Identification of novel potent and non-toxic anticancer, anti-angiogenic and antimetastatic rhenium complexes against colorectal carcinoma.

EuropeanJournalofMedicinalChemistry204(2020)112583 ContentslistsavailableatScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech Research paper Identification of novel potent and non-toxic anticancer, anti- angiogenic and antimetastatic rhenium complexes against colorectal carcinoma Joachim Delasoie a, Aleksandar Pavic b,* , Noe (cid:1) mie Voutiera, Sandra Vojnovic b, Aurelien Crochet a, Jasmina Nikodinovic-Runic b,** , Fabio Zobi a,*** aDepartmentofChemistry,FribourgUniversity,CheminDuMus(cid:1)ee9,1700,Fribourg,Switzerland bInstituteofMolecularGeneticsandGeneticEngineering,UniversityofBelgrade,VojvodeStepe444a,11042152,Belgrade,RepublicofSerbia a r t i c l e i n f o a b s t r a c t Articlehistory: Combinationtherapytargetingbothtumorgrowthandvascularizationisconsideredtobeacornerstone Received21April2020 forcolorectalcarcinomas(CRC)treatment.However,themajorobstaclesofmostclinicalanticancerdrugs Receivedinrevisedform aretheirweakselectiveactivitytowardscancercellsandinherentinnerorganstoxicity,accompanied 6June2020 with fast drug resistance development. In oureffort to discover novel selective and non-toxic agents Accepted14June2020 effective against CRC, we designed, synthesized and characterized a series of rhenium(I) tricarbonyl- Availableonline12July2020 based complexes with increased lipophilicity. Two of these novel compounds were discovered to possess remarkable anticancer, anti-angiogenic and antimetastatic activity in vivo (zebrafish-human Keywords: HCT-116xenograftmodel),beingeffectiveatverylowdoses(1e3mM).Atdosesashighas250mMthe Colorectalcarcinoma Rhenium complexesdidnotprovoketoxicityissuesencounteredinclinicalanticancerdrugs(cardio-,hepato-,and Zebrafish myelotoxicity). In vivo assays showed that the two compounds exceed the anti-tumor and anti- Xenograft angiogenic activity of clinical drugs cisplatin and sunitinib malate, and display a large therapeutic Angiogenesis window. Antimetastatic ©2020ElsevierMassonSAS.Allrightsreserved. 1. Introduction deaths [1]. In current oncological practice, combination therapy based on the use of cytostatic and anti-angiogenic drugs is a The latest report released by the International Agency for cornerstone for CRC treatment since it simultaneously targets Research on Cancer indicated that cancer burdenworldwide has malignant cells and tumour vasculature [3]. However, a major risen to 18.1 million new cases and 9.6 million cancer deaths in problemresponsibleforthestillhighdeathincidenceinCRCisthe 2018, while the statistical forecasts provided by WHO estimate poorefficacyandlowselectivityofclinicallyusedanticancerdrugs. morethan 21 million newcancercases and13 million deaths by Therefore, together with efficient prevention and early detection 2030[1].Amongallcancertypes,colorectalcarcinomas(CRC)are policies,newdrugsandtreatmentsareurgentlyrequiredtocom- amongst the most aggressive cancers globally, being ranked as plementandmaintainexistingtherapies.Withtheaimofidenti- thirdintermsofincidenceandsecondintermsofmortality,and fying potentially new anticancer agents and to guide the togetherwithlungandbreastcancers,areresponsibleforonethird formulation of new composite drug delivery systems, we have ofthecancerincidenceandmortality[1,2].Thissolidmalignancy, recentlybegunaprogramcenteredontheuseoffrustulesderived characterizedbyhighmetastaticpotential,accountsinEuropeand fromunicellularmicroalgaeforthetargeteddeliveryofanticancer theAmericasaloneformorethan40%oftheglobalcancercasesand transitionmetaldrugstocancers,inparticular,ofthecolorectum [4,5]. Among inorganic and organometallic anticancer compounds, cisplatinandotherplatinum-baseddrugshave beenusedforde- * Correspondingauthor. ** Correspondingauthor. cadesinthetreatmentofavarietyofneoplasticdiseases.However, *** Correspondingauthor. theclinicalsuccessofthesedrugsislimitedbysignificantsideef- E-mail addresses: sasapavic@imgge.bg.ac.rs (A. Pavic), jasmina.nikodinovic@ fects,especiallyinherentnephrotoxicityandototoxicityoriginating imgge.bg.ac.rs(J.Nikodinovic-Runic),fabio.zobi@unifr.ch(F.Zobi). https://doi.org/10.1016/j.ejmech.2020.112583 0223-5234/©2020ElsevierMassonSAS.Allrightsreserved. 2 J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 fromtheplatinumelement.Accordingly,theuseofRe(I)tricarbonyl ethanol.Compounds2e6werepreparedbynucleophilicsubstitu- complexes is gaining momentum due to the advantageous char- tionofthebromideatomofthea-diimineligandofcomplex1by acteristicsofthespecies(highstability,lowtoxicity,structuraldi- the corresponding amine. These complexes were prepared in versity, rich spectroscopic properties, different mechanisms of moderate(ca.40%)togood(>60%)yieldfollowingcrystallizationin action), making Re complexes potential anticancer therapeutics a dichloromethane:pentane mixture. Compounds 7 and 8 were whichmaybesuitabletoenterclinicalresearchanddevelopment. preparedrespectivelyinanaqueoussolutionfromthereactionof þ Compoundsofthefac-[Re(CO)3] core(facialisomeroftherhenium [Et4N]2fac-[Re(CO)3Br3]with0.5and1equivalentof thebpy-DPA tricarbonyl core) are robust, relatively straightforward to synthe- ligand which was in turn prepared according to a reported pro- sizeandcanbeproperlydesignedtotargetspecificcellularcom- cedure[52]. partments. Due to their luminescent properties, these molecules 1H NMR spectra (Fig. S1-S8) showed pure diamagnetic com- canoftenbetrackedintracellularlytocorrelatecellularandtissue pounds, according to the symmetry given by the facial-arranged distribution with their mode of action [6e10]. Furthermore, the CO’s and low-spin d6 nature of the metal ion. IR spectroscopy reported antiproliferative activity of these species on different analysis was in accordance with the typical tricarbonyl vibration cancercelllinesoftenequatesorexceedsthatofwell-established pattern. inorganicdrugs(e.g.cisplatin)[11]. Crystalssuitableforx-raydiffractionanalysiswereobtainedfor Themajorityofanticancerfac-[Re(CO)3] þ compoundstestedto fiveoftheeightnewspecies.Crystallographicdetailsarepresented dateisthatoffac-[Re(CO)3(a-diimine)L]ntypecomplexes,wherea- inTable1.Complexes1,4,3and8allcrystallizedinamonoclinic diimine are typically 2,20-bipyridine or phenantroline-based li- lattice and space groups C2/c, P21/n and P21/c (3 and 8) respec- gands and L is a monodentate ligand (most often a pyridine de- tively, whereas 7 was obtained in the triclinic space group P-1 rivative)orahalide(Cl,Br).However,examplesofantiproliferative (Fig.1). The five complexes all presenta distorted octahedral ge- þ fac-[Re(CO)3] complexesofotherbidentateligandtypes,including ometryaroundthecentralmetalionbutstructuralparametersare NeO [12,13], OeO [14], PeP [15] and SeeSe chelates [16e20], not significantly different from similar fac-[Re(CO)3] þ species N,N,N-tridentate chelates [21e26] or bridged di- or tri-nuclear (CCDCsearch). alkoxo/hydroxo are also known [27e29]. Additionally, but rarer, phototoxicRecomplexescapableofeithergeneratingreactiveox- 2.2. Relativelipophilicityofcomplexes ygen species (ROS) or liberating CO have also been identified [25,30e33]. The former type of complexes act as photodynamic The activity of drugs may often be correlated to their lip- therapy photosensitizers, the latter, of general formula fac- ophilicity as it is generally the case for the cytotoxicity of Re(I) [Re(CO)3(a-diimine)PR3] þ (PR3 ¼ phosphine type-ligands) have tricarbonylcomplexes.Thelipophilicityisdefinedbyoctanol-water been recently described by different authors [34e39]. Relatively partition coefficient (logP value) that can be determined by recentandcomprehensivereviewsbyGasseretal.[11],Policaretal. different experimental techniques [53,54]. In order to define the [40],Loetal.[41],Colleryetal.[42],Kühnetal.