, 2H, J = andpurifiedasdescribedabovefor7,startingfromcompound 3 5.7Hz),3.85(t,2H,J=5.7Hz),6.78(d,2H,3J=9Hz),7.23(d, 6 (200 mg, 0.55 mmol). Yield: 35% (68 mg, 0.19 mmol); R = f 1H,3J=8.1Hz),7.61(s,1H),7.85(d,1H,3J=8.1Hz),7.91(d, 0.48(CHCl –MeOH4:1);1HNMR(CDCl ,300MHz):δ2.42(s, 3 3 2H, 3J = 9 Hz); 13C NMR (CDCl , 75 MHz): δ 21.5, 39.0, 54.7, 3H),2.65(t,2H,J=7.5Hz),2.88(m,2H),2.93(s,3H),3.14(m, 3 60.1,111.9(2C);121.2,121.6,127.7,128.9,134.5,151.5,168. 2H),3.50(t,2H,J=7.5Hz),6.62(d,2H,3J=9Hz),7.19(d,1H, 2-[N-Methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-benzothiazole 3J = 8.4 Hz), 7.55 (s, 1H), 7.82 (m, 3H); 13C NMR (CDCl , 3 (5). To a solution of 3 (0.51 g, 1.8 mmol) in dichloromethane 75 MHz): δ 21.4, 21.3, 28.5, 29.5, 38.5, 39.2, 52.1, 111.7 (2C), (10 mL) was added carbon tetrabromide (0.65 g, 1.9 mmol) at 121.2, 121.6, 127.6, 128.9 (2C), 134.4, 150.3, 152.1, 167.8, 0°Cfollowedbytriphenylphosphine(0.517g, 1.8mmol).The 176.7. reactionmixturewasstirredfor3hand,thereafter,thesolvent Ethyl 2-[2′-(2″-((4″-(benzo[d]thiazol-2-yl)phenyl)(methyl)- was concentrated. The residue was subjected to column amino)ethylthio)ethylamino] acetate (9). To a solution of 7 chromatography on silica gel (EtOAc–n-hexane 1:6) to afford (100 mg, 0.29 mmol), KI (1.2 mg, 7.3 μmol), and K CO 2 3 5. Yield: 56% (350 mg, 1.0 mmol) as an yellow oil; R = 0.46 (20 mg, 0.15 mmol) in anhydrous CH CN (10 mL) was added f 3 (AcOEt–n-hexane 1:2); 1H NMR (CDCl , 300 MHz): δ 3.09 (s, ethyl 2-bromoacetate (8.2 μL, 0.145 mmol) under a nitrogen 3 3H),3.48(t,2H,J=7.5Hz),3.80(t,2H,J=7.5Hz),6.73(d,2H, atmosphere, and the reaction mixture was refluxed overnight. 3J=8.7Hz),7.30(dd,1H,3J=8.1Hz,3J=8.1Hz),7.43(dd,1H, Hencethesolventwasconcentrated.Themixturewastakenin 3J = 8.1 Hz, 3J = 8.1 Hz), 7.83 (d, 1H, 3J = 8.1 Hz), 7.96 (d, 2H, water (30 ml) and extracted with CH Cl (3 × 30 ml). The 2 2 3J = 8.7 Hz), 8.01 (d, 1H, 3J = 8.1 Hz); 13C NMR (CDCl , organic phasewasdried withNa SO , filtered,and the filtrate 3 2 4 75 MHz): δ 27.89, 38.87, 54.07, 111.56 (2C), 121.38, 122.27, was evaporated. The residue was subjected to column chrom- 124.41, 126.12, 129.17 (2C), 150.11; ESI/MS C H BrN S (346) atography on silica gel (CHCl –MeOH 95:5) to provide 9. 16 15 2 3 m/z347.0[M+H]+(97%),347.9[M+H]+(20%),348.9[M+H]+ Yield: 58%. (73 mg, 0.17 mmol) as a light yellow oil; R = 0.55 f (100%),349.9[M+H]+(20%). (CHCl –MeOH 93:7); 1H NMR (CDCl , 300 MHz): δ 1.19 (t, 3 3 6-Methyl-2-[N-methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-ben- 3H, J = 7.2 Hz), 2.68 (m, 4H), 2.77 (t, 2H, J = 6.3 Hz), 3.00 (s, zothiazole (6). Compound 6 was synthesized and purified as 3H), 3.36 (s, 2H), 3.55 (t, 2H, J = 6.2 Hz), 4.12 (q, 2H, J = 7.2 described above for 5, starting from compound 4 (300 mg, Hz), 6.67 (d, 2H, 3J = 9.0 Hz), 7.23 (ddd, 1H, 4J = 1.2 Hz, 3J = 1.00 mmol). Yield: 64% (231 mg, 0.64 mmol); R = 0.39 8.1Hz,3J=7.5Hz),7.36(ddd,1H,2J=1.2Hz,3J=8.1Hz,3J= f (n-hexane–AcOEt 1:4); 1H NMR (CDCl , 300 MHz): δ 2.46 (s, 8.1Hz),7.77(d,1H,3J=8.1Hz),7.88(d,2H,3J=9.0Hz),7.90 3 3H), 3.09 (s, 3H), 3.48 (t, 2H, J = 7.5 Hz), 3.80 (t, 2H, J = 7.5 (d, 1H, 3J = 7.5 Hz); 13C NMR (CDCl , 75 MHz): δ 14.0, 28.3, 3 Hz),6.73(d,2H,3J=8.7Hz),7.24(d,1H,3J=8.2Hz),7.62(s, 32.4,38.4,47.9,50.3,52.2,60.6,111.3(2C),121.1,121.3,122.0, 1H), 7.87 (d, 1H, 3J = 8.2 Hz), 7.95 (d, 2H, 3J = 8.7 Hz); 13C 124.0, 125.7, 128.8 (2C), 134.2, 150.2, 154.1, 168.4, 171.9 NMR (CDCl , 75 MHz): δ 21.1, 27.7, 38.2, 53.4 (CH ), 111.0 (CvO). 