Rhenium(I) tricarbonyl complexes of salicylaldehyde semicarbazones: synthesis, crystal structures and cytotoxicity.

JournalofInorganicBiochemistry119(2013)10–20 ContentslistsavailableatSciVerseScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio Rhenium(I) tricarbonyl complexes of salicylaldehyde semicarbazones: Synthesis, crystal structures and cytotoxicity ⁎ Junming Ho, Wan Yen Lee, Kelvin Jin Tai Koh, Peter Peng Foo Lee, Yaw-Kai Yan NaturalSciencesandScienceEducation,NationalInstituteofEducation,NanyangTechnologicalUniversity,1NanyangWalk,Singapore637616,Singapore a r t i c l e i n f o a b s t r a c t Articlehistory: A series of N,N-disubstituted salicylaldehyde semicarbazones (SSCs), HOCHCH_N-NHCONR, and their 6 4 2 Received23July2012 rhenium(I)tricarbonylcomplexes,[ReBr(CO)(SSC)],havebeensynthesisedandcharacterisedbyIRand1H 3 Receivedinrevisedform10October2012 NMRspectroscopy.Crystallographicanalysisofthecomplex[ReBr(CO)(HBu)](HBu =SSCwhereR=Bun) 3 2 2 2 2 Accepted23October2012 showed that the SSC acts as a bidentate ligand via its imino nitrogen and carbonyl oxygen atoms. The Availableonline31October2012 [ReBr(CO)(SSC)]complexesexhibitmoderatetohighcytotoxicitiestowardsMOLT-4cells(IC =1–24μM,cf. 3 50 18μMforcisplatin),andthemajorityofthemarevirtuallynon-toxicagainstnon-canceroushumanfibroblasts. Keywords: Apoptoticassaysof[ReBr(CO)(HBnz)](Bnz=benzyl)revealedthatitmediatescytotoxicityinMOLT-4cells Salicylaldehydesemicarbazone 3 2 2 Rhenium(I)carbonyl viaapoptosis.Thecomplex[ReBr(CO) 3 (H 2 Bnz 2 )]reactswithguanosinebyprotontransferfromthephenolic Cytotoxicity OHgrouptoN(7)ofguanosine.In(CD 3 ) 2 SO,[ReBr(CO) 3 (H 2 Bnz 2 )]undergoesfacileconversiontothedimeric Anti-cancer complex,[Re(CO)(HBnz)],viabromidedissociation. 3 2 2 Crystalstructure ©2012ElsevierInc.Allrightsreserved. Apoptosis 1.Introduction andaromaticN-substituentswerechosentogenerateSSCsandcom- plexes that span a wide range of lipophilicities. The cytotoxicity of Semicarbazones and thiosemicarbazones (Fig. 1) are versatile thesecompoundstowardstwohumancancercelllines(MOLT-4and compoundsthatexhibitawidespectrumofpharmacologicalactivi- MCF-7)andnon-canceroushumanfibroblastswasalsodetermined.In ties,includingantitumouractivity[1–8].Withtheincreasinginterest addition, 1H NMR experiments were conducted on a representative inmetal-baseddrugs,thereisalsoextensiveliteratureonthemedic- rheniumcomplextoinvestigateitsstabilityinsolutionandstudyits inalpropertiesofmetalcomplexesofthiosemicarbazones[9–19].The reaction with guanosine. This representative rhenium complex was chemistry and biological activity of semicarbazone complexes have alsoinvestigatedforitsabilitytotriggerapoptosisinhumanleukaemia receivedlessattention,however,althoughtheanti-cancerproperties cells(MOLT-4). ofvanadium,iron,copperandplatinumcomplexesofsalicylaldehyde semicarbazones have been reported [17,20–22]. Significant cancer 2.Experimental cell cytotoxicity has been reported for rhenium(I) tricarbonylcom- plexes of bis(diphenylphosphinomethyl)amines [23]. Rhenium(I) 2.1.Materialsandapparatus carbonyl2-(dimethylamino)ethoxidecomplexeswerealsofoundto be very active against suspended cancer cell lines (e.g. MOLT-4 and Allexperimentswerecarriedoutundernitrogenorargon,employing HL-60)andsolidtumours(e.g.MCF-7andSK2)[24].Wethereforedecid- standardSchlenktechniques.Chemicalreagents,unlessotherwisestat- ed to extend the above studies by investigating the cytotoxicity of ed, were used directly from commercial sources. Solvents were dried rhenium(I)tricarbonylsalicylaldehydesemicarbazonecomplexes.Whilst and distilled under nitrogen before use. The compound [ReBr(CO) ] rhenium(I)tricarbonylcomplexesofthiosemicarbazonesareknown(but 5 waspreparedaccordingtoapublishedmethod[29].ProtonNMRspectra not their biological activity) [25–28], rhenium(I) semicarbazone com- were recorded on a Bruker CRX400 spectrometer at 25°C. Elemental plexeshavenotbeenreported.Itisthusalsoofinteresttostudythestruc- analyseswerecarriedoutusinganElementarVarioMICROCUBEinstru- tureandsolutionchemistryofrhenium(I)salicylaldehydesemicarbazone ment.TheIRspectrawererecordedonKBrdiscsusingaPerkin–Elmer complexes. Spectrum100FT-IRspectrometer. Inthispaper,wereportthesynthesisandcharacterisationofase- riesofN,N-disubstitutedsalicylaldehydesemicarbazones(SSCs)and theirrhenium(I)tricarbonylcomplexes(Fig.2).Avarietyofaliphatic 2.2.PreparationofSSCligands ⁎ Correspondingauthor.Tel.:+6567903813;fax:+6568969414. The preparative protocol was adapted from published methods E-mailaddress:yawkai.yan@nie.edu.sg(Y.-K.Yan). [21,30]. For H 2 Hex 2 , H 2 Bnz 2 and H 2 Ph 2 , the carbamic chloride 0162-0134/$–seefrontmatter©2012ElsevierInc.Allrightsreserved. http://dx.doi.org/10.1016/j.jinorgbio.2012.10.011 J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 11 each)andthecombinedextractwasevaporatedunderreduced pressuretogivethesemicarbazide[R NC(O)NHNH ].Asolution 2 2 ofthesemicarbazideintoluene(5mL)wasplacedonamagnetic stirrer.Tothiswasaddedanequimolaramountofsalicylaldehyde andcatalyticamountsofp-toluenesulfonicacid.Themixturewas stirredatroomtemperaturefor30mintogiveayelloworwhite precipitateofthesemicarbazone,whichwasisolatedbyfiltra- tion.Afterwashingwithcoldtolueneanddryinginvacuo,the productwasrecrystallisedfromacetone–watermixture. 2.2.1.DataforH Bu Fig.1.Thegeneralstructureofsemicarbazones(X=O)andthiosemicarbazones(X=S); 2 2 Yield:(3.3g,76%).Anal.Calc.forC H N O :C,65.9;H,8.7;N, R1,R2,R3andR4=H,alkylorarylgroups. 14.4.FoundC,65.7;H,8.5;N,14.5.IR( 1 c 6 m 2 − 5 1) 3 :ν 2 (C_O)1640vs.1H NMR[(CD ) CO,ppm]:11.80(1H,s,phenolO\H),9.65(1H,s,hydra- 3 2 [R NC(O)Cl] was synthesised from the respective amines, whilst for zineN\H),8.28(1H,s,imineC\H),7.24(2H,m,Ph-H),6.87(2H,m, 2 theotherligands,thecarbamicchlorideswereobtainedcommercially. Ph-H),3.37(4H,t,J=7Hz,α-CH ),1.61(4H,m,β-CH ),1.35(4H,m, 2 2 γ-CH ),0.95(6H,t,J=6Hz,CH ). 