[43]andWilson relative lipophilicity of complexes 1e8, we decided to calculate etal. [44] havediscussed the subject in detail, and it is apparent theirmolecularpropertiesviatheALOGPS2.1andMolinspiration from these that the cytotoxicity of Re(I) tricarbonyl complexes is softwares [55,56]. Results for the latter are given in Table 2. The generally found to increase with lipophilicity [45e50] (most values calculated by Molinspiration software are similar to those probablyduetoanimprovedcellularuptake)althoughexceptions experimentallydeterminedfor relatedcomplexes[57].Both soft- doexist[51]. waresindicatethesamerelativelipophilicityof thespecies,with Withinthecontextabove,wethereforedecidedtoexplorethe thetrendbeingsimilartothatofthea-diimineligandonly.Because antiproliferativeefficacyofaseriesoffac-[Re(I)(CO)3] þ N-derivat- oftheirprimarycoordinationsphere,onlycomplexes1e6canbe ized N-([2,20-bipyridin]-6-ylmethyl)-complexes of increasing lip- directlycompared.Theirlipophilicityincreasesintheorder6<2< ophilicity against different cell lines, but with a focus on cells 1<3<5<4(4havingthelogPvalueof5.12,Table2).Overall,with derived from CRC. In this study we report the synthesis, charac- theexceptionoftheirmolecularweight,moleculesratewellinthe terization and in vitro evaluation of this series of rhenium(I) tri- drug-likenessassessment(Table2). carbonyl complexes as potential anticancer agents. In addition to that,wealsoreportthedetailedtoxicityprofileofthemoleculesin 2.3. Invitroanticanceractivityevaluation the zebrafish model (Danio rerio) as well as the anticancer/anti- metastatic efficacyand effective inhibition of angiogenesis in the The anticanceractivity of fac-[Re(CO)3] þ compounds has been zebrafish xenograft model of human CRC. We found that at least tested on several different cancer cell lines. The majority of the twonewmoleculespossessremarkableanticancer,anti-angiogenic studies were focused on antiproliferative effects on cervical and antimetastatic activity being effective at very low doses [30e33,47,51,57e61],ovarian[62,63],breast[12,20,25,64e66]and (1e3 mM) in vivo, and exceeding the potency of clinical drugs epithelial adenocarcinoma [24,49,67,68]_ENREF_50 cell lines cisplatin andsunitinib malate. The mostpotent compounds (vide showingIC50valuesaslowase.g.0.1(HeLa)[30],1(MCF-7)[15], infra,Scheme1,1and4)causenotoxicsideeffects(cardio-,hepato- 0.75(A549)[67],4(NB69andH4cells)mM[12].Toourknowledge, , and myelotoxicity) at concentrations as highas 250 mM, and all onlyafewrhenium(I)carbonylcomplexeshavebeentestedagainst xenografts receiving 3-days treatments survived and developed coloncancercelllines[16],[20,69e75](someofwhichareshown normally. in Fig. 2), the majority of them revealing weak or no anticancer þ activity. Only fac-[Re(CO)3] complexes of pentylcarbonato (Re-2 2. Resultsanddiscussion and Re-3 in Fig. 2) and mono- and multinuclear Re-4 complexes demonstratedpotentanticanceractivity.Theformerresultinginan 2.1. Synthesisandcharacterizationofcomplexes almostcompleteinhibitionofCCL-227coloncancercellsata4mM concentration [69], while the latter (Re-4) were active on DLD-1 Rheniumcomplexes1e8werepreparedaccordingtothesyn- colon carcinoma cells with IC50 values between 10 and 18 mM, theticprotocolillustratedinScheme1.Molecule1wasobtainedin butmainlywithalowselectivityindexwhencomparedtotheef- highyieldbytreatmentofthe[Et4N]2fac-[Re(CO)3Br3]saltwiththe fectonnormalcells[71]. 6-(bromomethyl)-2,20-bipyridineligandinamixtureofwaterand In order to address the anticancer potential and selective J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 3 Scheme1. Syntheticschemeforthepreparationofcomplexes1e8. activity towards carcinoma cells of newlysynthesized complexes toxicity towards healthy MRC-5 cells (Table 3) constrained it for 1e8, their antiproliferative effect was determined on a panel of further analysis. On the other side, complexes 1 and 4 showed cancer and normal cell lines (Table 3). As evident from Table 3, moderateselectivitybetweenhealthyandcancercelllineswithSi complexes 1e4 showed higher antiproliferative activity than of6.8and3.2,respectively,whiletheirtoxicityinvivowasmuch complexes5e8(allbut5bearingthe1-([2,20-bipyridin]-6-yl)-N,N- lower(Table3)makingthemsuitableforfurtherinvestigation.In þ bis(pyridin-2-ylmethyl)methanamine, bpy-DPA ligand), which comparisontootherfac-[Re(CO)3] complexestestedagainstcolon converselyhadweakornocytotoxiceffectoncancercelllinesand cancercelllines,1and4arecomparabletopentylcarbonatocom- lung fibroblasts. Complexes 1e4 exhibited the highest potency plexes[69]Re-2andRe-3(Fig.2)andshowgreaterantiproliferative against HCT-116 colorectal carcinoma cells and MIA PaCa-2 activitythantheotherknownrhenium(I)carbonylcomplexes. pancreatic carcinoma cells, with the IC50 values in the range of Fromabiologicalpointofview,tumorcellsresponsetovarious 5e10 mM (HCT-116) and 8e15 mM (MIA PaCa-2) range. Among drugsdependsontheuniquepropertiesofeachcellline,including them,3wasthemostactivecompoundonallthetestedcancercell various pathways and signals, as well as inherent and acquired lines except on HCT-116 cells; however, its equal or even higher mechanisms of drug resistance (target modifications, 4 J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 Table1 Crystallographicdetailsforcomplexes1,3,4,7and8. 1 3 4 7a 8 CCDC 1982188 1982189 1982190 1982191 1982192 Formula C14H9Br2N2O3Re C20H23BrN3O3Re C22H27BrN3O3Re C29.66H23.66Br2N5O6.67Re2 C26H27N6O9Re MW 599.25 619.52 647.57 1089.04 753.73 T[K] 250(2) 250(2) 250(2) 250(2) 250(2) Lattice monoclinic monoclinic monoclinic triclinic monoclinic Spacegroup C2/c P21/c P21/n P-1 P21/c Z 8 4 4 2 4 a[Å] 12.9334(8) 15.944(2) 15.9463(8) 8.4676(3) 12.6279(4) b[Å] 11.2986(6) 10.6655(9) 10.5244(3) 12.5978(5) 17.1561(4) c[Å] 22.2063(15) 13.2172(16) 16.1768(7) 17.7868(8) 13.2342(4) a[(cid:2)] 90 90 90 76.963(3) 90 b[(cid:2)] 92.263(5) 100.188(10) 114.598(3) 85.213(3) 101.189(2) g[(cid:2)] 90 90 90 72.830(3) 90 V[Å3] 3242.5(3) 2212.1(4) 2468.50(19) 1765.80(13) 2812.63(14) dcalcd[g/cm3] 2.455 1.860 1.742 2.048 1.780 R1,wR2 0.0405,0.0866 0.0624,0.1462 0.0349,0.0782 0.0511,0.1219 0.0181,0.0416 a aCH3OHmoleculewitha67%occupancyispresentintheunitcell. Fig.1. Crystalstructuresofcompounds1,3,4,7and8.Thermalellipsoidsareat30%probability.Hydrogenatomsareomittedforclarity;for7onebromideandonemethanol moleculeandfor8onenitrateandthreewatermoleculesarealsoomittedforclarity.Colourcode:C,grey,O,red,N,lightblue,Br,yellow,Re,turquoiseblue.(Forinterpretationof thereferencestocolorinthisfigurelegend,thereaderisreferredtotheWebversionofthisarticle.) overexpression of permeases or drug efflux transporters) [76]. study. In the current preclinical drug development pipeline, the Accordingly, distinctive characteristics of HCT-116 cells, including zebrafishemergedasaversatilebiotechnologicalplatformforthe mutation in the KRAS proto-oncogene, stem cell-like properties, toxicityassessmentandbioactivityevaluationofchemicallydiverse low differentiation level, fast division as well as epithelial molecules. Owing totheir high genetic, physiological and immu- morphology[77e79]couldcontributetotheirhighersensitivityto nologicalsimilaritytohumans,andgoodcorrelationinresponseto theappliedrhenium(I)-tricarbonylcomplexes. pharmaceuticals, zebrafish markedly simplify the path to clinical trialsandreducethefailureatlaterstagesoftesting[80,81].Inthe lastyears, the preclinical toxicityofavarietyofmetal complexes 2.4. Invivotoxicityassessmentandtherapeuticpotential (Ag,Au,Ir,Os,Pt,Ru,etc.)hasbeentestedinthezebrafishembryos determination [82e85], while, to the best of our knowledge, only one study exploredRe(I)tricarbonylcomplexesinthismodel[51]. Following in vitro anticancer activity assessment, the in vivo toxicity evaluation of compounds 1e8 was carried out using Herein,accordingtothedeterminedLC50values(Table3),active antiproliferative Re(I) complexes were ranked by toxicity as a zebrafish(Daniorerio)embryosastheanimalmodelsystem,with follow:3¼2[1>4.Amongthem,complexes2and3exerted the aim of determining their therapeutic potential early in this J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 5 Table2 Calculatedmolecularpropertiesofinvestigatedcompoundsfortheassessmentofdrug-likeness. Complex miLogPa(Ligand) TPSAb Natoms c MWd NON e NOHNH f Nviol. g Nrotb. h Voli 1 3.78(2.22) 61.08 22 599.25j 5 0 1 1 289.13 2 3.63(2.07) 64.32 26 591.48j 6 0 1 4 350.75 3 4.23(2.66) 64.32 28 619.53j 6 0 1 4 383.93 4 5.12j(3.56) 64.32 