3 2 (2C),120.8,121.4,121.7,127.1,128.4(2C),133.9,134.2,149.4, Ethyl 2-[2′-(2″-((4′′′-(6″″-methylbenzo[d]thiazol-2-yl)phenyl)- 151.9,166.9;ESI/MSC H BrN S(362)m/z363.2.0[M+H]+. (methyl)amino)ethylthio)ethylamino] acetate (10). Compound 17 17 2 2-[N-Methyl-N-(2′-(2″-aminoethylthio)ethyl)-4′-aminophenyl]- 10wassynthesizedandpurifiedasdescribedabovefor9,start- benzothiazole(7). To asolutionof2-aminoethanethiolhydro- ingfromcompound8(55mg,0.15mmol).Yield:55%(37mg, chloride (296 mg, 2.55 mmol) in anhydrous EtOH (8 mL) was 0.08 mmol); R = 0.48 (CHCl –MeOH, 97:3); 1H NMR (CDCl , f 3 3 added NaOH (163 g, 4 mmol) under a nitrogen atmosphere. 300MHz):δ1.24(t,3H,J=6.9Hz),2.44(s,3H),2.72(m,4H), After 1 h, compound 5 (448 mg, 1.29 mmol) was added and 2.82(t,2H,J=5.7Hz),3.03(s,3H),3.40(s,2H),3.59(t,2H,J= the reaction mixture was stirred for additional 20 h at room 7.2 Hz),4.16 (q,2H,J=6.9 Hz),6.70 (d,2H, 3J=9.0 Hz),7.23 temperature. Then, the solvent was concentrated. The pallid (d,1H,3J=8.4Hz),7.60(s,1H),7.83(d,1H,3J=8.4Hz),7.91 brown residue was taken in water (25 mL) and the pH was (d, 2H, 3J = 9.0 Hz); 13C NMR (CDCl , 75 MHz): δ 14.2, 21.4, 3 adjustedto7witha1MHClsolution.Theaqueousphasewas 28.6, 32.6,38.6, 48.2, 50.3,52.5, 60.8,111.5 (2C),121.1, 121.8, extracted with CH Cl (4 × 25 mL), dried over Na SO , filtered 127.4, 128.8 (2C), 134.2, 134.6, 150.2, 152.4, 167.5, 172.1 2 2 2 4 andthefiltratewasconcentrated.Theresiduewassubjectedto (CvO). columnchromatographyonsilicagel(CHCl –MeOH70:30)to 2-[N-Methyl-N-(2′-(4′-bromobutoxy)ethyl)-4′-aminophenyl]- 3 afford the desired amine as an oil. Yield: 76% (335 mg, benzothiazole (11). To a suspension of NaH (525 mg, 0.98 mmol); R = 0.64 (CHCl –MeOH 3:1); 1H NMR (CDCl , 13.2 mmol) in anhydrous THF (40 mL) was added 3 (2.0 g, f 3 3 300MHz):δ2.68(m,4H),2.87(t,2H,J=6.9Hz),3.04(s,3H), 8.05 mmol) under a nitrogen atmosphere. After 30 min, 1,4- 3.59(t,2H,J=6.9Hz),6.71(d,2H,3J=8.7Hz),7.28(dd,1H,3J dibromobutane (5.6 mL, 47.3 mmol) was added and the solu- = 7.8 Hz, 3J = 7.2 Hz), 7.41 (dd, 1H, 3J = 7.2 Hz, 3J = 7.8 Hz), tion was left on vigorous stirring for 3 days at room tempera- 7.82(dd,1H,3J=7.8Hz),7.93(d,2H,3J=8.7Hz),7.95(d,1H, ture.Thereafter,thesolventwasconcentratedandthereaction 5190 | Org.Biomol.Chem.,2015,13,5182–5194 Thisjournalis©TheRoyalSocietyofChemistry2015 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Organic&BiomolecularChemistry Paper residuewassubjectedtocolumnchromatographyonsilicagel 8.1Hz),7.80(dd,1H,4J=0.6Hz,3J=7.8Hz),7.90(d,2H,3J= (n-hexane–AcOEt 1:1) to afford 11. Yield: 45% (1.518 g, 9.0 Hz), 7.94 (d, 1H, 3J = 8.1 Hz); 13C NMR (CDCl , 75 MHz): 3 3.62 mmol); R = 0.54 (CHCl –MeOH 99:1); 1H NMR (CDCl , δ 13.9, 25.9, 28.5, 31.2, 31.4, 38.8, 47.4, 49.8, 51.8, 60.6, 67.8, f 3 3 300 MHz): δ 1.67 (m, 2H), 1.89 (m, 2H), 3.04 (s, 3H), 3.41 (m, 70.5, 111.2 (2C), 120.8, 121.1, 121.9, 123.9, 125.7, 128.6 (2C), 4H),3.58(s,4H),6.68(d,2H,3J=9.0Hz),7.23(dd,1H,3J=7.2 134.1, 150.8, 154.0, 168.5, 171.5 (CvO); ESI/MS C H N O S 26 35 3 3 2 Hz, 3J = 7.8 Hz), 7.37 (d, 1H, 3J = 7.2 Hz, 3J = 8.1 Hz), 7.77 (d, (501.2)m/z502.4[M+H]+. 1H, 3J = 7.8 Hz), 7.88 (d, 2H, 3J = 9.0 Hz), 7.92 (d, 1H, 3J = 8.1 Ethyl 2-[4′-((6″-methyl)benzo[d]thiazol-2-yl)phenyl]-5-oxa-10- Hz); 13C NMR (CDCl , 75 MHz): δ 28.2, 29.6, 33.7, 39.2, 52.2, thia-2,13-diazapentadecan-15-oate (16). Compound 16 was 3 68.2, 70.4, 111.6 (2C), 121.4, 121.9, 124.5, 126.3, 129.2, 131.9 synthesized and purified as described above for 9, starting (2C),151.4,164.2. fromcompound14(700mg,1.63mmol).Yield:36%(303mg, 6-Methyl-(2-(N-methyl-N-(2′-(4′-bromobutoxy)ethyl)-4′-amino- 0.59 mmol); R = 0.41 (CHCl –MeOH 97:3); 1H NMR (CDCl , f 3 3 