2 3 (a) Preparationofcarbamicchlorides A solution of the secondary amine (15mmol) in 2.2.2.DataforH Hex 2 2 dichloromethane(25mL)wasaddeddropwiseviaacannula Yield:(3.4g,65%).Anal.Calc.forC H N O :C,69.1;H,9.6;N, 20 33 3 2 toastirredsolutionoftriphosgene(5.25mmol)andpyridine 12.1.FoundC,69.2;H,9.4;N,12.3.IR(cm−1):ν(C_O)1636vs.1H (30mmol)indichloromethane(25mL)inanice-waterbath. NMR[(CD ) CO,ppm]:11.80(1H,s,phenolO\H),9.70(1H,s,hydra- 3 2 Theresultantorangesolutionwaslefttostandatroomtempera- zineN\H),8.27(1H,s,imineC\H),7.23(2H,m,Ph-H),6.88(2H,m, tureforthreedaysundernitrogentogiveayellowsolutionofthe Ph-H),3.37(4H,t,J=7Hz,α-CH ),1.63(4H,m,β-CH ),1.33(12H, 2 2 carbamicchloride. m,γ-toε-CH ),0.90(6H,t,J=6Hz,CH ). 2 3 (b) PreparationofSSCs To the solution of carbamic chloride was added diethyl ether 2.2.3.DataforH Bnz 2 2 (60mL).Theresultantmixturewasaddeddropwisetoasolution Yield:(4.0g,75%).Anal.Calc.forC H N O :C,73.6;H,5.9;N, 22 21 3 2 ofhydrazinemonohydrate(60mmol)inethanol(30mL),with 11.6.FoundC,73.7;H,6.3;N,11.6.IR(cm−1):ν(C_O)1645vs.1H vigorousstirring.Thereactionmixturewaslefttostirfor30min NMR[(CD ) SO,ppm]:11.55(1H,s,phenolO\H),10.85(1H,s,hy- 3 2 beforethesolventswereremovedunderreducedpressure.The drazine N\H), 8.35 (1H, s, imine C\H), 7.2-7.4 (12H, m, Ph-H), residuewasextractedthreetimeswithdichloromethane(10mL 6.88(2H,m,Ph-H),4.52(4H,s,CH ). 2 Fig.2.Thesalicylaldehydesemicarbazonesandrhenium(I)carbonylcomplexessynthesisedinthiswork. 12 J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 2.2.4.DataforH MePh 7.86(1H, s, imine C\H), 7.2-7.4 (12H, m, Ph-H), 6.85 (1H, d, J= 2 Yield:(2.4g,60%).Anal.Calc.forC H N O :C,66.9;H,5.6;N, 8Hz, Ph-H), 6.69 (1H, t, J=8Hz, Ph-H), 4.64 (2H, br d, J=16Hz, 15 15 3 2 15.6.FoundC,66.9;H,5.9;N,15.4.IR(cm−1):ν(C_O)1661vs.1H CH ),4.43(2H,brd,J=16Hz,CH ). 2 2 NMR[(CD ) CO,ppm]:11.65(1H,s,phenolO\H),9.35(1H,s,hydra- 3 2 zineC\H),8.10(1H,s,imineC\H),7.1–7.5(7H,m,Ph-H),6.82(2H, 2.3.4.Datafor[ReBr(CO) (H MePh)] 3 2 m,Ph-H),3.31(3H,s,CH ). Yield: (50mg, 40%). Anal. Calc. for C H BrN O Re: C, 34.9; H, 3 18 15 3 5 2.4; N, 6.8. Found C, 34.9; H, 2.4; N, 6.8. IR (cm−1): ν(C`O) 2025 2.2.5.DataforH Ph vs, 1916 vs, 1881 vs; ν(C_O) 1626 vs. 1H NMR [(CD ) CO, ppm]: 2 2 3 2 Yield:(3.0g,60%).Anal.Calc.forC H N O :C,72.2;H,5.1;N, 8.25(1H,s,imineC\H),7.4–7.6(7H,m,Ph-H),7.04(1H,m,Ph-H), 20 17 3 2 12.9.FoundC,72.3;H,5.3;N,12.7.IR(cm−1):ν(C_O)1691vs.1H 6.95(1H,m,Ph-H),3.47(3H,m,CH ). 3 NMR[(CD ) CO,ppm]:11.58(1H,s,phenolO\H),9.61(1H,s,hydra- 3 2 zine N\H), 8.18 (1H, s, imine C\H), 7.1–7.5 (12H, m, Ph-H), 6.84 2.3.5.Datafor[ReBr(CO) (H Ph )] 3 2 2 (2H,m,Ph-H). Yield: (49mg, 36%). Anal. Calc. for C H BrN O Re: C, 40.5; H, 23 17 3 5 2.5; N, 6.1. Found C, 40.3; H, 2.6; N, 5.9. IR (cm−1): ν(C`O) 2027 2.2.6.DataforH BF vs, 1920 vs, 1889 vs; ν(C_O) 1618m. 1H NMR [(CD ) CO, ppm]: 2 3 2 Yield:(4.3g,81%).Anal.Calc.forC H N O :C,73.9;H,5.4;N, 8.34 (1H, s, imine C\H), 7.3-7.6 (12H, m, Ph-H), 7.05 (1H, m, 22 19 3 2 11.8.FoundC,73.7;H,5.6;N,11.6.IR(cm−1):ν(C_O)1655vs.1H Ph-H),6.93(1H,m,Ph-H). NMR [(CD ) SO, ppm]: 11.45 (1H, s, phenol O\H), 10.10 (1H, s, 3 2 hydrazineN\H),8.32(1H,s,imineC\H),7.42(2H,m,Ph-H),7.27 2.3.6.Datafor[ReBr(CO) (H BF)] 3 2 (8H,m,Ph-H),6.87(2H,m,Ph-H),3.08(4H,s,CH ). Yield: (78mg, 55%). Anal. Calc. for C H BrN O Re: C, 42.4; H, 2 25 19 3 5 2.7; N, 5.9. Found C, 42.1; H, 2.9; N, 5.9. IR (cm−1): ν(C`O) 2028 2.3.Preparationof[ReBr(CO) (SSC)]complexes vs, 1915 vs, 1892 vs; ν(C_O) 1623m. 1H NMR [(CD ) CO, ppm]: 3 3 2 10.77 (1H, s, hydrazine N\H), 8.31(1H, s, imine C\H), 7.3-7.7 Amixtureof[ReBr(CO) ](0.2mmol),SSC(0.2mmol)andtoluene (10H,m,Ph-H),7.02(1H,m,Ph-H),6.92(1H,m,Ph-H),3.5(4H,s, 5 (3mL)wasplacedinaSchlenktubewithamagneticstirrerbar.The CH ). 2 mixturewasrefluxedandtheprogressofthereactionwasmonitored hourlybyIRspectroscopy.FortheH Hex ligand,thereactionmix- 2.4.Formationof[Re(CO) (HBnz )] and[Re(CO) (HHex )] 2 2 3 2 2 3 2 2 turewasstirredat105°C,slightlybelowtheboilingpointoftoluene, becausedecompositionoccurredatrefluxingtemperature.Generally, Hexane was layered over dilute solutions of the mononuclear thereactionwascompleteafter5h. complexes, [ReBr(CO) (H Bnz )] and [ReBr(CO) (H Hex )], respec- 3 2 2 3 2 2 Some the complexes (where L=H Bu , H Bnz and H MePh) tively, in dichloromethane. Crystals of the corresponding dinuclear 2 2 2 2 2 began to precipitate out within the first 2h of reaction. For these complexes,[Re(CO) (HBnz )] and[Re(CO) (HHex )] ,wereformed 3 2 2 3 2 2 complexes,thecrudeproductwasisolatedbyfiltrationandwashed aftertwoweeksatroomtemperature. withasmallvolumeofcoldtoluene.Thecrudeproductwasdissolved inaminimumamountofdichloromethaneandlayeredwithhexane 2.4.1.Datafor[Re(CO) (HBnz )] 3 2 2 toaffordyellowcrystalswithinthreedays. IR (cm−1): ν(C`O) 2021 vs, and 1902 vs; ν(C_O) 1573s. 1H In other cases (L=H Hex , H Ph and H BF), the crude product NMR [(CD ) SO, ppm]: 12.40 (2H, s, hydrazine N\H), 7.91 (4H, d, 2 2 2 2 2 3 2 wasobtainedasawaxysolidbyremovingthetolueneunderreduced J=9Hz, Ph-H), 7.88 (2H, s, imine C\H), 7.4-7.2 (20H, m, Ph-H), pressure.Recrystallisationwascarriedoutbydissolvingthesolidina 6.78(4H,q,J=9Hz,Ph-H),4.55(8H,q,J=16Hz,CH ). 2 minimumamountofdichloromethaneandmixingwithapproximate- lytwovolumesofhexanetoformahomogeneoussolution.Thevial 2.4.2.Datafor[Re(CO) (HHex )] 3 2 2 wassealedwithsealingfilmandstoredat8°Cforthreedaystoob- IR (cm−1): ν(C`O) 2018 vs, 1910 vs, and 1879 vs; ν(C_O) tainyellowcrystalsofthecomplex.Thecrystalswerewashedwith 1585m. 1HNMR(CDCl ,ppm):13.83(2H,s,hydrazineN\H),7.82 3 asmallvolumeofhexane,crushedanddriedunderreducedpressure. (2H,s,imineC\H),7.50(2H,m,Ph-H),7.1–7.3(4H,m,Ph-H),6.82 (2H, m, Ph-H), 3.65 (2H, m, α-CH ), 3.0–3.3 (4H, m, α-CH ), 2.55 2 2 2.3.1.Datafor[ReBr(CO) (H Bu )] (2H, m, α-CH ), 1.46–1.06 (32H, m, β- to ε-CH ), 0.96–0.84 (12H, 3 2 2 2 2 Yield: (58mg, 45%). Anal. Calc. for C H BrN O Re: C, 35.6; H, m,CH ). 