30 647.59j 6 0 2 6 417.53 5 4.44(2.88) 70.80 36 733.73j 8 0 1 12 510.26 6 3.34(1.77) 90.10 36 717.60j 8 0 1 6 452.14 7 0.97(1.77) 122.16 43 985.82j 11j 0 2 2 527.96 8 (cid:3)0.87(1.77) 90.10 35 635.68j 8 0 1 3 425.26 a Octanolewaterpartitioncoefficient(logPvalueobtainedusingMolinspirationmethod). b MolecularpolarsurfaceareainÅ2. c Numberofnonhydrogenatoms. d Molecularweight. e Numberofhydrogen-bondacceptors(OandNatoms). f Numberofhydrogen-bonddonors(OHandNHgroups). g Numberof“Ruleoffive”violations. h Numberofrotatablebonds. i MolecularvolumeinÅ3. j “Ruleoffive”violation. Fig.2. SelectedstructuresofRe(I)carbonylcomplexestestedagainstcoloncancercelllines. Table3 Invitrocytotoxicity(IC50,mM)andinvivotoxicity(LC50,mM)ofcomplexes1e8. Cells/Comp. 1 2 3 4 5 6 7 8 MRC-5 34±2 12.7±0.9 5±0.4 20±1 190±4 105±2 230±3 250±5 HCT-116 5.0±0.2(6.8) 10.0±0.9(1.3) 7.4±0.2(0.7) 6.2±0.1(3.2) 50±2(3.8) 60±4(1.8) 140±2(1.6) 180±4(1.4) MiaPaCa-2 10.7±0.7(3.2) 15±1(0.8) 8.1±0.8(0.6) 12±1(1.7) 200±6(1) 75±3(1.4) 185±6(1.2) 230±7(1.1) HeLa 27.5±0.9(1.2) 17±1(0.7) 5.0±0.3(1) 9.5±0.8(2.1) 143±2(1.3) 70±4(1.5) 190±5(1.2) 200±7(1.3) A549 38±1(0.9) 15.4±0.8(0.8) 10±1(0.5) 23.2±0.8(0.9) 75±2(2.5) 30±3(3.5) 185±7(1.2) 180±5(1.4) Zebrafish 244.4±10.2 <30 <30 271.4±4.8 >250 >250 261.2±9.7 >250 Thevaluesinbracketsrepresenttheselectivityindex(Si)determinedasaratiobetweentheIC50valuesforMRC-5cellsandcorrespondingcancercells.IC50ofcompounds showinggoodselectivitytowardsHCT-116coloncancercellsarebold. 6 J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 toxic side effects at the doses (cid:4)25 mM (especiallycardiotoxicity), anti-angiogenicactivity,Tg(fli1:EGFP)zebrafishembryoswithGFP- while1and4didnotaffectembryossurvival,norcausedterato- labelledendothelialcellswerechallengedtodifferentdosesofboth genic malformations even at doses of 250 mM (Fig. 3). Notably, compounds for 24 h, and imaged by fluorescent microscopy to neither 1 nor 4 caused cardiac dysfunctions (pericardial edema, directly visualize the effect of the applied agents on new vessel disturbedheartbeatingrate)(Fig.3AandB)ormyelotoxicresponse development.Innormallydevelopingembryos,5e9subintestinally (neutropeniaorinflammation)(Fig.3EandF)ataconcentrationas vessels(SIVs)and28e30intersegmentalvessels(ISVs)connected high as 250 mM, nor induced the liver failure (reduced liver area with dorsal lateral vessels (DLAVs) were present, while the anti- index,hepatomegaly,livernecrosisorreducedyolkconsumption) angiogenic phenotype is defined by reduced number/length of at200mMand250mM,respectively(Fig.3CandD).Ontheother ISVsordisruptedDLAVs. side,sunitinibanddoxorubicin,twowell-knownanticanceragents, We foundthatcomplexes1and4arehighlyeffective ininhi- provokedseverepericardialedema,markedlydecreasedheartbeat bitionofangiogenesis.Theywereactiveatverylowdosesandina rate,neutrophilsoccurrence,liversizeaswellastheliverareaindex dose-dependent manner, and the treated embryos displayed the (Fig. 3), which accompanied with the pronounced teratogenic anti-angiogenicphenotypealreadyat0.25mMand0.5mMdoses,for malformations, led to embryos’ death by 120 h post-fertilization 1and4respectively(Fig.4,c2test).Wepointtothefactthatthe (hpf),aspreviouslyshown[83,86]. overall anti-angiogenic effect of both Re(I) tricarbonyl complexes Herein,wecouldnotdetermineatrendoftheinvivotoxicityof exceededthatofsunitinib,aclinicalanti-angiogenicdrug.At1mM, complexes1e4asafunctionoftheirlipophilicity.Extensiveliter- sunitinib reduced subintestinal vessels (SIVs) and intersegmental ature data show a strong correlation between compound’s lip- vessels(ISVs)lengthby50.4± 2.1%and41.2±1.7%,respectively, ophilicity (logP) and their uptake by zebrafish embryos, proving while complex 1 showed higher inhibitory effect than this drug that lipophilic molecules are uptaken up by the embryos [87], decreasingtheSIVsangiogenesisby57.6±1.9%(P<0.001)andthe which is very important for their in vivo efficiency and toxicity ISVangiogenesisby49.1±2.6%(P<0.001)(Fig.4C,D),andcomplex evaluation. On the other side, the meta-analysis of toxicity of 4exertedcomparableactivityto1mMsunitinibatthedosesof2mM variouscompoundsfromdiversechemicalclassesinthezebrafish and 2.5 mM, respectively. Moreover, when applied at the doses modelprovednocorrelationbetweenthecompounds’toxicityand (cid:4)3mM,theoverallantivasculareffectof1and4wasmuchgreater lipophilicity (logP) [88,89], which we also have observed in this thanthatof1mMofsunitinib,achievingupto100%regressionin study. ISVsandSIVslength(P<0.001).Inaddition,theinvestigatedRe(I) Since cardio-, hepato- and immunotoxicity are the most complexes exerted much higher anti-angiogenesis potency than commonly encountered drawbacks of clinically approved anti- thatreportedforauranofin,agold-basedagent[83]. cancerdrugs,limitingtheirlong-termapplicationinchemotherapy, Itisimportanttoemphasizethatthetreatmentswitheffective dataobtainedinthisstudyindicateaverygoodtoxicityprofileof doses of 1 and 4 elicited notoxic response in zebrafish embryos, complexes1and4,whichcouldbeconsideredassafeagentswith whilesunitinibappliedatanti-angiogenicdoses(cid:4)1mMprovoked promising anticancer activity. Furthermore, we found that both life-threateningpericardialedema(Fig.4A)andmarkedlyreduced moleculespossessalargetherapeuticwindow(determinedasthe the heartbeat rate (P < 0.001) what progressively decreased the LC50/IC50 ratio), greater than 43 (Table 4), which is of particular embryos’survivalby120hpf.Thesetoxicityissueshaverestricted relevance since HCT-116 represents a CRC cell line thateasilyac- the application of sunitinib at higher doses and its overall anti- quires resistance to clinical anticancer drugs, including cisplatin, angiogenic potential. Cardiotoxicity, in particular, is one of the oxaliplatin,docetaxel,5-FU,andothers[79,90e92].Takentogether, majorobstaclesforalong-termuseofmanyclinicalanti-angiogenic theseresultspromptedustofurtheraddresstheefficacyof1and4 drugs,includingsunitinib[99]. againstcolorectalcarcinomainvivo.Weevaluatedbothanticancer Todeterminetheoveralltherapeuticanti-angiogenicpotential and anti-angiogenic activity of these molecules using various of1and4,andtounderstandwhetherthesecompoundshavemore zebrafishmodels. specific inhibitoryeffects on neovascularization in comparison to toxicity/teratogenicity, we determined their IC50ang doses (the 2.5. Inhibitionofangiogenesisinvivo concentration upon which 50% of embryos displayed anti- angiogenic phenotype), EC50 doses (the effective concentration Angiogenesis(newvesselsformation)isanessentialprocessfor resulting in toxic response at 50% of embryos) and therapeutic tumourgrowth,invasionandmetastasis[93].SinceCRCarehighly windows (Tw; the ratio between EC50 and IC50ang values). The vascularizedandmetastatictumors,theincreasedangiogenesisin therapeutic potential of complexes was evaluated in relation to CRC is associated with poor prognosis and tumour relapse [94]. sunitinibandresultsaregiveninTable5.ThedatashowthatIC50ang Accordingly,inhibitionoftumourvascularizationisaprovenclin- dosesof1and4arefarbelowtheirrespectiveEC50doses,contrary icalstrategyforCRCtreatment[3],wherethecombinationofanti- tothatof sunitinib,for whichEC50 and IC50ang doses areclose to angiogenicdrugswithcytotoxicdrugsincreasesanti-tumoureffi- eachother,indicatingthushighriskoffetaltoxicity[100].Basedon cacy and provides significantly better survival of the cancer pa- therespectiveTwvalues,1and4displayed349-foldand211-fold tients [3,95]. Compared to conventional chemotherapy with higher anti-angiogenic potential than this FDA-approved drug cytostatic agents, a treatment targeting angiogenesis brings rela- (Tw¼1.41),implyingtheirpossibleuseaseffectiveandsafeanti- tively less side effects associated with prolonged administration angiogenicagentsinoncologicalpractice. and less risk of developing tolerance because such treatment is focusedonvascularcells. 