phenyl))-benzothiazole (12). Compound 12 was synthesized 300MHz):δ1.23(t,3H,J=7.2Hz),1.60(m,4H),2.42(s,3H), and purified as described above for 11, starting from com- 2.49 (m, 2H), 2.61 (t, 2H, J = 6.3 Hz), 2.76 (t, 2H, J = 6.3 Hz), pound 4 (1.0 g, 3.35 mmol). Yield: 40% (581 mg, 1.34 mmol); 3.02(s,3H),3.39(m,4H),3.55(s,4H),4.14(q,2H,J=7.2Hz), R = 0.64 (n-hexane–AcOEt 1:1); 1H NMR (CDCl , 300 MHz): 6.70(d,2H,3J=9.0Hz),7.20(d,1H,3J=8.4Hz),7.58(s,1H), f 3 δ1.64(m,2H),1.86(m,2H),2.42(s,3H),3.00(s,3H),3.37(m, 7.82 (d, 1H, 3J = 8.1 Hz), 7.88 (d, 2H, 3J = 9.0 Hz); 13C NMR 4H), 3.53 (s, 4H), 6.69 (d, 2H, 3J = 8.7 Hz), 7.2 (d, 1H, 3J = (CDCl , 300 MHz): δ 13.2, 20.5, 25.2, 27.8, 30.2, 30.7, 38.1, 3 8.2Hz),7.57(s,1H),7.82(d,1H,3J=8.2Hz),7.87(d,2H,3J= 46.9,48.8,51.2,60.3,67.2,69.8,110.6(2C),120.2,120.5,120.8, 8.7 Hz); 13C NMR (CDCl , 75 MHz): δ 21.4, 28.1, 29.5, 33.7, 126.5, 127.8 (2C), 133.2, 133.7, 150.0, 151.5, 166.7; ESI/MS 3 39.0, 52.0, 70.2, 111.4 (2C), 121.1, 121.3, 121.6, 127.3, 128.7 C H N O S (515.2)m/z516.3[M+H]+. 27 37 3 3 2 (2C), 134.1, 134.5, 150.8, 152.3, 167.6; ESI/MS C H BrN OS 2-[(2-((2″-((4″-(Benzo[d]thiazol-2-yl)phenyl)(methyl)amino)- 21 25 2 (433)m/z434.4[M+H]+. ethyl)thio]ethyl)amino) acetic acid (L1). A solution of 9 2-[N-Methyl-N-(2′-(4″-(2′′′-aminoethylthio)butoxy)ethyl)-4′-amino- (148 mg, 0.35 mmol) in THF (10 mL) and water (15 mL) was phenyl]-benzothiazole(13). Compound 13 was synthesized and refluxed with NaOH (136 mg, 3.40 mmol) for 17 h. After purified as described above for 7, starting from compound 11 cooling down, the reaction mixture was neutralized with 1 M (1.2g,2.86mmol).Yield:46%(547mg,1.32mmol);R =0.42 HCl, followed by extraction with CHCl (2 × 25 mL). The f 3 (CHCl –MeO 4:1); 1H NMR (CD OD, 300 MHz): δ 1.62 (m, organic phase was dried over Na SO , filtered and the filtrate 3 3 2 4 4H),2.51(m,2H),2.63(t,2H,J=6.6Hz),2.9(t,2H,J=6.6Hz), wasconcentrated.Recrystallizationoftheprecipitateinmetha- 3.08(s,3H),3.46(m,2H),3.64(s,4H),6.73(d,2H,3J=9.0Hz), nol afforded L1 as a white solid. Yield: 58% (80 mg, 7.28(dd,1H,3J=7.5Hz,3J=7.8Hz),7.41(dd,1H,3J=7.9Hz, 0.20 mmol); R = 0.29 (CHCl –MeOH 3:1); 1H NMR (DMSO, f 3 3J = 7.5 Hz), 7.82 (d,1H, 3J = 7.9 Hz), 7.92 (d, 2H, 3J = 9.0 Hz), 300MHz):δ2.80(m,4H),2.98(t,2H,J=8.1Hz),3.03(s,3H), 7.96 (d, 1H, 3J = 7.8 Hz); 13C NMR (CD OD, 75 MHz): δ 27.3, 3.21 (s, 2H), 3.62 (t, 2H, J = 8.1 Hz), 6.84 (d, 2H, 3J = 9.0 Hz), 3 29.8, 32.2,39.3, 40.5, 52.9,69.5, 71.7,112.9 (2C),122.6, 122.6, 7.39 (ddd, 1H, 4J = 1.2 Hz, 3J= 7.8 Hz, 3J= 7.5 Hz), 7.51 (ddd, 122.7,125.7,127.4,129.9(2C),135.3,153.2,155.1,171.1. 1H, 4J = 1.2 Hz, 3J = 7.5 Hz, 3J = 8.1 Hz), 7.89 (d, 2H, 3J = 9.0 6-Methyl-2-[N-methyl-N-(2′-(4″-(2′′′-aminoethylthio)butoxy)- Hz),7.92(d,1H,3J=8.1Hz),8.04(d,1H,3J=7.8Hz);13CNMR ethyl)-4′-aminophenyl]-benzothiazole (14). Compound 14 (DMSO, 75 MHz): δ 25.7, 46.4, 49.6, 51.3, 56.1, 111.8 (2C), wassynthesizedandpurifiedasdescribedabovefor7,starting 120.3, 122.0, 124.5, 126.4, 128.7 (2C), 133.8, 150.7, 153.9, fromcompound12(900mg,2.08mmol).Yield:86%(767mg, 167.8;ESI/MSC H N O S (401.55)m/z402.2[M+H]+,424.2 20 23 3 2 2 1.79 mmol); R = 0.58 (CHCl –MeOH 3:1); 1H NMR (CDCl , [M + Na]+; Anal. calcd for C H N O S ·1.4H O: C 56.38, H f 3 3 20 23 3 2 2 2 300 MHz): δ 1.56 (m, 4H), 2.39 (s, 3H), 2.44 (m, 2H), 2.59 (t, 6.09,N9.86;foundC56.54,H6.15,N9.61. 