19 25 3 5 3 3.9; N, 6.6. Found C, 35.3; H, 3.9; N, 6.4. IR (cm−1): ν(C`O) 2028 vs, 1914 vs, 1902 vs; ν(C_O) 1625s. 1H NMR [(CD ) CO, ppm]: 2.5.X-raycrystallography 3 2 8.30(1H, s, imine C\H), 7.75 (1H, m, Ph-H), 7.55 (1H, m, Ph-H), 7.22 (1H, d, J=8Hz, Ph-H), 7.15 (1H, t, J=8Hz, Ph-H), 3.40 (4H, Crystals of [ReBr(CO) (H Bu )] were grown by layering hexane 3 2 2 m, α-CH ), 1.64 (4H, m, β-CH ), 1.37 (4H, m, γ-CH ), 0.94 (6H, t, overadilutesolutionofthecomplexindichloromethane.Singlecrys- 2 2 2 J=6Hz,CH ). tals of [Re(CO) (HBnz )] and [Re(CO) (HHex )] were obtained as 3 3 2 2 3 2 2 describedin Section 2.4.The crystalsweremounted on glassfibres 2.3.2.Datafor[ReBr(CO) (H Hex )] for data collectionat 298(2) K. The data werecollectedin the θ/2θ 3 2 2 Yield: (59mg, 42%). Anal. Calc. for C H BrN O Re: C, 39.6; H, modeusingaSiemensP4diffractometerwithMoKαradiation(λ= 23 33 3 5 4.8; N, 6.0. Found C, 39.6; H, 4.9; N, 6.1. IR (cm−1): ν(C`O) 2029 0.71073Å), and were corrected for absorption effects using ψ-scan vs, 1930 vs, 1887 vs; ν(C_O) 1626s. 1H NMR [(CD ) CO, ppm]: data.Thestructuresweresolvedbytheheavyatommethodandre- 3 2 8.30(1H, s, imine C\H), 7.75 (1H, m, Ph-H), 7.55 (1H, m, Ph-H), fined by full-matrix least-squares on F2. All non-hydrogen atoms 7.17(2H,m,Ph-H),3.44(4H,m,α-CH ),1.67(4H,m,β-CH ),1.35 wererefinedanisotropically.Hydrogenatomswereintroducedincal- 2 2 (12H,m,γ-toε-CH ),0.89(6H,t,J=6Hz,CH ). culatedpositionsandallowedtorideontheircarrieratoms.Crystal 2 3 andrefinementdataaregiveninTable1. 2.3.3.Datafor[ReBr(CO) (H Bnz )] ThecarbonatomsC(17),C(18),C(19)andC(18′)of[ReBr(CO) (H 3 2 2 3 2 Yield: (57mg, 40%). Anal. Calc. for C H BrN O Re: C, 42.3; H, Bu )]showedverylargedisplacementparameters,suggestingdisorder. 25 21 3 5 2 3.0; N, 5.9. Found C, 42.4; H, 3.1; N, 5.9. IR (cm−1): ν(C`O) 2030 Refinement was performed with two disordered positions (65% and vs, 1923 vs, 1878 vs; ν(C_O) 1616s. 1H NMR [(CD ) SO, ppm]: 35% occupancies, respectively) for each of these atoms, with the 3 2 J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 13 disorderedbutylchainsbeingrestrainedtohaveC\Cbondlengthsof whiletheadherentMCF-7cellsandfibroblastswereseededat20,000 1.5400±0.0001Å.TheC\C\Canglesinvolvingthedisorderedatoms cellsperwellandculturedfor24hpriortotreatmentwithcompounds. arerestrainedtobe109.50±0.01°.Adjacentcarbonatomsofeachdis- AllcompoundsweredissolvedinabsoluteDMSOataconcentra- orderedchainarerestrainedtohavesimilarU components,allowing tionof100mM.Thesesolutionswerethendilutedtovariousconcen- ij foragradualchangeinmagnitudeanddirectionoftheanisotropicdis- trations,usinga10%v/vsolutionofDMSOintheappropriateculture placementparametersfromtheα-carbonoutward. medium,beforebeingaddedtothecellculturewells.ThefinalDMSO Thehexylchainsof[Re(CO) (HHex )] wererestrainedtohaveC\C concentration was 1.25% v/v in each well. Six serial dilutions were 3 2 2 bond lengths of 1.5400±0.0001Å and C\C\C angles of 109.50± made for each compound in order to generate a dose–response 0.01°.Adjacentcarbonatomsofeachchainarerestrainedtohavesimi- curve, and six replicate wells were set up for each concentration of lar U components,allowing for a gradualchange in magnitude and thecompound.Eachplatecontainedablankwell(cell-freemedium- ij directionoftheanisotropicdisplacementparametersfromtheα-carbon onlywell),solventcontrolwellswhichcontainedcellsand1.25%v/v outward. DMSO,drugcolourcontrolwellswhichcontaineddrugandmedium only,andgrowthcontrolwellswhichcontainedonlycellsinmedium. Followingincubationofcellswithtestfractionsfor24hat37°C 2.6.Celltypesandcultureconditions and5%CO ,20μLofa5mg/mLsolutionofMTTwasaddedtoeach 2 well (final MTT concentration 1mg/mL). Three hours later, 100μL Thehumancancercelllines,MOLT-4(T-lymphoblasticleukaemia) of lysing solution (20% sodium dodecyl sulfate dissolved in 50% N, and MCF-7 (breast carcinoma), were obtained from the American N-dimethylformamide, pH adjusted to 4.7 with acetic acid) was TypeCultureCollection.Humanforeskinfibroblasts(asapassage1 addedtoeachwell.Afterthemicrotitreplatewaslefttostandover- culture) were obtained from the Department of Surgery, National night, the absorbance of the solution in each well was read at UniversityofSingapore.Growthofthefibroblastswassubsequently 570nm.Thepercentinhibitionofgrowthforeachconcentrationof expanded and a passage 4 culture was used for the cytotoxicity compound was calculated from the absorbance [31] and plotted assay. An RPMI-1640 based medium containing 10% foetal bovine againsttheconcentrationtogiveagraphfromwhichtheIC value serum wasused toculture MOLT-4 cells,while Dulbecco'sMinimal 50 (concentration of compound required to inhibit the growth of the EssentialMedium/Ham'sF12medium(1:1)supplementedwith10% cellsby50%)wasdetermined. foetalbovineserumwasusedforMCF-7cells.Humanfibroblastswere culturedinDulbecco'sMinimalEssentialMediumsupplementedwith 2.8.Assessmentofapoptosisinduction 20%foetalbovineserum.Allmediaalsocontained100units/mLofpen- icillin and 100μg/mL of streptomycin. The cells were maintained at Theabilityof[ReBr(CO) (H Bnz )]toinduceapoptosisinMOLT-4 37°Cina5%CO incubator. 3 2 2 2 cellswasdeterminedbythefollowingexperiments.Eachexperiment wascarriedoutthreetimestoensurethereproducibilityofresults. 2.7.Cytotoxicityassay 2.8.1.AnnexinV-FITCstainingassay Cytotoxicity of the compounds was determined using the MTT MOLT-4 cells (106 for each experiment) were grown in 5cm2 [3(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay sterile petri dishes and treated with [ReBr(CO) (H Bnz )] (7.3μM) 3 2 2 [31]onmicrotitreplates.ThesuspendedMOLT-4cellswereseededat orsolventcontrol(1%DMSO)for3,6,12and24h,respectively.Ap- 40,000 cells per well and exposed to compounds on the same day, optotic cell death was determined by staining treated cells with Annexin-V-FITC and counterstaining with propidium iodide (PI). Annexin-V-FITC binds to the exposed phosphatidylserine (PS) on theouterplasmamembraneofapoptoticcells,whilePIisexcludedbyvi- Table1 ablecellswithintactmembranes.Cellspositiveforannexin-V-FITCbut Crystallographic data and structure refinement details for [ReBr(CO)3H2Bu2], negativeforPIaredefinedasapoptotic,whilecellsstainedwithPIalone [Re(CO)3(HBnz2)]2and[Re(CO)3(HHex2)]2. isindicativeofnecrosis[32,33].Treatedcellswerewashedtwicewith Complex [ReBr(CO)3(H2Bu2)] [Re(CO)3(HBnz2)]2 [Re(CO)3(HHex2)]2 PBS,stainedwithannexin-V-FITC(BDPharmingen,51-65874X)andPI Chemicalformula C19H25BrN3O5Re C50H40N6O10Re2 C46H64N6O10Re2 (Sigma,20μg/mL),andanalysedonaDakoCytomationCyanLXflow Formulaweight 641.53 1257.28 1233.44 cytometerwith488nmlaserexcitation.EmissionfromFITCwasdetected Crystalsystem Monoclinic Monoclinic Triclinic at525nmandthatforPIat575nm. Spacegroup P2(1)/n C2/c P−1 a(Å) 12.525(4) 19.281(5) 10.031(2) 2.8.2.Detectionofcaspase3activity b(Å) 19.21(1) 13.684(3) 10.3750(9) c(Å) 19.297(5) 17.610(2) 13.953(1) Thecaspase3activitywasquantifiedaccordingtoinstructionsof α(°) 90 90 108.958(5) thecolorimetricassaykit(Clontech636217).MOLT-4cells(106for β(°) 94.68(2) 99.31(1) 109.44(1) each experiment) were treated with [ReBr(CO) (H Bnz )] (7.3μM) γ(°) 90 90 93.310(9) 3 2 2 orsolventcontrol(1%DMSO)forvariousdurations,afterwhichthe Volume(Å3) 4627(4) 4585(2) 1272.5(3) Z 8 4 1 cellswerecollectedbycentrifugationat200gfor5min.Thecellpel- Crystalsize 0.30×0.20×0.10 0.30×0.20×0.10 0.30×0.20×0.10 letwaslysedin50μLcelllysisbuffer.Aliquotsof50μLofcelllysate (mm3) were mixed with 50μL of reaction buffer and incubated with the θRange(°) 1.87to25.01 1.83to25.00 2.11to25.00 caspase 3 substrate Ac-DEVD-pNA (50μM) at 37°C for 1h. The Max.andmin. 0.9835and 0.8037and0.5103 0.9301and0.6603 caspase3activitywasdeterminedspectrophotometricallyat405nm. transmission 0.4515 μ(mm−1) 7.012 5.343 4.810 Reflections 9893 4832 5199 2.8.3.Westernblotanalysis collected MOLT-4 cells (106 for each experiment) were incubated with Ind re e fl p e e c n t d io e n n s t 8 = 0 0 9 . 3 03 [R 8 ( 4 i ] nt) 4 = 0 0 3 . 0 02 [R 9 ( 9 i ] nt) 4 = 3 0 7 . 5 02 [R 2 ( 3 i ] nt) [ReBr(CO) 3 (H 2 Bnz 2 )](7.3μM)forvariousdurations,afterwhichthe FinalRindices R1=0.0495, R1=0.0322, R1=0.0378, cellpelletwascollectedbycentrifugationat200gfor5minat4°C. [I>2sigma(I)] wR2=0.0974 wR2=0.0804 wR2=0.1004 The cells were lysed by resuspension in lysis solution (7M urea, Rindices R1=0.1075, R1=0.0436, R1=0.0461, 2M thiourea, 4% CHAPS, 30mM Tris, pH 9.0) and brief sonication (alldata) wR2=0.1119 wR2=0.0851 wR2=0.1044 on ice for 2min at 30% amplitude, 30s intervals. The cell lysates 14 J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 were centrifuged at 200g for 5min, after which the protein- Thefacileformationofthesedinuclearcomplexesissomewhatun- containing supernatants were quantified using the Bradford protein expected since the soft rhenium(I) centre is expected to bind more assayreagent(Biorad,500–0201).Equalamountsofproteins(20μg) stronglytobromidethantothephenoxideoxygen.Presumably,thefor- wereboiledat100°Cwith10μLof2×Laemmlisamplebuffer(Biorad, mationof[Re(CO) (HBnz )] and[Re(CO) (HHex )] isdrivenbyen- 3 2 2 3 2 2 161–0737)for2min,andloadedintoeachlaneofaSDS-PAGEgel(5% tropy rather than enthalpy, with the formation of 2mol of gaseous stacking, 10% resolving). After electrophoresis, the separated pro- HBrcontributingtotheincreaseinentropy.Thereactionmostlikely teinsweretransferredtoanitrocellulosemembrane(GEHealthcare, proceeds via the dissociation of bromide as the rate-limiting step, RPN2020D). The blots were blocked in phosphate buffered saline followedbycoordinationofthehydroxyl group,andthendeproton- (PBS) containing 0.05% Tween 20 and 5% non-fat milk for 1h at ation. That bromide dissociation is rate-limiting is supported by the roomtemperature.Afterblocking,theblotswereprobedwithrabbit fact that, whereas only 50% of [ReBr(CO) (H Bnz )] is converted to 3 2 2 polyclonal caspase 3 antibody at 1:2500 dilution (US Biological, [Re(CO) (HBnz )] after 24h at37°Cin (CD ) SO (seeSection3.6), 3 2 2 3 2 C2087-16). After washing twice with PBS Tween (PBS containing quantitative conversion occurs within 30min after addition of one 0.05% Tween 20), the blots were incubated with horseradish molarequivalentofAgNO to[ReBr(CO) (H Bnz )]atambienttemper- 3 3 2 2 peroxidase-conjugated polyclonal goat anti-rabbit secondary antibody ature (as shown by 1H NMR spectroscopy). Carballo et al. have ob- (1:5000, US Biological, 11904-39) for 1h at room temperature. Blots served the similar formation of a dinuclear [ReL(CO) ] complex 32 werewashedagain(3×5min)inPBSTweenandvisualisedusinganen- duringslowconcentrationofanacetonesolutionof[ReBr(CO) (HL)] 3 hancedchemiluminescencekit(GEhealthcare,RPN2124),inaccordance (HL=ferrocenylcarbaldehyde N-methylthiosemicarbazone) [27], in withthemanufacturer'srecommendations. which case deprotonation occurs at the hydrazidic nitrogen and Re-S-Rebridgesareformed. 3.Resultsanddiscussion 3.2.Infraredspectroscopy 3.1.Syntheses AlltheSSCssynthesisedshowanintenseν(C_O)absorptionband ThesyntheticprotocolforSSCsreportedbyLee[14]wasmodified at 1636–1691cm−1. The carbonyl stretching frequencies are consis- toimprovetheefficiencyofreactionbetweenthesemicarbazideand tentlyhigherfortheSSCsbearingaromaticsubstituentsontheterminal salicylaldehyde. In Lee's procedure, this reaction was carried out in nitrogen(H Ph ,H MePhandH BF).Thismayberationalisedinterms 2 2 2 2 ethanolat0°C,andtheSSCproductprecipitatedfromthesolution. ofresonanceeffects(Scheme1),wherethephenylringstabilisesreso- SomeoftheSSCsusedinthisworkarequitesolubleinethanol,how- nancestructureIIIbycompetingwiththecarbonylgroupforconjuga- ever, and did not precipitate out even after a significant portion of tionwiththeamidicnitrogenlonepair. ethanol was evaporated off. This resulted in poor yields for these Thecarbonylstretchingfrequenciesdecreaseoncoordinationofthe ligands (b50%). A protocol by Noblia [21] was thus used, in which SSCstorhenium(Table2).Thiscanbeattributedtothestabilisationof thereactionbetweenthesemicarbazideandsalicylaldehydewascar- resonance structures IIa and IIb through stabilisation of the negative riedoutinthepresenceofcatalyticamountsp-toluenesulfonicacidin charge on the carbonyl oxygen