2.6. Anticanceractivityinthezebrafishxenograftmodelof Tothebestofourknowledge,theanti-angiogenicpotentialof colorectalcarcinoma rhenium tricarbonyl complexes remains mostly unexplored, in contrasttocomplexesofotherthirdrowtransitionmetalslikegold, The potent antiproliferative activity of complexes 1 and 4 osmium, and iridium [44,83,96,97]. Yang et al. [98] recently re- (Tables 3 and 4) accompanied with the effective inhibition of ported a theranostic rhenium(I) tricarbonyl-dichloroacetate con- angiogenesis(Fig.4)fortifiedustoevaluatethemagainstcolorectal jugate, which at a dose of 5 mM destroyed already formed carcinomainvivo.TodatetheanticanceractivityofRe(I)tricarbonyl vasculatureinzebrafishembryos,leadingtotheirdeathwithin48h complexes has rarely been evaluated in vivo. To our knowledge, post-treatment. To address whether complexes 1 and 4 possess only three studies have reported the efficiency of fac-[Re(CO)3] þ J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 7 Fig.3. Complexes1and4arenotcardio-,hepato-ormyelotoxicinthezebrafishmodel.Cardiotoxicitywasaddressedinwildtype(wt)embryos(n¼30)accordingtothe appearanceofpericardialedema(arrow)(A)anddecreasedheartbeatingrate(B).HepatotoxicitywasassessedinTg(-2.8fabp10a:EGFP)embryoswithfluorescentlylabelledliver (n¼10)accordingtotheliverfluorescence(C)andtheliverareaindex(D),whilemyelotoxicitywasevaluatedinTg(mpx:GFP)embryoswithfluorescentlylabelledneutrophils (n¼30)accordingtoneutrophilsfluorescence(E)andoccurrence(F).Alltoxicityendpointsanalysedinthezebrafishembryostreatedwith1and4werecomparedtothoseinthe control(DMSO-treated)groupandthegroupstreatedwithclinicallyapproveddrugs-cisplatin,doxorubicinandsunitinibmalate.Complexes1and4didnotprovokeanysideeffect atdosesupto250mM(excepthepatotoxicityof1at250mM).Cisplatin,sunitinibanddoxorubicinappearedtobecardiotoxic,hepatotoxicandmyelosupressiveatthetested concentrations.P<0.05;**P<0.01;***P<0.001. 8 J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 complexesinanimalmodelsofhumantumors.Colleryetal.have Table4 reportedaverypromising Re(I) diselenoethercomplexcandidate Therapeuticwindows(Tw)ofcomplexes1e8. for breast-tumor treatment, which completely inhibits tumor Cells/Compound 1 2 3 4 5 6 7 8 development in MDA-MB-231-bearing mice xenografts, but it is HCT-116 48.9 3.0 4.1 43.8 5.0 4.2 1.9 1.4 inactiveinlungandcolorectalcarcinomacelllines[17,101].More MiaPaca-2 22.8 2.0 3.7 22.6 1.3 3.3 1.4 1.1 þ recently, Konkankit [62] and He [68] described fac-[Re(CO)3] HeLa 8.9 1.8 6.0 28.6 1.8 3.6 1.4 1.3 complexes with 1,10-phenthroline and b-carboline derivatives, A549 6.4 2.0 3.0 11.7 3.3 8.3 1.4 1.4 respectively,whichinducedupto60%regressionofovarianorlung TheTwvaluesintherangefrom5to20areitalic,whilethoseabove20arebold. tumors in carcinoma mice models. So far, none of the fac- þ [Re(CO)3] complexeswiththereportedanticanceractivityagainst CRCcelllineshasbeenevaluatedinvivoinanimaltumormodels. disseminationforaperiodofthreedayspostinjection(treatment). Herein, we investigated the activity of complexes 1 and 4 Considering that: a) tumor mass was reduced in all xenografts against human colorectal carcinoma in vivo using the zebrafish- receiving1/8(cid:5)IC50doses(~40%;Fig.5B);b)~60%ofthesexeno- HCT-116 xenograft model. Zebrafish xenografts have emerged in grafts still had ectopic vessels for HCT-116 cells dissemination last years as a new platform for translational research in human (Fig.6D);c)metastasesoccurredonlyin16%(complex1)and36% carcinomas demonstrating crucial hallmarks of cancer biology, (complex 4) of embryos (Fig. 5C) and at a very low number such as tumour cells proliferation, dissemination, metastasis and (1e5 cells perembryo)(Fig. 5D); wesuggest that the anti-tumor tumourangiogenesis,andprovidingdifferentialdiscriminationin and antimetastatic effects of 1 and 4 can be initially assigned to anticancertherapyresponseswithsingle-cellresolution[102,103]. theircytotoxicactivityonHCT-116cells,whileathigherdoses,this Accordingly,HCT-116cellswerefluorescentlylabelledandinjected activity is accompanied with inhibition of tumor neo- intotheyolkofTg(fli1:EGFP)embryos,andat3dayspostinjection vascularization and results in markedlydecreased tumor growth. (dpi), xenografts were processed for fluorescence microscopy These findings are in a line with the potential of complexes to evaluating the effects of applied complexes on the tumour mass inhibitnewvesselformationintheangiogenesisassay(Fig.4),and development, tumour neo-angiogenesis, as well as cancer cells indicate their non-detrimental effect on already established disseminationandmetastasis. vascularization[106]. Ourresults(Fig.5)showthattreatmentswith1and4signifi- In addition to the anti-tumor activity attained in vivo, it is cantly inhibited both colorectal tumour growth (P < 0.001) and importanttonotethatafter3-daystreatmentsallxenograftssur- cancer cells dissemination (P < 0.001), the complexes being vivedanddevelopedwithoutsideeffects(Fig.5A).Cardiovascular noticeablyeffective even at a concentration 8-fold lower of their toxicity, induced by different mechanisms such as mitochondrial respectiveinvitroIC50values(Table3).BydeterminingtheED50- dysfunction, DNA damage in cardiomyocytes, damage of heart HCT116values(effectivedrugconcentrationreducingtumourmass endothelial cells [107], is one of the most common drawbacks of by50%inrelationtothatinuntreatedxenografts)wefoundthat1 neoplastic drugs. In our study, we did not observe any signs of exerted 2.3-fold higher anticancer potency than 4 (ED50-HCT116 of cardiac dysfunction (edema, hypo/hypertension) or a decrease of 1.23mMvs.2.81mM).Comparisonoftumourgrowthtountreated endothelialcellsfluoresceinthepericardiumoftreatedxenografts. HCT-116 xenografts at 3 dpi (120 hpf) indicated that 1 and 4 The tested Re(I) tricarbonyl complexes exerted much higher reducedtumourmassinthetreatedxenograftsby38.2±5.18%and anticancer activity in the zebrafish-human CRC cells xenografts 41.2±9.11%at1/8(cid:5)IC50doses,respectively(P<0.0001,forboth thanonthecancercellsinculture,implyingthattheoverallther- compounds), while tumors almost completely disappeared upon apeuticpotentialof1and4againstcolorectalcarcinomaishigher ½(cid:5)IC50dosesofcomplexes(correspondingto2.5mMand3.1mM, invivothanwhatweexpectedfrominvitroassay.Variousfactors respectively,Fig.5B). mayaccountforthisdiscrepancy,includinge.g.tumorcellsdensity, In addition to the anti-tumor effect, dissemination of HCT- durationofinvitrotreatments,numberofinjectedtumorcellsin 116cellswassignificantlyreducedalreadyat1/8(cid:5)IC50dosesof1 embryos,siteoftheirinjectionanddurationofinvivotreatment.In and4(P<0.001,forbothcompounds),andcompletelyabolishedat additiontothedirectantiproliferativeeffectofRe(I)complexeson higherdoses(Fig.5CandD).Moreover,complexesefficientlysup- the implanted HCT-116 cells and a possible effect on the tumor pressed tumor neo-angiogenesis in a dose-dependent manner microenvironment,thehighefficacyinrestrictingtumorgrowthin (P < 0.001), preventing ectopic vessels formation from the SIV xenografts could be attributed to the activity of their innate im- basket,especiallyat½(cid:5)IC50doses(Fig.6CandD),incontrastto mune cells, especially of neutrophils. A study of Collery demon- relativelyrobustangiogenicresponseinuntreatedHCT-116xeno- stratedtheimportanceofpreservingimmunityfortheanticancer grafts(Fig.6A).ItiswellknownthatHCT-116cellsinjectedcloseto activity of Re-diselenoether complexes since the investigated the SIV basket release pro-angiogenic factors locally, and thus complexwascompletelyineffectiveifmicewerepre-treatedwith change normal developmental pattern of SIVs bystimulating the irradiation[17].Theimportanceofneutrophilsindeterminingthe endothelial cells migration and the growth of sprouting vessels efficacy and toxicity of clinical anticancer therapies has become towards the implant [104]. Unlike the complexes, cisplatin increasinglyapparentoverthepastdecade[108].Neutrophilsare appearedinactiveagainstgraftedCRCcellssinceitfailedtoinhibit predominant immune cells that protect the host from microbial tumour development and HCT-116 cells dissemination at its infection,andtheirrolesintumorhavelongbeenignoreddueto maximal non-toxic dose of 34 mM (Fig. S13), and decreased the theirshortlifespanandterminaldifferentiationphenotype.Recent ectopicvesselslengthonlyinfewtreatedembryos(Fig.6B),inline studieshaveshownthatneutrophilsmakeupasignificantportion withstudyofStenton[105]. of