2H, J = 6.4 Hz), 2.86 (t, 2H, J = 6.4 Hz), 2.97 (s, 3H), 3.35 (m, 2-[2′-(2″-((4′′′-(6″″-Methylbenzo[d]thiazol-2-yl)phenyl)(methyl)- 2H),3.51(s,4H),3.76(s,2H),6.67(d,2H,3J=8.8Hz),7.17(d, amino)ethylthio)ethylamino] acetic acid (L2). L2 was syn- 1H,3J=8.4Hz),7.54(s,1H),7.80(d,1H,3J=8.4Hz),7.85(d, thesizedand purifiedasdescribedabovefor L1,startingfrom 2H,3J=8.8 Hz);13CNMR(CDCl , 75MHz):δ21.3,26.1,28.6, compound 10 (30 mg, 0.067 mmol). Yield: 65% (18 mg, 3 31.4, 34.0,38.8, 40.3, 52.0,68.0, 70.6,111.4 (2C),121.0, 121.2, 0.04 mmol); 1H NMR (CD OD, 300 MHz): δ 2.46 (s, 3H), 3.09 3 121.5, 127.3, 128.6 (2C), 134.0; 134.4, 150.8, 152.3, 167.5; ESI/ (s, 2H), 2.85 (m, 4H), 3.42 (s, 3H), 3.69 (m, 4H), 6.84 (d, 2H, MSC H N OS (429.2)m/z430.3[M+H]+. 3J=9.0Hz),7.28(d,1H,3J=8.4Hz),7.70(s,1H),7.76(d,1H, 23 31 3 2 Ethyl 2-[4′-(benzo[d]thiazol-2-yl)phenyl]-5-oxa-10-thia-2,13- 3J = 8.4 Hz), 7.87 (d, 2H, 3J = 9.3 Hz); 13C NMR (CD OD, 3 diazapentadecan-15-oate (15). Compound 15 was synthesized 75 MHz): δ 22.5, 25.3, 28.3, 29.2, 37.8, 67.7, 111.7 (2C), 121.0, andpurifiedasdescribedabovefor9,startingfromcompound 121.2, 127.7, 128.7 (2C), 134.9; ESI/MS C H N O S (415.14) 21 25 3 2 2 13(500mg,1.20mmol).Yield:66%(397mg,0.79mmol);R = m/z 416.3 [M + H]+, 438.3 [M + Na]+; Anal. calcd for f 0.53 (CHCl –MeOH 95:5); 1H NMR (CDCl , 300 MHz): δ 1.23 C H N O S ·1.6H O:C56.75,H6.12,N 9.46;foundC55.98, 3 3 21 25 3 2 2 2 (t, 3H, J = 7.2 Hz), 1.61 (m, 4H), 2.50 (m, 2H), 2.62 (t, 2H, J = H6.48,N9.27. 6.2Hz),2.77(t,2H,J=6.2Hz),3.04(s,3H),3.40(m,4H),3.57 [4-(Benzo[d]thiazol-2-yl)phenyl]-5-oxa-10-thia-2,13-diazapen- (s, 4H), 4.15 (q, 2H, J = 7.2 Hz), 6.72 (d, 2H, 3J = 9.0 Hz), 7.26 tadecan-15-oic acid (L3). L3 was synthesized and purified as (dd,1H,3J=7.2Hz,3J=7.8Hz),7.40(dd,1H,3J=7.2Hz,3J= described above for L1, starting from compound 15 (350 mg, Thisjournalis©TheRoyalSocietyofChemistry2015 Org.Biomol.Chem.,2015,13,5182–5194 | 5191 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Paper Organic&BiomolecularChemistry 0.70 mmol). Yield: 52% (172 mg, 0.36 mmol); 1H NMR C H N O S Re (685.07) (m/z) 686.3 [M + H]+ (100); Anal. 24 24 3 5 2 (CD OD,300MHz):δ1.63(m,4H),2.55(t,2H,J=7.2Hz),2.75 calcd for C H N O S Re: C 42.09, H 3.53, N 6.14; found C 3 24 24 3 5 2 (t, 2H, J = 7.2 Hz), 3.08 (s, 3H), 3.14 (t, 2H, J = 6.9 Hz), 3.47 42.25,H3.75,N6.09. (m, 4H), 3.64 (s, 4H), 6.86 (d, 2H, 3J = 7.2 Hz), 7.34 (ddd, 1H, Re3 was obtained in 76% yield (78 mg, 0.11 mmol); 1H 3J=7.8, 7.5Hz, 4J=1.2,0.6 Hz),7.46(td, 1H,3J= 7.8;7.5 Hz, NMR (DMF-d , 300 MHz, T = 20 °C): δ 1.71 (m, 4H), 3.08 (s, 7 4J = 1.2; 0.6 Hz), 7.89 (m, 4H); 13C NMR (CD OD, 75 MHz): 4H),3.50(s,3H),3.65(m,5H),6.90(d,2H,J=8.4Hz),7.39(m, 3 δ 27.1, 28.6, 29.8, 32.1, 39.3, 52.9, 66.9, 69.5, 71.7, 112.9 (2C), 1H), 7.50 (m, 1H), 7.95 (m, 3H); 13C NMR (DMF-d , 300 MHz, 7 121.6, 122.6, 122.7, 125.7, 127.4, 129.9 (2C), 135.3, 153.2, T = 20 °C): δ 25.01, 28.32, 37.10, 38.54, 51.53, 53.74, 55.76, 155.1, 171.1 (CvO); ESI/MS C H N O S (473.18) m/z 472.2 68.13, 70.10, 111.90, 120.18, 121.82, 121.97, 124.78, 126.56, 24 31 3 3 2 [M − H]+; Anal. calcd for C H N O S ·2.2H O: C 56.16, H 128.79, 134.10, 151.86, 154.14, 168.93, 181.51, 192.31, 196.12, 24 31 3 3 2 2 6.95,N8.19;foundC56.63,H7.09,N8.02. 197.75; IR (KBr) ν: 4008, 2923, 2027, 1896, 1748, 1606, 1559, 2-[4′-((6″-Methyl)benzo[d]thiazol-2-yl)phenyl]-5-oxa-10-thia- 1541,1508,1487,1456,1384,1350,1261,1183,1101,816,691, 2,13-diazapentadecan-15-oic acid (L4). L4 was synthesized 651, 567, 516 cm −3; ESI/MS (+) C H N O S Re (743.0) (m/z) 27 30 3 6 2 and purified as described above for L1, starting from com- 744.4 [M + H]+, 766.4 [M + Na]+; Anal. calcd for pound 16 (250 mg, 0.48 mmol). Yield: 65% (154 mg, C H N O S Re: C 43.65, H 4.07, N 5.66; found C 43.48, H 27 30 3 5 2 0.32 mmol); 1HNMR (CD OD,300 MHz): δ1.60 (m,4H), 2.52 4.24,N5.54. 