by the Lewis-acidic rhenium centre. tolueneatroomtemperature.Carryingoutthereactionatroomtem- Amongst the complexes, there is negligible difference between the peratureratherthaninanice-bathalsoincreasedtherateofreaction, ν(C_O)oftheSSCligandswithaliphaticsubstituentsandthatofthe andthecrudeproductusuallyprecipitatedoutofsolutionalmostin- SSCswitharomaticsubstituents.Thisimpliesthatresonancestructures stantaneously. This approach resulted in significantly higher yields IIaandIIbaremuchpreferredoverIIIinthecomplexes.Interestingly, (atleast60%)foralltheSSCs. the C_O stretching frequencies of [Re(CO) (HBnz )] and 3 2 2 Therhenium(I)tricarbonylcomplexesoftheSSCswerepreparedby [Re(CO) (HHex )] areeachlowerthanthoseof[ReBr(CO) (H Bnz )] 3 2 2 3 2 2 refluxinganequimolarmixtureoftheligandand[ReBr(CO) ]intoluene. and [ReBr(CO) (H Hex )], respectively, by ca. 40cm−1. Presumably, 5 3 2 2 Toluene was used because it is a non-coordinating solvent with high thephenoxideoxygen,beingapoorerσ-donorthanbromide,makes boiling point. Coordinating solvents such as tetrahydrofuran were therheniumatomsinthedinuclearcomplexesmoreLewis-acidicthan foundtocompetewiththeSSCsforcomplexationandresultedinpoor thoseinthemononuclearcomplexes,therebyfurtherfavouringreso- yieldsofthedesiredcomplexes. nancestructuresIIaandIIb. During an attempt to grow single crystals of the complexes [ReBr(CO) (H Bnz )]and[ReBr(CO) (H Hex )],crystalsofthedinuclear 3.3.ProtonNMRspectroscopy 3 2 2 3 2 2 complexes [Re(CO) (HBnz )] and [Re(CO) (HHex )] were obtained 3 2 2 3 2 2 instead(Fig.3).Thesecrystalsweregrownunderconditionssimilarto ThesignalsoftheSSCprotonsgenerallyshiftdownfieldoncoordi- thoseforrecrystallisingthemononuclearcomplexes,butmorediluteso- nation.Asingleimineprotonpeakappearsinthespectrumofeach lutionswereusedandconsequentlycrystalsonlyappearedafterapprox- complex,indicatingthattheSSCligandiscoordinatedtotherhenium imatelytwoweeks.Crystallographicanalysis(videinfra)revealedthat atominasingleconfiguration.Interestingly,themethyleneprotons thephenolicoxygenatomshavebeendeprotonated,andhavedisplaced of the H Bnz ligand give two broad doublets in the spectrum of 2 2 thebromideligandsoftherheniumcentrestoformabridgedstructure. [ReBr(CO) (H Bnz )],asopposedtoasharpsingletinthespectrum 3 2 2 Fig.3.Formationofthedinuclearcomplexes[Re(CO)3(HBnz2)]2and[Re(CO)3(HHex2)]2. J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 15 Table3 Selectedbondlengths(Å)andangles(°)for[ReBr(CO)3(H2Bu2)]. Bondlengths Re(1)-C(1) 1.90(1) Re(1′)-C(1′) 1.89(1) Re(1)-C(2) 1.90(1) Re(1′)-C(2′) 1.89(1) Re(1)-C(3) 1.90(1) Re(1′)-C(3′) 1.91(1) Re(1)-Br(1) 2.615(1) Re(1′)-Br(1′) 2.627(1) Re(1)-N(1) 2.167(7) Re(1′)-N(1′) 2.172(7) Re(1)-O(5) 2.160(6) Re(1′)-O(5′) 2.138(6) C(10)-N(1)a 1.28(1) C(10′)-N(1′)a 1.28(1) C(11)-O(5)b 1.25(1) C(11′)-O(5′)b 1.25(1) C(11)-N(3)c 1.32(1) C(11′)-N(3′)c 1.33(1) C(11)-N(2)d 1.37(1) C(11′)-N(2′)d 1.37(1) Bondangles C(11)-N(3)-C(12) 119.9(9) C(11′)-N(3′)-C(12′) 118.7(8) C(11)-N(3)-C(16) 123.6(8) C(11′)-N(3′)-C(16′) 123.8(7) Scheme1.Fouroftheresonancestructuresofthecarbamidegroupofsemicarbazones. C(12)-N(3)-C(16) 116.5(7) C(12′)-N(3′)-C(16′) 117.4(7) a ImineC_N. b AmideC_O. T C a o b m le pa 2 rison of carbonyl stretching frequencies (ν˜/cm−1) between SSCs and c d A H m yd id ra e z C id \ e N C . \N. [ReBr(CO)3(SSC)]complexes. Ligand ν˜(ligand) ν˜(complex) ν˜(ligand)–ν˜(complex) semicarbazone, HOC 6 H 4 CH_N\NHCONH 2 [34]. The three angles abouttheterminalnitrogenatomofbothmoleculesintheunitcell H H2 2 B H u e 2 x2 1 1 6 6 4 3 0 6 1 1 6 6 2 2 5 6 1 1 5 0 [N(3)andN(3′)]sumupto360°,indicatingatrigonalplanargeometry H2Bnz2 1645 1616 29 forthenitrogenatoms.Thisisconsistentwiththedominanceofreso- H2MePh 1661 1626 35 nance structure IIa (Scheme 1), where the terminal nitrogen is sp2 H2Ph2 1691 1618 73 hybridised.Thechelateringsarevirtuallyplanar,withmaximumdevi- H2BF 1655 1623 32 ations of 0.01 [for C(11)] and 0.03Å [for N(1′) and N(2′)] from the respectivemeanplanes.Thecoordinationplaneofeachterminalnitro- ofthefreeligand.Thismaybeattributedtothestabilisationofreso- genisalmostparalleltothemeanplaneofthechelateringtowhichthe nancestructureIIaoncomplexation(seeSection3.2),whichresults nitrogenisattached(anglesofdeviationare3°each),providingfurther in hindered rotation of the Bnz N−CO bond and hence non- evidencefortheconjugationofthenitrogenlonepairwiththeC_O 2 equivalence of the methylene protons. Similar effects are observed doublebond.Notably,anintramolecularhydrogenbondexistsbetween in the methylene proton signals of [ReBr(CO) (H Hex )] and the phenolic oxygen and the hydrazine NH group of each molecule 3 2 2 [ReBr(CO) (H Bu )]. (Fig.4).Thisismadepossiblebytheadoptionofthecisconfiguration 3 2 2 bytheiminegroupinthecomplex(asopposedtothetransconfigura- tionexpectedforthefreeligand[34]).Theiminegroupsarenotcopla- 3.4.Crystalstructures narwiththephenylringsbearingthem,thustheiminoprotonsdeviate significantlyfromthemeanplanesofthephenylrings[by0.30Åforthe 3.4.1.Crystalstructureof[ReBr(CO) 3 (H 2 Bu 2 )] protononC(10)and0.29ÅforthatonC(10′)].TheRe-Br,Re-COand There are two independent molecules of the complex Re-NC(imine) distances are close to those observed in analogous [ReBr(CO) 3 (H 2 Bu 2 )]intheunitcell(Fig.4).TheH 2 Bu 2 ligandactsas rhenium(I)tricarbonylcomplexes[25,26,28]. abidentateligand,bindingtotherheniumcentreviatheiminenitro- genandcarbonyloxygen.Thisbindingmodetothe{Re(CO) }unitis 3 analogous to that of thiosemicarbazones, wherein the sulfur and 3.4.2.Crystalstructuresof[Re(CO) (HBnz )] and[Re(CO) (HHex )] 3 2 2 3 2 2 imine nitrogen atoms are coordinated to rhenium [25,26,28]. The The deprotonated SSC ligands adopt a tridentate coordination lengthsoftheC_O, C_NandC\NbondswithintheH Bu ligand modein[Re(CO) (HBnz )] (Fig.5)and[Re(CO) (HHex )] (Fig.6), 2 2 3 2 2 3 2 2 (Table3)aresimilartothoseobservedintheparentsalicylaldehyde withtheiminenitrogenandcarbonyloxygenbindingtoonerhenium Fig.4.Thetwoindependentmoleculesinthecrystalstructureof[ReBr(CO)3(H2Bu2)](40%probabilityellipsoids);hydrogenatomsbondedtothebutylandphenylgroupsare omittedforclarity.AnintramolecularhydrogenbondexistsbetweenO(4)andN(2)andbetweenO(4′)andN(2′)[O(4)∙∙∙H(2A)1.98Å,O(4)∙∙∙N(2)2.64Å,∠O(4)∙∙∙H(2A)-N(2) 133°;O(4′)∙∙∙H(2′A)2.03Å,O(4′)∙∙∙N(2′)2.65Å,∠O(4′)∙∙∙H(2′A)-N(2′)128°,N\H0.860Å]. 