the inflammatory cell infiltrate in cancer, whereby they show As stated above, the size of HCT-116 tumors was markedly high functional plasticity and display both antitumor and pro- reducedinxenograftsreceiving1/8(cid:5)IC50dosesofcomplexes,as tumor activities. Moreover, the number of neutrophils in blood compared to untreated group. Initially, tumor cells were injected and tumor tissues of cancer patients is associated with disease anddevelopedintotheyolk(120hpf,3dpi),anenvironmentrichin progression and patient outcome. From the one side, anti-tumor nutrients. The ISV and the SIV vasculature of these xenografts neutrophils (N1 neutrophils) eliminate malignant cells by stayedfunctionalandvisibleduringentiretreatment(Fig.5A),thus releasingthecytotoxiccontentsintheirgranules(reactiveoxygen enablingadditionalnutrientsperfusiontoHCT-116cellsandtheir species-ROS,myeloperoxidase,hydrogenperoxide,andproteases) J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 9 Fig.4. Complexes1and4effectivelyinhibitedangiogenesisinTg(fli1:EGFP)zebrafishembryoswithfluorescentlylabelledendothelialcells(n¼30).Embryoswereexposedto differentdosesof1and4,andevaluatedfortheinhibitionoftheISVandtheSIVvasculatureat48hpfand72hpf,respectively.At1mM,theinhibitoryactivityofcompound1was greaterthanthatofclinicalanti-angiogenicdrugsunitinib.Thelatter,however,wascardiotoxicattheeffectivedose,while1didnotelicitanytoxicresponseinthetreatedembryos. Compound4atadoseof3mMdisplayedcomparableactivitytosunitinibat1mM,and,as1,causednosideeffects.Representativeimagesofembryosareshown. Table5 2.7. Cellularlocalizationandinteractionof1and4with Toxicologicalparametersderivedfromtheconcentration-responsecurvesforthe nucleobasesandaminoacids toxicityassessmentandanti-angiogenicpotentialofcomplexes1and4incom- parisontotheclinicaldrugsunitinibmalate. It is generally accepted that for rhenium-based anticancer Treatment LC50 EC50 IC50ang Tw EC50 agentstoexhibittheirantiproliferativeproperties,theyhavetobe ISVs SIVs internalized into the cell. In order to provide preliminary in- 1 244.4 244.4 <0.5 >488.8 1.6 0.9 dications of possible target sites and mechanism of action of 4 271.4 271.4 0.9 295 2.6 2.1 complexes1and4,wefirstinvestigatedtheirdistributionwithin Sunitinib 1.1 0.7 0.5 1.4 1.8 1.1 HCT-116 cells by fluorescent microscopy. The complexes exhibit LC50 etheconcentrationinducingthelethaleffectof50%embryos,EC50 ethe weak fluorescence with maximum emission wavelength (l em) concentrationaffecting50%embryos(survivalanddevelopmentaldefects),IC50ang- centeredat624and606nm,respectively(for1and4,maximum theconcentrationuponwhich50%ofembryosdisplayedanti-angiogenicpheno- exctitation wavelength (l ex) was 400 nm, Fig. S12). Fluorescent type,TwetherapeuticwindowdeterminedastheEC50/IC50angratio,EC50(ISVs)- microscopyinvestigationofHTC-116cellsincubatedwith1and4at theeffectiveconcentrationresultingin50%decreaseISVslengthcomparedtothe 4(cid:2)Cshowednofluorescentsignaldetection,contrarytothefluo- c e o x n p t r r e o s l se (D d M in SO mM -tr . eated)group.LC50,EC50,IC50angandEC50(ISVsorSIVs)valuesare rescevisibleincellsincubatedat37(cid:2)C,suggestingthattheinter- nalizationoftheinvestigatedcomplexesacrossthemembranemay occur via an energy-dependent process [60,111,112]. The intracel- orsecretingimmunemediatorstorecruitandactivateotheranti- lularlocalizationofthecomplexeswasfurtherprobedbydeterm- tumoreffectorcells[109].Ontheotherside,tumorderivedfactors ingthecolocalizationoftheirfluorescencewithorganelle-specific released within the tumor environment can convert anti-tumor fluorescent probes. As shown in Fig. 7, complexes 1 and 4 were neutrophils into a pro-tumor neutrophils (N2 phenotype) which distributedinthecytoplasmofHCT-116cells(visibleasbluediffuse promotetheproliferation,migration,andinvasionoftumorcells, emissionoverthewholecell),whilethenucleishowedveryweak and stimulate angiogenesis. Accordingly, to preserve neutrophils ornoemissionincontrasttostrongstainingwithDAPI(Fig.7a,a’,c during chemotherapy is very important for microbial infection andc’).Inadditiontocytosolicdistributionof1and4,co-staining defence, while preventing the switch of the anti-tumor into the experimentwithLysoTrackerDeepReddyeshowedthatthecom- pro-tumor phenotype could be an important strategy for an plexeswerepariallylocalizedinthelysosomes,beingapparentas effective anti-tumor therapy [108,109]. Herein, our finding that largeperinuclearcytoplasmicvesicles(Fig.7b’,b’’,d’andd’’). Re(I)tricarbonylcomplexes1and4didnotprovokeneutropeniain Such cytosolic-lysosomal distribution of the rhenium(I) tri- zebrafishembryosover5-daystreatments(unlikeothercytostatics carbonyl complexes has previously been reported in molecules ofclinical relevance,Fig.3Cand D)is ofaparticular significance, bearingb-carboline[68],1,10-phenanthroline[49,113]anddiimine especially due to the fact that neutropenic cancer patients are ligands[57].Preferentialaccumulationinlysosomes,mitochondria highlysusceptibletomicrobialinfections[110]. orlocalizationonlyincytoplasmhasalsobeenevidencedforother 10 J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 Fig.5. Anticanceractivityofcomplexes1and4againsthighlymetastatichumanHCT-116cellsinzebrafishxenografts.Tg(fli1:EGFP)xenografts(n¼20)wereexposedtocomplexes 1and4atdosescorrespondingtotheirrespective1/8(cid:5)IC50-1/2(cid:5)IC50values,andanalysedafter3-daystreatmentsfortumourprogressionandmetastasis.Representative fluorescentmicroscopyimagesareshown(A);whitesolidarrowsindicatedisseminatedcells.Theappliedtreatmentsmarkedlyreducedthetumorgrowth(B),incidenceofcancer cellsdissemination(C)andthenumberofdisseminatedHCT-116cellsperxenograft(D)comparedtothoseinthecontrolgroup(P<0.001,forallhallmarks).Dataarenormalizedin relationtothecontrolgroup(B,C).*P<0.05;**P<0.01;***P<0.001. Fig.6. Inhibitionoftumor-inducedneoangiogenesisinthezebrafishxenograftsbearingHCT-116tumors.LateralviewsofthetransgenicTg(fli1:EGFP)embryosat72hpf(24hpi) showingtheectopicvessels(arrow)sproutingfromtheSIVplexusuponstimulationbycloselyinjectedHCT-116cells(AeC).Newlyformedectopicvesselsinuntreated(A)and cisplatin-treatedxenografts(B)aredenoted,whilevesselswerenoformedupontreatmentwithcomplex1.(D)Dose-dependentinhibitionofneovascularizationbycomplexes1 and4,andcisplatinatitsthehighestsafedoseof34mM.Scalebar¼100mm. J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 11 Fig.7. FluorescentmicroscopeimagesofHCT-116cellstreatedwith1and4(5mM,30min,37(cid:2)C/5%CO2).Theintracellulardistributionofcomplex1(aandb)andcomplex4(cand d),aswellasofDAPI-labellednuclei(a’andc’)andLysoTracker-labelledlysosomes(b’andd’).Theco-localizationoftheappliedcomplexeswithDAPIorLysoTrackerdyeisshown onimagesa"-d".Arrowsindicatemagnifiedcellsinthelastcolumn.Magnifications(cid:5)100wasused.Scalebars¼10mm. rhenium(I) tricarbonyl molecules [114,115]. It is noteworthy to clearlyalkylatedthesidechainFmoc-Lys-OH(Fig.S15).Inaddition pointoutthatthelysosomesareemergingasveryattractivetargets to that, both metal species showed unspecific interaction with in anticancer therapy, and drugs affecting the integrity of lyso- Fmoc-Lys-OH(i.e.