3 (t,2H,J=7.2Hz),2.74(t,2H,J=7.2Hz),3.06(s,2H),3.12(t, Re4 was obtained in 81% yield (86 mg, 0.11 mmol); 1H 2H, J = 7.2 Hz), 3.45 (s, 3H), 3.62 (s, 4H), 6.80 (d, 2H, 3J = 9.0 NMR(DMF-d ,400MHz,T=20°C):1.74(s, 2H),1.83(s,2H), 7 Hz),7.24(d,1H,3J=8.4Hz),7.68(s,1H),7.72(d,1H,3J=8.4 2.47 (s,3H), 3.12 (s,5H), 3.41 (q,2H,J=3.2 Hz), 3.52(s, 2H), Hz), 7.82 (d, 2H, 3J = 9.0 Hz); 13C NMR (CD OD, 75 MHz): 3.68(s,5H),6.93(d,2H,J=7.4Hz),7.33(s,1H),7.85(m,2H), 3 δ 27.5, 28.9, 30.1, 32.5, 39.7, 53.4, 67.3, 69.9, 72.1, 112 (2C), 7.94 (d, 2H, J = 7.4 Hz); 13C NMR (DMF-d , 400 MHz, T = 7 123.0, 123.2, 129.8, 129.0 (2C), 135.2.2, 153.4, 155.3, 171.4 20 °C): δ 15.94, 21.68. 26.28, 35.02, 35.23, 37.86, 39.30, 39.59, (CvO);ESI/MSC H N O S (487.2)m/z488.4[M+H]+,510.4 52.69,54.80,56.08,66.38,69.19,71.13,112.90,121.80,122.65, 25 33 3 3 2 [M + Na]+. Anal. calcd for C H N O S ·1.5H O: C 58.34, H 128.79, 129.61, 135.52, 152.61, 153.55, 168.26, 171.92, 177.35, 25 33 3 3 2 2 6.75,N8.17;foundC57.24,H6.88,N8.24. 180.44, 194.18, 197.49; IR (KBr) ν: 3490, 2920, 2028, 1895, 1653,1606,1558,1506,1457,1384,1261,1100,921,688cm −3; Generalprocedureforthesynthesisoftherheniumcomplexes ESI/MS (+) C H N O S Re (757.0) (m/z) 758.6 [M + H]+ (100), (Re1–Re4) 780.5[M+N 2 a 8 ]+ 3 (8 2 0) 3 ;A 6 na 2 l.calcdforC H N O S Re:C44.43, 28 32 3 6 2 fac-[Re(H O) (CO) ]Br was reacted with equimolar amounts of H4.26,N5.55;foundC43.97,H4.6,N5.43. 2 3 3 L1–L4 in refluxing methanol for 16 h. Thereafter, the solvent was removed under vacuum and the desired complexes were Generalprocedureforthesynthesisof99mTccomplexes purified by successive washing with water, diethyl ether and n-hexane. The obtained yellow solids were further recrystal- Ina nitrogen-purgedglassvial, 120 μLof a1.1 ×10 −3 Msolu- lized from methanol to afford pure samples of Re1–Re4, as tion of L1–L4 in propanediol-1,3 was added to 1.2 mL of a checkedbyHPLCanalysis. solution of the organometallic precursor fac-[99mTc- Re1 was obtained in 62% yield (54 mg, 0.08 mmol); 1H (H O) (CO) ]+ (1–2mCi) in salineat pH 7.4. The reactionmix- 2 3 3 NMR(DMF-d ,300MHz,T=20°C):2.64(m,2H),3.20(s,4H), tures were then heated to 100 °C for 30 min, cooled to room 7 3.38 (m, 2H), 3.71 (m, 2H), 3.96 (m, 2H), 7.05 (d, 2H, J = 9.0 temperature and analyzed by RP-HPLC, using the method Hz), 7.40 (m, 1H), 7.52 (m, 1H), 7.98 (m, 3H), 8.10 (d, 1H, J = describedabove.Followingthisprocedure,all99mTccomplexes 7.8Hz);13CNMR(DMF-d ,300MHz,T=20°C):δ39.1,51.50, (Tc1–Tc4) were synthesized in radiochemical yields >95% 7 53.32, 54.40, 55.43, 56.40, 113.09, 113.47, 122.93, 123.27, withouttheneedforfurtherpurification.Theirchemicaliden- 125.64, 127.40, 129.95, 135.60, 152.30, 155.71, 169.13, 180.16, tity was ascertained by HPLC comparison with the Re conge- 193.72,197.17; IR(KBr) ν: 3410, 2922, 1894,1699, 1606, 1486, ners(seeTable1). 1455, 1384, 1261, 1191, 1104, 817 cm −3; ESI/MS (+) C H N O S Re(671.0)(m/z):672.2[M+H]+(100);Anal.calcd 23 22 3 5 2 Determinationofpartitioncoefficients forC H N O S Re:C41.18,H3.31,N6.27;foundC41.07,H 23 22 3 5 2 3.47,N6.19. The logD values of complexes Tc1–Tc2 were determined by o/w Re2 was obtained in 57% yield (32 mg, 0.05 mmol); 1H the “shake flask” method.28 A mixture of octanol (1 mL) and NMR (DMF-d , 300 MHz, T = 20 °C): δ 2.47 (m, 6H), 3.19 (s, 0.1 M PBS pH = 7.4 (1 mL) was stirred vigorously, followed by 7 9H), 6.99 (s, 2H), 7.32 (s, 1H), 7.83 (m, 2H), 7.95 (m, 2H); 13C the addition of 25 μL of the aqueous solutions of each NMR (DMF-d , 300 MHz, T = 20 °C): δ 21.18, 32.33, 38.51, complex. The