16 J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 Fig.5.Crystalstructureof[Re(CO)3(HBnz2)]2;allhydrogenatoms,exceptthoseoftheiminogroups[onC(7)andC(7A)],areomittedforclarity.Themoleculehasacrystallographic centreofsymmetry. Fig.6.Crystalstructureof[Re(CO)3(HHex2)]2;hydrogenatomsareomittedforclarity.Themoleculehasacrystallographiccentreofsymmetry. J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 17 Table4 aretheonlyoneswithappreciabletoxicitytowardsMCF-7cells.They Selected bond lengths (Å) and angles (°) for [Re(CO)3(HBnz2)]2 and arealsosignificantlymoreactivethancisplatinagainstbothMOLT-4 [Re(CO)3(HHex2)]2. andMCF-7cells. [Re(CO)3(HBnz2)]2 [Re(CO)3(HHex2)]2 ThecytotoxicityofthecomplexestowardsMOLT-4cellsappears tobecorrelatedwiththelipophilicityoftheligands(Table5).Cyto- Bondlengths Re(1)-C(11) 1.909(7) Re(1)-C(1) 1.899(8) toxicity increases with increasing lipophilicity from H 2 MePh to Re(1)-C(12) 1.911(6) Re(1)-C(2) 1.925(8) H Bnz /H BF,anddecreasesthereafterwithafurtherincreaseinlipo- 2 2 2 Re(1)-C(13) 1.904(6) Re(1)-C(3) 1.897(8) philicity.Thissuggeststhatabsorptionacrosslipidmembranesinthe Re(1)-O(2A) 2.145(5) Re(1)-O(5) 2.144(5) cellsisanimportantfactordeterminingthecytotoxicityofthecom- Re(1)-N(1A) 2.189(4) Re(1)-N(1) 2.190(5) Re(1)-O(1) 2.158(4) Re(1)-O(4A) 2.156(4) pounds[39]. C(7)-N(1)a 1.300(7) C(10)-N(1)a 1.272(9) Allthecompoundstestedexcept[ReBr(CO) 3 (H 2 BF)]exhibitnegligi- C(8)-O(2)b 1.269(6) C(11)-O(5)b 1.247(9) bletoxicityagainstfibroblasts,showingthatmostofthesecompounds C(8)-N(3)c 1.322(7) C(11)-N(3)c 1.335(9) areselectiveagainstcancercells.Thisisanimportantpropertyforany C(8)-N(2)d 1.373(7) C(11)-N(2)d 1.352(9) chemotherapeutic agent, since harmful side effects of the treatment Bondangles will be minimised. The complex [ReBr(CO) 3 (H 2 Bnz 2 )] stands out as C(8)-N(3)-C(9) 118.5(5) C(11)-N(3)-C(21) 119.9(6) the only one with appreciable activity against both MOLT-4 and C(8)-N(3)-C(10) 123.9(5) C(11)-N(3)-C(31) 123.4(6) MCF-7cells,butnegligibletoxicitytowardsfibroblasts. C(9)-N(3)-C(10) 117.3(5) C(21)-N(3)-C(31) 116.7(5) a ImineCN. 3.6.Solutionchemistry b AmideCO. c AmideCN. d HydrazideCN. Thesolutionchemistryof[ReBr(CO) 3 (H 2 Bnz 2 )]wasinvestigated by 1HNMRspectroscopy.Thiscomplexwaschosenasitshowsthe centreandthephenoxooxygentotheother.Ingeneral,thelengthsof bestactivityprofileinthecytotoxicityassays.Sincethesolubilityof the C_O, C_N and C\N bonds within the SSC ligands (Table 4) [ReBr(CO) (H Bnz )]inwateristoolowforNMRstudies,investiga- 3 2 2 do not differ significantly from those in HOC H CH_N\NHCONH tionswereconductedin(CD ) SO. 6 4 2 3 2 [34], although there is noticeable variation in the lengths of these The spectral changes that occurred after incubating a solution bondsbetween[Re(CO) (HBnz )] and[Re(CO) (HHex )] .Likethose of [ReBr(CO) (H Bnz )] at 37°C for 24h (Fig. 7) are consistent 3 2 2 3 2 2 3 2 2 of[ReBr(CO) (H Bu )],theamidenitrogensofboth[Re(CO) (HBnz )] with the conversion of the compound to the dinuclear complex 3 2 2 3 2 2 and [Re(CO) (HHex )] have trigonal planar coordination geometry [Re(CO) (HBnz )] . In particular, signals attributed to the dinuclear 3 2 2 3 2 2 (sumofbondangles=360°).Theiminoprotonsof[Re(CO) (HBnz )] complex (quartets at 4.54 and 6.78ppm) appeared while those of 3 2 2 and [Re(CO) (HHex )] deviate more from the mean planes of the the mononuclear complex (broad doublets at 4.43 and 4.64, triplet 3 2 2 salicylaldehyde phenyl rings than those of [ReBr(CO) (H Bu )], with at 6.70, and doublet at 6.83ppm) decreased in intensity. Since the 3 2 2 the proton of [Re(CO) 3 (HBnz 2 )] 2 deviating by 0.41Å and that of quartet at 6.78ppm overlaps with the doublet at 6.83ppm but not [Re(CO) 3 (HHex 2 )] 2 by0.42Å. thetripletat6.70ppm,thepercentconversionof[ReBr(CO) 3 (H 2 Bnz 2 )] to [Re(CO) (HBnz )] could be calculated to be about 50% from the 3 2 2 3.5.Cytotoxicity expression[(I −I )/(I +I )],whereI isthetotalintegratedintensity a b a b a oftheoverlappingsignalsat6.78and6.83ppmandI istheintegrated b ThecytotoxicitiesoftheSSCsandtheirrheniumtricarbonylcom- intensityofthetripletat6.70ppm.Theseobservationsareconsistent plexeswereevaluatedagainstMOLT-4(cisplatin-sensitive[35])and withthefacileconversionof[ReBr(CO) (H Bnz )]to[Re(CO) (HBnz )] 3 2 2 3 2 2 MCF-7(cisplatin-resistant[36–38])cells.Toassesstheselectivityof duringcrystalgrowingexperiments(Section3.1). these compounds towards cancer cells, their cytotoxicities against To investigate the possible interaction of [ReBr(CO) (H Bnz )] 3 2 2 fibroblasts (non-cancerous) were also determined. Cisplatin was with DNA, we also examined the reaction of [ReBr(CO) (H Bnz )] 3 2 2 included in the cytotoxicity assays for comparison. The results are withguanosine.Additionofanequimolaramountofguanosinetoa showninTable5. freshly-preparedsolutionof[ReBr(CO) (H Bnz )]causesimmediate 3 2 2 Generally, the [ReBr(CO) (SSC)] complexes exhibit similar or changes to the 1H NMR signals of [ReBr(CO) (H Bnz )] (Fig. 8), 3 3 2 2 higher activity than the corresponding SSC ligands against MOLT-4 mostnotablyanupfieldshiftoftheimineprotonsignalfrom7.87to cells, but are generally less active than the ligands against MCF-7 7.77ppmandanupfieldshiftofthedoubletat6.83ppm(assigned cells. The complexes [ReBr(CO) (H Bnz )] and [ReBr(CO) (H BF)] totheprotonorthotothehydroxylgroup)to6.78ppm.Theseshifts 3 2 2 3 2 are consistent with the deprotonation of the hydroxyl group of Table5 [ReBr(CO) (H Bnz )]. Correspondingly, the N(1)-H (10.63ppm), 3 2 2 IC50valuesoftheSSCligandsandtheirrheniumcarbonylcomplexes.Standarderrors C(8)-H (7.93ppm) and NH (6.46ppm) signals of guanosine are