-NH2or-COO-coordinationtorhenium,Fig.S15- somes have been proven to be effective in cancer treatment S16). [116e120]. The lackof clearevidence of nuclearaccumulation of 1 and 4 Due to the fact that the fluorescence of 1 and 4 is visible doesnotexcludethe possibilitythata smallfraction of thecom- throughout the entire cytosplasm of HCT-116 cells, it is possible plexes enters the nucleus or that the same mayexert their cyto- that some cytosolic proteins could be targetsof the tested mole- toxicity via DNA or RNA interactions or nucleobases binding. þ cules, as recently demonstrated for a Re(I) tricarbonyl isonitrile Severalfac-[Re(CO)3] areshowntobindtonucleobasesanddou- complex which exerted anticancer activity via the endoplasmic blestranded(ds)DNA[57,132e139].Therefore,weprobednextthe reticulumstressandtheaccumulationofmisfoldedproteins[113]. reactivityofthecomplexeswithdslambdabacteriophageDNA(l- Accordingly, we probed the reactivityof complexes 1 and 4 with DNA) and the model nucleobases adenine and guanine. Gel elec- heneggwhitelysozyme(HEWL),amodelproteinoftenemployed trophoresisexperimentsrevealedthatonly1,andonlyatconcen- toinvestigatemetaleproteinadducts[121e127].Theinteractionof trations >250 mM, significantly prevented ethidium bromide the complexes with HEWL was examined by HPLC and ESI-MS. intercalation in l-DNA (Fig. S17). Furthermore,1H NMR spectros- Similar rhenium complexes have beenpreviously shown to pref- copyprovidednoevidenceofadenineorguaninecoordinationto erentiallybindtotheonlyhistidineresidueoftheenzyme(His15) the metal center (Fig. S18-S19). Collectively, the results indicate givinge.g. fac-[His15-Re(CO)3(X)2] type adducts [128e130]. Com- thatDNAisanunlikelytargetforthemodeofactionof1and4.The plex 4 showed no binding to HEWL. On the other hand, ESI-MS complexes,therefore,arelikelytoactatthecytoplasmaticlevelby analysisoftheinteractionof1withHEWLrevealed,alreadyafter altering key cellular processes possibly via alkylation of one or 1 day of incubation, the formation of a new lysozyme-complex severalproteins(complex1). adduct with a mass increased by 440 mass units (Fig. S14). This masscorrespondtocomplex1lackingtwobromides.Theadductis 3. Conclusions consistentwiththepossibilitythat1mayhavealkylatedHEWLby reactingwiththeaminegroupsofbasicaminoacids(aa)residuesof We have reported the synthesis, characterization and the e.g.lysine,arginine,asparagineorglutamine(collectively35aaof evaluation of a small library of rhenium(I) tricarbonyl complexes the129composingtheprotein),viathesamenucleophilicsubsti- foranticanceractivity,anddiscoveredtwocomplexes,1and4,as tution of the bromide atom of the a-diimine ligand as shown in promising agents against CRC with a good therapeutic profile. Scheme1.WecannotexcludethepossibilityofHis15coordination, Thesetwocomplexesarefoundtopossesstripleanticancer,anti- however, lackof directHis15 bindingof fac-[Re(CO)3(L2)Br] com- angiogenicandantimetastaticactivityinvivo,notyetreportedfor plexes(L2¼bidentateligand)wasconsistentlyobservedinprevi- þ ous studies [128,131]. In order to confirm aa alkylation by 1, we a do f s a e c s -[ f R r e o ( m CO 1 )3] to m 3 o m le M cu . l T e h , e be n in o g vel ef r fe h c e t n iv iu e m in (I) th tr e ica ra rb n o g n e yl of co lo m w - furthertestedthereactivityofthecomplexeswithFmoc-protected plexes displayed up to 349-fold higher anti-angiogenic potential Lys-OH,Asn-OHandGln-OH(allbearingunprotectedsidechains). than clinical drug sunitinib malate, while in the zebrafish xeno- Complexes1and4showednointeractionwithAsnandGln,but1 graft model of colorectal carcinoma achieved almost complete 12 J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 inhibition of tumour growth, tumor vascularization and tumor 4.3. Generalsynthesisprocedures cells metastasis, contrary to cisplatin. Moreover, the investigated complexes showed a large therapeutic window and at doses as 4.3.1. fac-[Re(CO)3L2Br](L2 ¼6-(bromomethyl)-2,20-bipyridine),1 high as 200e250 mM did not induce toxicity issues encountered To a stirring 50 mM solution of [Re(CO)3Br3](Et4N)2 in water in clinical anticancer drugs (cardio-, hepato-, and myelotoxicity), (850 mg in 22 mL H2O) a 450 mM solution of 6-(bromomethyl)- appearing to be much safer and more effective than FDA- 2,20-bipyridine (0.9 eq.) in ethanol (247 mg in 2.2 mL EtOH) was approved sunitinib, doxorubicin or cisplatin, and demonstrating added. The resulting mixturewas stirred two days at room tem- a strong potential for the application in CRC therapy. Invitro co- perature (RT) after which time a yellow precipitate of 1 was localization studies suggested that the investigated complexes collected by filtration and rinsed several times with a cold 9:1 localize preferentially in the cytoplasm, and, to some extent, water:ethanolmixture.Theresultingpowderwasthendriedunder within the lysosomes and may act by altering cellular processes vacuumfortwodays.Yield:220mg,80%.1HNMR(300MHz,CDCl3) possibly via alkylation of one or several key proteins. Future ef- d H ¼9.17(1H,qd,J¼5.5Hz),8.18(2H,m,J¼1.3Hz),8.07(2H,m, forts will be directed towards studies aimed at verifying anti- J¼1.6Hz),7.89(1H,dd,J¼1.3Hz),7.56(1H,m,J¼5.5Hz),5.03 tumour efficacy and therapeutic potential of the molecules on (2H, dd, J ¼ 12 Hz) ppm. ESI-MS analysis (positive mode) m/ primary tumorcells fromthe patients with CRC and the detailed z ¼ 518.9 [M-Br] þ . IR (KBr pellet), n C≡O: 2021 cm (cid:3)1, 1905 cm (cid:3)1, elucidation of their targets and mechanism of action. 1896cm (cid:3)1.UVeVis(CHCl3,nm):301,386.CrystalssuitableforX- ray diffractionwere obtained from slowevaporation of a deuter- atedchloroformsolution. 4. Materialsandmethods Complexes2e6werepreparedaccordingtothefollowinggen- eralprocedure.UnderinertconditionsatRT,toastirringsolutionof 4.1. Reagentsandchemicals 1(50mg,0.083mmol)in8mLdichloromethanethecorresponding secondaryamine(1eq.)andN,N-diisopropylethylamine(DIPEA1 AllreagentandsolventswerepurchasedfromsigmaAldrichand eq.)wereadded.Thereactionmixturewasthenrefluxedovernight TCI and used without further purification. 6-(bromomethyl)-2,20- under argon. After it had cooled to RT, the reaction mixture was bipyridine ligand was synthesized from commercially available washed 3 times with bi-distilled water and thenwith brine. The 2,20-bipyridine [140].Ligand 1-([2,20-bipyridin]-6-yl)-N,N-bis(pyr- organic phase was separated, dried over MgSO4, filtered and the idin-2-ylmethyl)methanamine (DPA-bpy) was synthesized solvent removed under reduced pressure. The obtained powder followingtheprocedureofRadarametal.from6-(bromomethyl)- wasthendissolvedinaminimumamountofdichloromethaneand 2,20-bipyridineandcommerciallyavailabledipicolylamine[52].The 40mLpentanewereaddedbeforeplacingtheflaskinthefridge. [Re(CO)3Br3](Et4N)2 salt was synthesized from dirhenium deca- Afterafewdays,acrystallinepowderwasfilteredtoyieldthepure carbonylintwostepsfollowingtheprocedureofAlbertoetal.[141]. compounds. For compound 3 sodium tert-butoxide was used insteadofDIPEA. 4.2. Instrumentsandanalysis 4.3.2. fac-[Re(CO)3L2Br](L2 ¼N-([2,20-bipyridin]-6-ylmethyl)-N- ethylethanamine),2 NMR spectra were measured on a Bruker Advance III 500 or 400MHz.Thecorresponding1Hchemicalshiftsarereportedrela- Yield:23mg,47%.1HNMR(300MHz,CDCl3)d H ¼9.13(1H,md, J¼7.8Hz),8.18(2H,d,J¼8.2Hz),8.03(3H,m),7.50(1H,m),4.18 tive to residual solvent protons. Mass analyses were performed (2H,dd,J¼25.7Hz),2.71(4H,q,J¼7.1Hz)1.10(6H,t,J¼7.1Hz) eitherusingESI-MSonaBrukerFTMS4.7-TApexIIinpositivemode orMALDIwithaBrukerUltrafleXtremeMALDI-TOF.UVeVisspectra ppm.ESI-MSanalysis(positivemode)m/z¼512.2[M-Br] þ .IR(KBr weremeasuredonaJascoV730spectrophotometer.IRspectrawere pellet), n C≡O: 2018 cm (cid:3)1, 1910 cm (cid:3)1, 1894 cm (cid:3)1. UVeVis (CHCl3, nm):300,385. recordedonaPerkinElmerFTIRFrontierSerie99155equippedwith a PIKE TECHNOLOGIES Glad-iATRTM system. Preparative and analyticalHPLCswereperformedwithaMerckHitachiHitachiL- 4.3.3. fac-[Re(CO)3L2Br](L2 ¼N-([2,20-bipyridin]-6-ylmethyl)-N- 7000system,whichcomprisesaPumpL-7100andaUV-DetectorL- isopropylpropan-2-amine),3 7 C 4 1 0 8 0 H . T F e o c r ( p 5 re m p m ara p t a iv rt e ic H le P s L i C z , e a ,1 c 1 o 0 lu Å m p n or M e a s c i h ze e , re 2 y 5 - 0 Na (cid:5) ge 2 l 1 N m uc m le ) o w du as r J¼ Y 5 i . e 5 ld H : z) 1 , 5 8. m 27 g, (1 3 H 0% ,d .1 , H J¼ N 7 M .1 R H ( z 4 ) 0 ,8 0 .1 M 7 H (1 z H ,C ,d D , C J l ¼ 3) 8 d . H 2 ¼ Hz 9 ) . , 1 8 4 .0 ( 3 1H (2 , H d, , used.Aqueoustrifluoroaceticacid0.1%solutionandpuremethanol m),7.97(1H,m),7.50(1H,m),4.27(1H,d,J¼17.7Hz),4.15(1H,d, were respectively used as solvents (A) and (B). The compounds J ¼ 17.8 Hz), 3.15 (2H, m),1.09 (12H, d, J ¼ 6.4 Hz) ppm. ESI-MS were separated using the following gradient: 0e5 min (75% A), analysis (positive mode) m/z ¼ 540.9 [M-Br] þ . IR (KBr pellet), 5 5 e e 3 3 5 0 ( ( 7 5 5 0 % % A A / / 0 0 % % A A ) ) , , 3 3 5 0 e e 4 4 5 5 m m i i n n ( ( 1 1 0 0 0 0 % % B B ) ), o t r h 0 e e fl 5 ow mi r n at ( e 50 s % et A t ) o , n C≡O:2019cm (cid:3)1,1910cm (cid:3)1,1894cm (cid:3)1.UVeVis(CHCl3,nm):301, 388.CrystalssuitableforX-raydiffractionwereobtainedbyslow 5mLmin (cid:3)1andthecompoundsdetectedat320nm.Singlecrystal evaporationofdeuteratedchloroform. diffraction