mixtures were vortexed and centrifuged 7 38.66, 47.99, 50.39, 50.83, 52.65, 54.30, 54.95, 55.66, 55.96, (3000 rpm, 10min, RT) to allow phaseseparation. Aliquots of 112.36, 112.72, 121.41, 122.09, 128.35, 129.17, 134.90, 135.24, 25μLoftheoctanolandPBSphaseswerecountedinagamma 151.32,152.88,167.69,181.17,192.76,196.56,198.16;IR(KBr) counter. The partition coefficient (P ) was calculated by o/w ν: 3395, 2924, 2028, 1893, 1700, 1604, 1509, 1483, 1437, 1384, dividing the number of counts of the octanol phase by those 1261, 1193, 1108, 817, 759, 698, 650 cm −3; ESI/MS (+) fromthePBSphase,andtheresultsareexpressedaslogD . o/w 5192 | Org.Biomol.Chem.,2015,13,5182–5194 Thisjournalis©TheRoyalSocietyofChemistry2015 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Organic&BiomolecularChemistry Paper Cellculture Cellularuptake The human MCF7 breast and PC3 prostate cells (American Cellular uptake assays of the 99mTc complexes (Tc1–Tc4) were Type Culture Collection, ATCC) were grown respectively in performed using PC-3 and MCF-7 cells seeded at a density of DMEM and RPMI media, supplemented with 10% (v/v) fetal 4 × 104 cells in 500 μL medium per well in 24-well plates and bovine serum and 1% penicillin–streptomycin (all from Invi- allowedtoattachovernight.Afterthatperiod,themediumwas trogen). Cells were cultured at 37 °C in a 5% CO incubator removedandcellsweretreatedwithfreshmediumcontaining 2 (Heraus, Germany) under a humidified atmosphere, with the approximately 25 kBq mL −1 of each 99mTc complex and incu- mediumchangedeveryotherday. bated undera humidified 5% CO atmosphere, at 37 °C fora 2 period of 30 min to 18 h. Cells were washed twice with cold PBS,lysedwith0.1MNaOHandthecellularextractswereana- Cytotoxicityassays lyzed for radioactivity. Each experiment was performed in Thecellviabilitywasevaluatedbyusingacolorimetricmethod duplicate with each point determined in at least four repli- based on the tetrazolium salt MTT ([3-(4,5-dimethylthiazol-2- cates. Cellular uptake data were expressed as an average plus yl)-2,5-diphenyltetrazolium bromide]), which is reduced by thestandarddeviationof%oftotalpermillionofcells. livingcellstoyieldpurpleformazancrystals.Cellswereseeded in 96-well plates at a density of 1–1.5 × 104 cells per well in Biodistributionstudies 200 μL of culture medium and incubated overnight. After All animal experiments were performed in compliance with careful removal of the medium, 200 μL of a dilution series of Portuguese regulations for animal treatment. The animals the compounds in fresh medium were added and incubation were housed in a temperature- and humidity-controlled room was performed at 37 °C/5% CO 2 for 72 h. The percentage of with a 12 h light/12 h dark schedule. Biodistribution of com- DMSO in cell culture medium did not exceed 1%. At the end plexes Tc1–Tc4 was evaluated in CD-1 mice (randomly bred, of the incubation period, the compounds were removed and obtained from IFFA, CREDO, Spain) weighing approximately the cells were incubated with 200 μL of MTT solution (500 µg 20–25 g. Animals were intravenously injected in the tail vein ml −1).After3h,themediumwasremovedandthepurplefor- withthetestcomplex(1.8–7.8MBq)dilutedin100μLofNaCl mazan crystals were dissolved in 200 μL of DMSO byshaking. 