areshowninparentheses(N=6).TheLogPvalues(logarithmofthepartitioncoeffi- 2 shifteddownfieldto10.98,8.46andca.6.7ppm,respectively,indi- cientbetweenn-octanolandwater)werecalculatedusingtheChemDrawUltra11.0 software. catingtheprotonationofguanosine,predominantlyatN(7).Addition of 1.0, 6.7 and 13.4 molar equivalents of HCl (aq) to guanosine in Ligand(L) LogP IC50(μM) (CD ) SO causes the chemical shift of C(8)-H to plateau at 3 2 L [ReBr(CO)3L] 9.33ppm. Taking this to indicate 100% protonation of guanosine, MOLT-4 MCF-7 Fibroblast MOLT-4 MCF-7 Fibroblast theextentofprotonation of guanosineobservedin thepresence of [ReBr(CO) (H Bnz )] is calculated to be ca. 36%, assuming that the H2MePh 2.59 56(3) >125 >125 24(6) >125 >125 3 2 2 H2Ph2 3.63 >63a >63a >63a 15(1) >125 >125 C(8)-Hsignalat8.46ppmistheweightedaverageofunprotonated H2Bu2 4.43 5(3) 5(4) >125 15(1) >125 >125 andprotonatedguanosine.NochangeisobservedfortheC\Hproton H2Bnz2 4.69 11(1) 8(3) 100(10) 7.3(0.4) 24(4) >125 resonances of the ribose moiety [5.75, 4.39, 4.12 and 3.92ppm for H2BF 5.02 b b b 1.0(0.1) 35(4) 9(1) protonsonC(1′)–C(4′),respectively;signalsofprotonsonC(5′)are H2Hex2 6.55 56(6) 8(4) >125 22(2) >125 >125 Cisplatin – 18(1) 71(8) 28(2) 18(1) 71(8) 28(2) maskedbythewaterpeak],hencethereisprobablynegligibleinter- actionbetweentheriboseunitand[ReBr(CO) (H Bnz )]. a Highestconcentrationtestedasthecompoundhaspoorsolubilityinthemedium 3 2 2 Interestingly,noreactionwasobservedbetweenguanosineandthe usedforthecytotoxicityassay. b Notsolubleinthemediumusedforthecytotoxicityassay. free ligand, H 2 Bnz 2 , within 24h, suggesting that H 2 Bnz 2 is a much 18 J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 Fig.7.ProtonNMRspectraof[ReBr(CO)3(H2Bnz2)](12mM)in(CD3)2SO:(a)obtainedfromafreshly-preparedsolution,(b)recordedafterthesolutionwasincubatedat37°Cfor 24h.Thepeakat5.76ppmisduetoadventitiousCH2Cl2. weaker acid than [ReBr(CO) (H Bnz )]. The higher acid strength of shieldingofthebenzylprotons.The{Re(CO) }groupprobablyalsocon- 3 2 2 3 [ReBr(CO) (H Bnz )]maybeduetothepresenceofanintramolecular tributestothehigheracidityofthecomplexsincethenegativecharge 3 2 2 (hydrazine)NH···O(phenol)hydrogenbondinthecomplex,assuming on the phenoxo oxygen can be delocalised onto the imino nitrogen, thathydrogenbondingsimilartothatobservedin[ReBr(CO) (H Bu)] whereitisstabilisedbytheLewisacidicrhenium(I)centre. 3 2 2 (seeSection3.4.1)occursin[ReBr(CO)(H Bnz )].Intramolecularhydro- Incubating the guanosine-[ReBr(CO) (H Bnz )] mixture at 37°C 3 2 2 3 2 2 genbondingbetweenphenolicoxygenatomsandNHprotonsisknown for 24h resulted in the appearance of resonances due to to increase the acid strength of phenols [40]. The existence of the [Re(CO) (HBnz )] (Fig.8).Takingthemultipletat4.39ppmtobecom- 3 2 2 NH···Ointeractionissupportedbythefactthatthebenzylprotonsignals posedofresonancesoftheguanosineC(2′)-Hprotonandtwooftheben- at4.43and4.64ppmshiftupfieldto4.39and4.58ppm,respectively(see zylprotonsof[ReBr(CO)(H Bnz )],andthatat4.54ppmtobecomposed 3 2 2 Fig.8),whenguanosineisaddedto[ReBr(CO)(H Bnz )]:deprotonation ofresonancesduetoallfourofthebenzylprotonsof[Re(CO)(HBnz)] 3 2 2 3 2 2 ofthephenolicoxygenincreasestheelectrondensityonthehydrazine and two of the benzyl protons of [ReBr(CO) (H Bnz )], the percent 3 2 2 nitrogen, which increases the dominance of resonance structure IIb conversionof[ReBr(CO) (H Bnz )]to[Re(CO) (HBnz )] wascalculated 3 2 2 3 2 2 (Scheme 1) at the expense of structure IIa, thereby increasing the tobeabout50%fromtheintensityratioofthemultiplets.Thesimilar Fig.8.ProtonNMRspectraof:(a)guanosine,(b)[ReBr(CO)3(H2Bnz2)],(c)freshly-preparedmixtureofguanosineand[ReBr(CO)3(H2Bnz2)],(d)guanosine-[ReBr(CO)3(H2Bnz2)] mixtureincubatedat37°Cfor24h.Allsolutesweredissolvedin(CD3)2SOat12mMconcentration.TheguanosineC(1′)-HsignaloverlapswiththatofadventitiousCH2Cl2at 5.76ppm. J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 19 complex to be present in significant amounts in the cells, however, since its rate of formation would be negligible under conditions of high dilution in the assays. Hence, the unsaturated intermediate [Re(CO) (H Bnz )]+islikelytoacceptelectronpairsfromdonorgroups 3 2 2 ofbiomoleculesinstead.TheNMRspectraof[ReBr(CO) (H Bnz )]and 3 2 2 guanosine-[ReBr(CO) (H Bnz )]mixtureshownoapparentsignalsas- 3 2 2 signabletouncomplexedH Bnz ,henceliganddisplacementdoesnot 2 2 occurtoasignificantextentwithin24hat37°C.GiventhatDMSOis astrongdonorsolvent,thelackofliganddisplacementinDMSOsug- geststhatliganddisplacementisnotinvolvedinthemechanismofcy- totoxicityofrheniumsalicylaldehydesemicarbazonecomplexes. 3.7.Assessmentofapoptosisinductionby[ReBr(CO) (H Bnz )] 3 2 2 MOLT-4cellstreatedwith[ReBr(CO) (H Bnz )]showedanincreas- 3 2 2 ing degree of Annexin-V-FITC positivity over time (Fig. 9), indicating thatthecellswereundergoingapoptosis.Occurrenceofapoptosiswas furtherconfirmedbymeasuringthecaspase3activityoftreatedcells. Activationofcaspase3activityisintegraltotheinitiationofapoptosis [41,42]. MOLT-4 cells treated with [ReBr(CO) (HBnz )] showed in- 3 2 2 creasedcaspase3activityoverthe24-hperiodoftreatment(Fig.10a), with activity reaching its maximum at 6h. Correspondingly, western blottingwithantibodiesagainstcaspase3alsorevealedthepresenceof anactivecleaved17kDacaspase3subunit(Fig.10b). Fig. 9. Flow cytometric assessment of apoptosis in MOLT-4 cells treated with [ReBr(CO)3(H2Bnz2)] for 3, 6, 12 and 24h, respectively: (a) cells stained with 4.Summaryandconclusion Annexin-V-FITConly;(b)cellsstainedwithpropidiumiodideonly. A series of N,N-disubstituted salicylaldehyde semicarbazones percentconversiontothatobservedintheabsenceofguanosineisconsis- (SSCs)andtheirrhenium(I)tricarbonylcomplexes,[ReBr(CO) (SSC)], 3 tentwiththeearlierdeduction(Section3.1)thattherate-limitingstepfor havebeensynthesisedandscreenedforcytotoxicityagainstMOLT-4, dimerisationisbromidedissociation(andnotdeprotonation). MCF-7 and human fibroblast cells. The dinuclear complexes, Therelativelystrongacidityof[ReBr(CO) (H