collections were done on Stoe IPDS2 diffractometer (MoKa 1 (l ¼ 0.71073 Å)) equipped with a cryostat from Oxford Cryosystems.ThestructureweresolvedwiththeShelXTstructure 4.3.4. fac-[Re(CO)3L2Br](L2 ¼N-([2,20-bipyridin]-6-ylmethyl)-N- solutionprogram[142]usingIntrinsicPhasingandrefinedwiththe isobutyl-2-methylpropan-1-amine),4 ShelXLrefinementpackage[143]usingLeastSquaresminimisation. Yield:32mg,60%.1HNMR(300MHz,CDCl3)d H ¼9.12(1H,d, All thecrystal structures are deposited at theCambridge Crystal- J ¼ 5.5 Hz), 8.2 (2H, m), 8.03 (3H, m), 7.50 (1H, m), 4.28 (1H, d, lographicDataCentre.CCDCnumbers1982188to1982192contain J¼16Hz),4.03(1H,d,J¼16Hz),2.32(4H,m),1.86(2H,m),0.94 thesupplementarycrystallographicdataforthispaper.Thesedata (12H, m) ppm. ESI-MS analysis (positive mode) m/z ¼ 568.0 [M- canbeobtainedfreeofchargefromtheCambridgeCrystallographic Br] þ .IR(KBrpellet),n C≡O:2019cm (cid:3)1,1911cm (cid:3)1,1896cm (cid:3)1.UVeVis DataCentreviawww.ccdc.cam.ac.uk/structures.Thepurityof the (CHCl3,nm):300,384.CrystalssuitableforX-raydiffractionwere synthesized materials was determined to be (cid:4) 95% by RP-HPLC obtained by vapor diffusion of pentane into a dichloromethane analysis. solution. J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 13 4.3.5. fac-[Re(CO)3L2Br](L2 ¼N1-([2,20-bipyridin]-6-ylmethyl)-N1- HEWL)[144].Thereactionmixturesweregentlyvortexed5s,and (2-(diethylamino)ethyl)-N2,N2-diethylethane-1,2-diamine),5 thenincubatedat37(cid:2)Cfor2days.Themixtureswerepurifiedin Yield:30mg,50%.1HNMR(300MHz,CDCl3)d H ¼9.07(1H,d, portions by HPLC. In order to avoid possible denaturation of the J ¼ 5.5 Hz), 8.23 (2H, dd, J ¼ 18 Hz, J ¼ 6.8 Hz), 8.03 (3H, m, enzyme,thefractionswerecollectedinaflaskcontainingdistilled J¼12.3Hz),7.47(1H,m),4.28(2H,dd,J¼20.8Hz),2.71(8H,m, water, always maintaining the 4MeOH fraction below enzymatic J¼6.5Hz),2.54(8H,q,J¼7.1Hz),0.99(12H,t,J¼7.1Hz)ppm.ESI- denaturation conditions (4MeOH ¼ 0.3 at pH 1.9) [145]. The MSanalysis(positivemode)m/z¼654.4[M-Br] þ .IR(KBrpellet), different fractions were directly analysed by ESI-MS (positive n C≡O:2019cm (cid:3)1,1911cm (cid:3)1,1894cm (cid:3)1.UVeVis(CHCl3,nm):300, mode). 379. 4.3.6. fac-[Re(CO)3L2Br](L2 ¼1-([2,20-bipyridin]-6-yl)-N,N- 4.5. Nucleobaseandaminoacidbinding bis(pyridin-2-ylmethyl)methanamine),6 Yield:36mg,60%.1HNMR(300MHz,CDCl3)d H ¼9.11(1H,m), The interaction of complexes 1 and 4 with DNA nucleobase 8.59(2H,m),8.43(1H,dd,J¼3.0Hz),8.15(1H,d,J¼8.2Hz),7.99 models guanine (G) and adenine (A) was investigated by NMR (3H,m),7.65(2H,m),7.49(3H,m),7.16(2H,m),4.45(2H,bs),4.04 spectroscopy in a DMSO‑d6 solution (0.5 mM 1 or 4, þ2 eq. DNA (4H,bs)ppm.ESI-MSanalysis(positivemode)m/z¼637.8[M-Br] þ . base).NobindingofthecomplexestoeitherGorAwasobserved IR (KBr pellet), n C≡O: 2019 cm (cid:3)1,1912 cm (cid:3)1,1896 cm (cid:3)1. UVeVis after 48 h incubation at 37 (cid:2)C. The interaction of the same com- plexes with Fmoc-protected Lys-OH, Asn-OH and Gln-OH was (CHCl3,nm):299,385. probedbyESI-MS(positivemode).Solutionscontainingcombina- N 4. , 3 N .7 -b . is fa (p c y -[ r R id e i ( n C - O 2 ) - 3 y B lm r]L e 2 t [ h R y e l) (C m O e )3 th ] a (L n 2 am ¼ in 1- e ( ) [ , 2 7 ,20-bipyridin]-6-yl)- t 0 i . o 5 n m s o L f w 1 a a te n r d /D 4 M ( S 1 O 0 , 0 4 m D M M ) S w O it ¼ h 0 th .7 e , p aa H ( ¼ 2 e 7 q .3 .) a w n e d re in p cu re b p a a te re d d fo in r 48hat37(cid:2)Cbeforeanalysis. 165mgof[Re(CO)3Br3](Et4N)2(0.214mmol)weredissolvedin 5mLwateratroomtemperature.0.5eq.(36mg,0.098mmol)ofthe ligand DPA-bpy in 5 mL ethanol were slowly added, and the 4.6. Cytotoxicityevaluation mixturewasthenstirredovernight.Afterfiltration,yellowcrystals were obtained of the pure compound. Crystals suitable for X-ray Antiproliferative activity was tested in a panel of tumor cells diffractionwereobtainedbyvapordiffusionofetherintoasolution including A549 (ling adenocarcinoma cells), HCT-116 (colorectal ofthecompoundindichloromethane.Yield:42mg,40%.1HNMR carcinomacells),MiaPaCa-2(pancreaticcarcinomacells)andHeLa, (300 MHz, CD3OD) d H ¼ 9.13 (1H, md, J ¼ 5.55 Hz), 8.92 (1H, d, (cervixcarcinoma cells), as well as on normal human lung fibro- J¼13.6Hz),8.87(1H,d,J¼13.6Hz),8.80(1H,d,J¼1.1Hz),8.67 blasts(MRC-5),allfromATCCcollection.Compoundswerefreshly (1H,d,J¼8.2Hz),8.50(1H,t,J¼7.9Hz),8.41(1H,dd,J¼7.1Hz), dissolved in DMSO and used for the bioactivity assessments. 8.30 (1H, td, J ¼ 1.5 Hz), 7.92 (2H, m, J ¼ 7.8 Hz), 7.73 (1H, m, Cytotoxicityintermsofantiproliferativeeffectswastestedbythe J¼7.1Hz),7.55(1H,d,J¼13.8Hz),7.5(1H,d,J¼7.9Hz),7.39(2H, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium m),5.87(2H,s),5.63(1H,d,J¼17.2Hz),5.06(1H,d,J¼15.6Hz), bromide (MTT) assay [146]. The assay was carried out after 48 h 4.77 (1H, d, J ¼ 15.7 Hz), 4.62 (1H, d, J ¼ 17.2 Hz) ppm. ESI-MS of cell monolayer incubation in the RPMI-1640 medium supple- analysis (positive mode) m/z ¼ 987.8 [M] þ . IR (KBr pellet), n C≡O: mentedwith100mgmL-1streptomycin,100UmL-1penicillinand 2028cm (cid:3)1,1943cm (cid:3)1,1921cm (cid:3)1,1889cm (cid:3)1.UVeVis(CHCl3,nm): 10% (v/v) fetal bovine serum (FBS) (all from Sigma, Munich, Ger- 299,385. many), containing different concentrations of the compounds in fourreplicates, twotimes.Cells weregrownin humidified atmo- 4 bi .3 s( .8 p . yr f i a d c i - n [ - R 2 e - ( y C lm O) e 3 t L h 2 y ]( l) N m O3 e ) th (L a 2 na ¼ m 1 in -( e [ ) 2 , ,2 8 0-bipyridin]-6-yl)-N,N- sphereof95%airand5%CO2at37(cid:2)C.TheextentofMTTreduction was measured spectrophotometrically at 540 nm using Tekan 385mg(0.500mmol)of[Re(CO)3Br3](Et4N)2weredissolvedin Infinite 200 Pro multiplate reader (Tecan Group Ltd., Ma€nnedorf, 10 mL H2O to which a 10 mL aqueous solution of silver nitrate Switzerland). Cytotoxicity was expressed as the concentration of (255 mg) was added. The mixture was refluxed for 2.5h and the AgBr precipitate filtered over paper. The filtrate was added to a thecompoundinhibitingcellgrowthby50%(IC50)incomparisonto theuntreatedcontrol. solution of the ligand DPA-bpy (184 mg, 0.501 mmol, in 1:1H2O:MeOH)andheatedto80(cid:2)Covernight.Afterremovalofthe methanol by rotary evaporation, 137 mg of yellowish crystals 4.7. Cellularuptakeandfluorescentmicroscopy formed over 2 days in the remaining aqueous mother liquor and were filtered to yield the pure compound. These crystals were Approximately2(cid:5)105HCT-116cellsweregrowninRPMI-1640 foundsuitableforX-raydiffractionmeasurements.Yield:137mg, medium on glass cover slips in 6-well microtiter plates, up to 40%.1HNMR(300MHz,CD3OD)d H ¼8.87(2H,d,J¼5.5Hz),8.68 reachingtheconfluency.Toaddresswhetherthecellularuptakeof (1H,dm,J¼2.1Hz),8.47(1H,d,J¼0.9Hz),8.39(1H,d,J¼1.0Hz), the complexes was mediated by energy-dependent process, 8.10 (1H, t, J ¼ 7.8 Hz), 7.92 (3H, m, J ¼ 1.5 Hz), 7.78 (1H, d, monolayerofHCT-116cellswastreatedwith1and4(5mM)and J¼0.9Hz),7.51(2H,d,J¼7.9Hz),7.46(1H,m,J¼2.7Hz),7.36(2H, incubatedat4(cid:2)Cor37(cid:2)Cfor30mininRPMImedium.Toaddress m,J¼13.2Hz),5.47(2H,d,J¼16.5Hz),5.12(2H,s),4.78(2H,d, intracellularlocalizationof1and4,HCT-116cellsmonolayerwas J ¼ 16.5 Hz) ppm. ESI-MS analysis (positive mode) m/z ¼ 638.0 incubatedfor 30min at37 (cid:2)Cand 5%CO2 in RPMImediumwith [M] þ . IR (KBr pellet), n C≡O: 2025 cm (cid:3)1, 1925 cm (cid:3)1, 1898 cm (cid:3)1. LysoTracker®DeepRed(25nM,ThermofisherScientific)and/or1 UVeVis(CHCl3,nm):283,332(shoulder). and4(5mM).Cellsweresubsequentlyfixedwith4%formaldehyde for20minatroomtemperature,washedtwicewithPBSandana- 4.4. Interactionwithheneggwhitelysozyme(HEWL) lysed or stained with DAPI [4,6-diamidino-2-phenylindole, dihy- drochloride](14.3mM,ThermofisherScientific)andlateranalysed. Stock solutions of HEWL and of 1 or 4 were mixed to a final Stained cells were observed under a fluorescence microscope concentrationof100mMHEWLand1mM1or4(ca.10eq.,2mL (OlympusBX51,AppliedImagingCorp.,SanJose,CA,UnitedStates) water/DMSO,4DMSO¼0.6,belowthedenaturationconditionsof under(cid:5)100magnification. 