0.9%.Miceweresacrificedbycervicaldislocationat2minand The cell viability was evaluated by the measurement of the 1hafterinjection.Theadministereddoseandtheradioactivity absorbanceat570nmusingaplatespectrophotometer(Power in the sacrificed animals were measured using a dose calibra- WaveXs,Bio-Tek).Thecellviabilitywascalculatedbytheratio tor (Curiemeter IGC-3, Aloka, Tokyo, Japan). The difference between the absorbance of each well and that of the control between the radioactivity in the injected and sacrificed wells (cells treated with medium containing 1% DMSO). Each animals was assumed to be due to excretion. The tissues of experiment was repeated at least three times and each point interest were dissected, rinsed to remove excess blood, was determined in at least six replicates. Data were analyzed weighed, and their radioactivity was measured using a withGraphPadPrismsoftware. γ-counter (LB2111, Berthold, Germany). The uptake in the tissues was calculated and expressed as a percentage of the injectedradioactivitydosepergramoftissue. Fluorescencespectraandfluorescencemicroscopyanalysis TheexcitationandemissionwavelengthsofL3andRe3(25μM Invivostabilitystudies inPBS)wererecordedusingaplatereader(TecanInfinite200, The in vivo stability of Tc1–Tc4 was evaluated by urine and Männedorf, Switzerland). The maximum excitation and emis- blood serum HPLC analysis, using the elution conditions sionwavelengthsweredeterminedaccordingtothemaximum abovedescribedfortheanalysisofthese99mTccomplexes.The fluorescenceintensitylevel. urine was collected at the sacrifice time and centrifuged for MCF7 and PC3 cells, cultured in 35 mm imaging dishes 5minat1500gbeforeRP-HPLCanalysis.Bloodcollectedfrom (Ibidi),weretreatedwithavehicle(DMSO)orwiththeligands micewasalsocentrifugedfor10minat1000gat4°C,andthe L3 or the complex Re3 (100 μM), for 24 h. Cells washed with supernatant (serum) was collected. Aliquots of 100 μL of PBS were fixed and permeabilized in 100% ice-cold methanol serumweretreatedwith200μLofcoldethanolforproteinpre- (−20 °C, 5 min). Cells, washed with PBS 3 times, were incu- cipitation.Sampleswerecentrifugedat1500gforanadditional batedwith20μgml −1propidiumiodide(nuclearstaining)for 10min,at4°C.Theremainingsupernatantwasseparatedand 4minatRT.CellswerewashedwithPBS3timesandwerekept injected through a RP-HPLC column (Nucleosil 100-10, 250 × at 4 °C until analysis. Samples were imaged on a widefield 3mm)foranalysis. fluorescence microscope Zeiss Axiovert 200M (Carl Zeiss MicroImaging) using a 63× Plan-Apochromat using the Red (λ = 540–552 nm, λ = 575–640 nm) and Blue (λ = Acknowledgements ex em ex 359–371 nm, λ > 397 nm) filter sets. Images were acquired em using a cooled CCD camera (Roper Scientific Coolsnap HQ ThisworkwasfundedbytheFundaçãoparaaCiênciaeTecno- CCD) using Zeiss Metamorph software. All images were pro- logia (FCT), Portugal (EXCL/QEQ-MED/0233/2012). The cessedusingImageJ–ImageProcessingandAnalysisinJava.29 researchwascarriedoutwithintheframeworkoftheEuropean Thisjournalis©TheRoyalSocietyofChemistry2015 Org.Biomol.Chem.,2015,13,5182–5194 | 5193 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online Paper Organic&BiomolecularChemistry Cooperation COSTAction CM1105. FCT is also acknowledged 11 C. G. Mortimer, G. Wells, J.-P. Crochard, E. L. Stone, for “Ciência 2008” program (GRM), FCT Investigator Grant T.D.Bradshaw,M.F.G.StevensandA.D.Westwell,J.Med. (FM) and grants SFRH/BPD/80758/2011 (EP), SFRH/BD/47308/ Chem.,2006,49,179. 2008(FS)andSFRH/BPD/64702/2009(HVM).TFOissupported 12 A. I. Loaiza-Pérez, V. Trapani, C. Hose, S. S. Singh, by the DFG Center for Nanoscale Microscopy and Molecular J. B. Trepel, M. F. G. Stevens, T. D. Bradshaw and Physiology of the Brain (CNMPB). The authors thank Dr E.A.Sausville,Mol.Pharmacol.,2002,61,13. Joaquim Marçalo and Dr Célia Fernandes for Mass Spec- 13 V. Trapani, V. Patel, C.-O. Leong, H. P. Ciolino, troscopy analysis, which was carried out on a QITMS instru- G. C. Yeh, C. Hose, J. B. Trepel, M. F. G. Stevens, ment, acquired with the support of the Programa Nacional de E. A. Sausville and A. I. Loaiza-Pérez, Br. J. Cancer, 2003, Reequipamento Científico (Contract REDE/1503/REM/2005- 88, 599–605. ITN) of FCT and is part of RNEM-Rede Nacional de Espectro- 14 E.W.PriceandC.Orvig,Chem.Soc.Rev.,2014,43,260. metriadeMassa. 15 C.-T. Yang and K.-H. Chuang, Med. Chem. Commun., 2012, 3,552–565. 16 S. Tzanopoulou, I. C. Pirmettis, G. Patsis, M. Paravatou- Notes and references Petsotas, E. Livaniou,M. Papadopoulosand M. Pelecanou, J.Med.Chem.,2006,49,5408. 1 I. Hutchinson, M.-S. Chua, H. L. Browne, V. Trapani, 17 S. Tzanopoulou, M. Sagnou, M. Paravatou-Petsotas, T.D.Bradshaw,A.D.WestwellandM.F.G.Stevens,J.Med. E. Gourni, G. Loudos, S. Xanthopoulos, D. Lafkas, Chem.,2001,44,1446. H. Kiaris, A. Varvarigou, I. C. Pirmettis, M. Papadopoulos 2 X.Peng,G.Xie,Z.Wang,H.Lin,T.Zhou,P.Xiang,Y.Jiang, andM.Pelecanou,J.Med.Chem.,2010,53,4633. S.Yang,Y.Wei,L.YuandY.Zhao,CellDeathDis.,2014,5, 18 H. K. Kim, M. K. Kang, K. H. Jung, S. H. Kang, Y. H. Kim, e1143. J.C.Jung,G.H.Lee,Y.ChangandT.J.Kim,J.Med.Chem., 3 G. R. Morais, A. Paulo and I. Santos, Eur. J. Org. Chem., 2013,56,8104. 2012,1279. 19 R. Alberto, R. Schibli, A. Egli and A. P. Schubiger, J. Am. 4 W.E.Klunk,H.Engler,A.Nordberg,Y.Wang,G.Blomqvist, Chem.Soc.,1998,120,7987. D. P. Holt, M. Bergstrom, I. Savitcheva, G. F. Huang, 20 R. Alberto, K. Ortner, N. Wheatley, R. Schibli and S. Estrada, B. Ausen, M. L. Debnath, J. Barletta, J. C. Price, A.P.Schubiger,J.Am.Chem.Soc.,2001,123,3135. J. Sandell, B. J. Lopresti, A. Wall, P. Koivisto, G. Antoni, 21 G.R.Morais,A.PauloandI.Santos,Organometallics,2012, C.A.MathisandB.Langstrom,Ann.Neurol.,2004,55,306. 31,5693. 5 H. Engler, A. Forsberg, O. Almkvist, G. Blomquist, 22 C. Moura, L. Gano, I. C. Santos, I. Santos and A. Paulo, E. Larsson, I. Savitcheva, A. Wall, A. Ringheim, Eur.J.Inorg.Chem.,2011,5405. B.LangstromandA.Nordberg,Brain,2006,129,2856. 23 C. Moura, L. Gano, F. Mendes, P. D. Raposinho, 6 S. M. Landau, B. A. Thomas, L. Thurfjell, M. Schmidt, A. M. Abrantes, M. F. Botelho, I. Santos and A. Paulo, R. Margolin, M. Mintun, M. Pontecorvo, S. L. Baker and Eur.J.Med.Chem.,2012,50,350. W.Jagust,Eur.J.Nucl.Med.,2014,41,1398. 24 I. Fichtner, A. Monks, C. Hose, M. F. G. Stevens and 7 S. Iikuni, M. Ono, H. Watanabe, K. Matsumura, T.D.Bradshaw,BreastCancerRes.Treat.,2004,87,97. M. Yoshimura, N. Harada, H. Kimura, M. Nakayama and 25 C.Fernandes,L.Maria,L.Gano,I.C.Santos,I.Santosand H.Saji,Mol.Pharmaceutics,2014,11,1132. A.Paulo,J.Organomet.Chem.,2014,760,138. 8 J.Jia,M.Cui,J.Dai,X.Wang,Y.-S.Ding,H.JiaandB.Liu, 26 M.Q.Zheng,D.Z.Yin,J.P.Qiao,L.ZhangandY.X.Wang, Med.Chem.Commun.,2014,5,153. J.FluorineChem.,2008,129,210. 9 A. F. Martins, J. F. Morfin, A. Kubickova, V. Kubicek, 27 N. Lazarova, S. James, J. Babich and J. Zubieta, Inorg. F. Buron, F. Suzenet, M. Salerno, A. N. Lazar, Chem.Commun.,2004,7,1023. C. Duyckaerts, N. Arlicot, D. Guilloteau, C. F. G. 28 D. E. Troutner, W. A. Volkert, T. J. Hoffman and C.GeraldesandE.Toth,ACSMed.Chem.Lett.,2013,4,436. R.A.Holmes,Int.J.Appl.Radiat.Isot.,1984,35,467. 10 T.D.BradshawandA.D.Westwell,Curr.Med.Chem.,2004, 29 M.D.Abramoff,P.J.MagelhaesandS.J.Ram,Biophotonics 11,1241. Int.,2004,11. 5194 | Org.Biomol.Chem.,2015,13,5182–5194 Thisjournalis©TheRoyalSocietyofChemistry2015 .25:04:91 5102/40/92 no MAHGNIMRIB TA AMABALA FO YTISREVINU yb dedaolnwoD .5102 hcraM 62 no dehsilbuP View Article Online