14 J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 4.8. DNAinteractions 2.8fabp10a:EGFP)zebrafishembryoswiththefluorescentlylabelled liver [150]. Embryos were exposed to the non-toxic doses of the DNAinteractionassayusinggelelectrophoresiswasconducted tested complexes at the 72 hpf (a stage when the liver is fully accordingtothepreviouslypublishedprocedureusingcommercial functional, vascularized and started metabolic transformation of lambda bacteriophage DNA (100 ng, Thermo Scientific™) [147]. absorbedcompounds)andprocessedbyfluorescentmicroscopyat DNA solution of the final concentration 20 ng/mL was incubated 120hpf.Experimentwasperformedtwotimesusing10embryos with50,100,250and500mMof1and4in10mMTris-Cl,(pH8.5) perconcentration.Thelivertoxicitywasevaluatedinrelationtothe in15mLreactionvolume.Controlcontainedanappropriatevolume control groupaccording tovarioushepatotoxicityendpoints such ofDMSO.After2hincubationat37(cid:2)C,samplesweremixedwith astheliverareaindex(therationbetweenliverareaandembryonic loading dye and run on 0.8% agarose gel with ethidium bromide lateralarea x 100%) [151], the livercolourand theyolkretention (EtBr)againsta1kbDNALadderPlus(NIPPONGeneticsEurope)at [152]. In all mentioned toxicity assays, sunitinib malate (Suten, 60Vfor2h.GelswerevisualizedandanalysedusingtheGelDocEZ Pfizer,NewYork),doxorubicin(Actavis,S.C.Sindan-PharmaS.R.L., system(Bio-Rad,LifeSciences,Hercules,USA),equippedwiththe Romania) and cisplatin (Merck KGaA, Darmstadt, Germany) were ImageLab™Software. used as the control, clinically approved drugs at the previously reportedeffectivedoses. 4.9. Invivotoxicityassessment 4.10. Anti-angiogenicactivityevaluation ToxicityevaluationofRe(I)-tricarbonylcomplexeswascarriedin thezebrafish(Daniorerio)modelaccordingtothegeneralrulesof The angiogenesis inhibitory activity of anticancer Re(I) tri- theOECDGuidelinesfortheTestingofChemicals(OECD,2013,Test carbonyl complexes with the best therapeutic profile was No.236)[148].Allexperimentsinvolvingzebrafishwereperformed addressedintransgeniczebrafishTg(fli1:EGFP)embryoswithEGFP- in compliance with the European directive 2010/63/EU and the labelled endothelial cells, as previously described [83]. Briefly, ethicalguidelinesoftheGuideforCareandUseofLaboratoryAn- zebrafishembryosat6hpfstagewereexposedtotherangeofnon- imalsoftheInstituteofMolecularGeneticsandGeneticEngineer- toxic concentrations of the selected complexes, and incubated at ing, University of Belgrade. Wild type (AB) zebrafish were kindly 28(cid:2)Cby48hpf.At48hpf,thetreatedembryoswereanesthetized providedbydrAnaCveji(cid:1)c(WellcomeTrustSangerInstitute,Cam- with 0.02% tricaine, imaged under a fluorescence microscope bridge, UK), raised to adult stage in a temperature- and light- (Olympus BX51, Applied Imaging Corp., San Jose, CA, USA) and controlled zebrafish facility at 28 (cid:2)C and standard 14:10-h light- analysed for the development of intersegmental blood vessels dark photoperiod, and regularly fed with commercially dry flake (ISVs),subintestinalvessels(SIVs)anddorsallongitudinalanasto- food (TetraMin™ flakes; Tetra Melle, Germany) twice a day and motic vessels (DLAVs). Sunitinib malate was used as a positive Artemia nauplii once daily. Embryos wereproduced bypair-wise control. The experiments were performed three times using 10 mating, collected and distributed into 24-well plates containing embryos per concentration. ISV and SIV lengths were measured 10 embryosperwelland 1 mL embryos water (0.2 g/L of Instant using Image J programme and expressed as mean value with Ocean® Salt in distilled water) and raised at 28 (cid:2)C. Forassessing standard deviation. Inhibitory effect of applied treatments was lethalityanddevelopmentaltoxicity,theembryosstagedat6hpost determinedinrelationtothecontrolgroup,arbitrarilysetto100%. fertilization(hpf)wereexposedtodifferentconcentrationsofthe tested compounds, and inspected for apical endpoints (Table S1) 4.11. AnticanceractivityevaluationinhumanCRC-zebrafish every day by 120 hpf upon an inverted microscope (CKX41; xenografts Olympus, Tokyo, Japan). Dead embryos were counted and dis- carded every 24 h. DMSO (0.25%) was used as negative control. ThehumancolorectalHCT-116celllinewasculturedinRPMI- Experiments were performed three times using 30 embryos per 1640 supplemented with 10% FBS, 100 mg/mL streptomycin and concentration. At 120 hpf, embryos were inspected for heartbeat 100 UmL (cid:3)1 penicillin, and grown as a monolayer in humidified rate, anesthetized by addition of 0.1% (w/v) tricaine solution atmosphereof95%airand5%CO2at37(cid:2)C.Priortomicroinjection, (Sigma-Aldrich,St.Louis,MO),photographedandkilledbyfreezing the cells were washed once with PBS and trypsinized (0.25% at(cid:3)20(cid:2)Cfor(cid:4)24h. trypsin/0.53 mM EDTA) to obtain a single cell suspension. After In addition to developmental toxicity, anticancer Re(I)tri- centrifugationat1200rpmfor5min,thecellswereresuspendedin carbonylcomplexeswiththebesttherapeuticprofile,1and4,were serum-free RPMI medium and labelled with 2 mM CellTracker™ selectedandevaluatedformyelotoxicityandhepatoxicity.Myelo- RedCMTPX (Thermofisher Scientific) according to the manufac- toxicity (neutropenia and inflammation) was addressed in the turer’sinstructions. transgenic Tg(mpx:GFP) zebrafish embryos, which express green fluorescentprotein(GFP)inneutrophils[149],enablingthusdirect 4.12. Zebrafishxenograftsinjectionandtreatment visualizationoftheeffectofappliedtreatmentsontheneutrophils occurrence. Embryos of Tg(mpx:GFP) zebrafish were kindly pro- The zebrafish xenografts with human HCT-116 cells were vided by Dr. Ana Cvejic (Wellcome Trust Sanger Institute, Cam- establishedaccordingtothepreviouslydescribedprocedure[153]. bridge,UK)andraisedinourzebrafishfacilitytoadultstageunder Before the microinjections, Tg(fli1:EGFP) embryos were kept at previouslydescribedlifeconditions.At6hpfstage,embryoswere 28(cid:2)Candmanuallydechorionatedfewhoursbeforetheinjection. exposedtovariousnon-toxicconcentrationsoftheselectedcom- At48hpf,5nLofcellssuspensioncontaining150labelledcellswas plexesandincubatedat28(cid:2)Cby120hpf.At120hpf,embryoswere microinjected into the yolk of anesthetized embryos by a pneu- imaged for the neutrophils occurrence under a fluorescence mi- maticpicopump(PV820,WorldPrecisionInstruments,USA).Exact croscope(OlympusBX51,AppliedImagingCorp.,SanJose,CA,USA), numberofcellswasconfirmedbydispensingtheinjectedvolume and the neutrophils occurrence (fluorescence intensity) was onto a microscope slide and by visual counting. After injection, determined using ImageJ programme (NIH public domain soft- embryoswereincubatedtorecoverforatleast1hat28(cid:2)C,dead ware; NIH is National Institutes of Health). The experiment was embryos wereremoved,andaliveembryoswere transferredinto performed three times using 10 embryos per a concentration. 24-wellplatescontaining1mLofembryowaterand10embryos Hepatotoxicity was determined using transgenic Tg(- perwell.Theinjectedxenograftsweretreatedwithdifferentdoses J.Delasoieetal./EuropeanJournalofMedicinalChemistry204(2020)112583 15 ofcomplex1and4(1/2,1/4and1/8ofIC50values),andmaintained IC50ang theconcentrationuponwhich50%ofembryos at33(cid:2)Cby120hpf.DMSO(0.25%)wasusedasanegativecontrol displayedanti-angiogenicphenotype andcisplatinasapositivecontrolatthemaximumtoleratedcon- ISVs intersegmentalvessels centration (34 mM) [105]. The survival and development of the LC50 concentrationofthecompoundinfeed(orwaterincase xenografted embryos was recorded every day until the end of offish)thatislethalfor50%ofexposedpopulation experiment.At3dayspostinjection(dpi),anesthetizedxenografts Tw therapeuticwindow were processed by fluorescent microscopy. We quantified tumor size, the number of xenografts with disseminated cells in caudal AppendixA. Supplementarydata region, and the number of disseminated cells per embryo. The tumour size was determined by the fluorescent images using Supplementary data to this article can be found online at ImageJprogramme.Theexperimentwasrepeatedtwotimes. https://doi.org/10.1016/j.ejmech.2020.112583. 4.13. Statisticalanalysis References Theexperimentalresultswereexpressedasmeanvalues±SD. [1] W.I.A.F.R.O.Cancer,LatestGlobalCancerData,2018. The differences in anti-angiogenic phenotypes between the un- [2] N. Keum, E. 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