Bnz )]suggeststhat [Re(CO) (HBnz )] and [Re(CO) (HHex )] , are formed during 3 2 2 3 2 2 3 2 2 thedeprotonatedcomplex[ReBr(CO) (HBnz )]−isanimportantspe- slow evaporation of dichloromethane-hexane solutions of 3 2 ciesinteractingwithcellsduringthecytotoxicityassay,whilethefacile [ReBr(CO) (H Bnz )] and[ReBr(CO) (H Hex )], respectively.Crys- 3 2 2 3 2 2 formation of [Re(CO) (HBnz )] suggests that bromide dissociation tal structures have been determined for [ReBr(CO) (H Bu )], 3 2 2 3 2 2 (i.e., solvolysis) occurs quite readily. It is unlikely for the dinuclear [Re(CO) (HBnz )] and [Re(CO) (HHex )] . On the whole, the 3 2 2 3 2 2 Fig.10.Detectionofcaspase3activityinMOLT-4cellstreatedwith[ReBr(CO)3(H2Bnz2)]:(a)colorimetricmeasurementofcaspase-specificcleavageofAc-DEVD-pNA;(b)western blotanalysisofproteinsseparatedbySDS-PAGE. 20 J.Hoetal./JournalofInorganicBiochemistry119(2013)10–20 [ReBr(CO) (SSC)]complexesexhibitsimilarorhigheractivitythan [9] M. Belicchi-Ferrari, F. Bisceglie, G. Pelosi, P. Tarasconi, Polyhedron 27 (2008) 3 1361–1367. thecorrespondingSSCligandsagainstMOLT-4cells,withtwocom- [10] M.Baldini,M.Belicchi-Ferrari,F.Bisceglie,G.Pelosi,S.Pinelli,P.Tarasconi,Inorg. plexesshowingparticularlyhighactivities.Mostofthetestedcom- Chem.42(2003)2049–2055. pounds demonstrate negligible activity against non-cancerous [11] M. Belicchi-Ferrari, F. Bisceglie, C. Casoli, S. Durot, I. Morgenstern-Badara, G. fibroblasts. Of these compounds, [ReBr(CO) (H Bnz )] shows the Pelosi,E.Pilotti,S.Pinelli,P.Tarasconi,J.Med.Chem.48(2005)1671–1675. 3 2 2 [12] M.Belicchi-Ferrari,G.Gasparri-Fava,E.Leporati,G.Pelosi,R.Rossi,P.Tarasconi,R. best cytotoxicity profile, being the only one that has high activity Albertini,A.Bonati,P.Lunghi,S.Pinello,J.Inorg.Biochem.70(1998)145–154. against both MOLT-4 and MCF-7 cells, and negligible toxicity towards [13] M.Belicchi-Ferrari,F.Bisceglie,G.Pelosi,P.Tarasconi,R.Albertini,A.Bonati,P. fibroblasts. It also induces apoptosis in MOLT-4 cells. In (CD) SO, Lunghi,S.Pinelli,J.Inorg.Biochem.83(2001)169–179. 3 2 [14] Z.Afrasiabi,E.Sinn,J.Chen,Y.Ma,A.L.Rheingold,L.N.Zakharov,Inorg.Chim.Acta [ReBr(CO) 3 (H 2 Bnz 2 )] reacts with guanosine by proton transfer from 357(2004)271. thephenolicOHgrouptoN(7)ofguanosine.Therelativelystrongacidity [15] N.C. Kasuga, K. Sekino, M. Ishikawa, A. Honda, M. Yokoyama, S. Nakano, N. of the OH group may be attributed to intramolecular (hydrazine) Shimada,C.Koumo,K.Nomiya,J.Inorg.Biochem.96(2003)298–310. [16] C.R.Kowol,R.Berger,R.Eichinger,A.Roller,M.A.Jakupec,P.P.Schmidt,V.B.Arion, NH···O(phenol)hydrogenbonding,whichismadepossiblebytheadop- B.K.Keppler,J.Med.Chem.50(2007)1254–1265. tionofthecisconfigurationbytheiminegroupin[ReBr(CO) 3 (H 2 Bnz 2 )]. [17] J.Patole,S.Padhye,M.S.Moodbidri,N.Shirsat,Eur.J.Med.Chem.40(2005) DisplacementoftheSSCligandisprobablynotinvolvedinthemechanism 1052–1055. [18] U.K.Mazumder,M.Gupta,S.S.Karki,S.Bhattacharya,S.Rathinasamy,S.Thangavel, ofcytotoxicityofrheniumsalicylaldehydesemicarbazonecomplexes,but Chem.Pharm.Bull.52(2004)178–185. interactionwithbiomoleculesafterlossofbromideisplausible. [19] S.Grguric-Sipka,C.R.Kowol,S.-M.Valiahdi,R.Eichinger,M.A.Jakupec,A.Roller,S. Shova,V.B.Arion,B.K.Keppler,Eur.J.Inorg.Chem.(2007)2870–2878. [20] P.Noblia,E.J.Baran,L.Otero,P.Draper,H.Cerecetto,M.Gonzalez,O.E.Piro,E.E. Acknowledgements Castellano,T.Inohara,Y.Adachi,H.Sakurai,D.Gambino,Eur.J.Inorg.Chem. (2004)322–328. TheauthorswouldliketothankDr.ShuUinGan(Departmentof [21] W.Y.Lee,P.P.F.Lee,Y.K.Yan,M.Lau,Metallomics2(2010)694–705. [22] J.Rivadeneira,D.A.Barrio,G.Arrambide,D.Gambino,L.Bruzzone,S.B.Etcheverry, Surgery,NationalUniversityofSingapore)fortheprovisionofhuman J.Inorg.Biochem.103(2009)633–642. fibroblasts.FinancialsupportfromtheNationalInstituteofEducation, [23] J. Zhang, J.J. Vittal, W. Henderson, J. Wheaton, I.H. Hall, T.S. Hor, Y.K. Yan, J. Singapore(grantnumberRI4/05LPF)isgratefullyacknowledged.We Organomet.Chem.650(2002)123–132. alsothankMs.Ying-ChangTingforthetechnicalassistance. [24] W.Wang,Y.K.Yan,T.S.Hor,J.J.Vittal,J.R.Wheaton,I.H.Hall,Polyhedron 21 (2002)1991–1999. [25] A.Nunez-Montenegro,R.Carballo,E.M.Vazquez-Lopez,Polyhedron27(2008) AppendixA.Supplementarydata 2867–2876. [26] G. Pereiras-Gabian, E.M. Vazquez-Lopez, U. Abram, Z. Anorg. Allg. Chem. 630 (2004)1665–1670. Crystallographic data (excluding structure factors) for the com- [27] R.Carballo,J.S.Casas,E.Garcia-Martinez,G.Pereiras-Gabian,A.Sanchez,J.Sordo, plexes[ReBr(CO) 3 (H 2 Bu 2 )],[Re(CO) 3 (HBnz 2 )] 2 and[Re(CO) 3 (HHex 2 )] 2 E.M.Vazquez-Lopez,J.C.Garcia-Monteagudo,U.Abram,J.Organomet.Chem.656 may be obtained from the Cambridge Crystallographic Data Centre (2002)1–10. [28] I.G.Santos,U.Abram,R.Alberto,E.V.Lopez,A.Sanchez,Inorg.Chem.43(2004) (CCDC,12UnionRoad,CambridgeCB21EZ,UK;fax:_/44-1223-336- 1834–1836. 033; e-mail: deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam. [29] S.P.Schmidt,W.C.Trogler,F.Basolo,Inorg.Synth.28(1990)160–165. ac.uk)onquotingthedepositorynumbersCCDC763433,763434and [30] P.F.Lee,C.T.Yang,D.Fan,J.J.Vittal,J.Ranford,Polyhedron22(2003)2781–2786. [31] M.B.Hansen,T.Fojo,J.Immunol.Methods119(1989)203–210. 763435,respectively. [32] J.F.Kerr,A.H.Wyllie,A.R.Currie,Br.J.Cancer26(1972)239–257. Supplementarydatatothisarticlecanbefoundonlineatdoihttp:// [33] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, J. Immunol. dx.doi.org/10.1016/j.jinorgbio.2012.10.011. Methods184(1995)39–51. [34] J. Valdes-Martinez, R.A. Toscano, R. Salcedo, R. Cea-Olivares, A. Melendez, Monatsh.Chem.121(1990)641–647. References [35] D.M.Fan,C.T.Yang,J.D.Ranford,J.J.Vittal,P.F.Lee,J.Chem.Soc.DaltonTrans. (2003)2680–2685. [1] H.Beraldo,D.Gambino,Minirev.Med.Chem.4(2004)31–39. [36] W. Friebolin, G. Schilling, M. Zoller, E. Amtmann, J. Med. Chem. 48 (2005) [2] A.Quiroga,C.Ranninger,Coord.Chem.Rev.248(2004)119–133. 7925–7931. [3] Z.Afrasiabi,E.Sinn,W.Lin,Y.Ma,C.Campana,S.B.Padhyé,J.Inorg.Biochem.99 [37] Y.J.Chua,C.Steer,D.Yip,CancerTreat.Rev.30(2004)521–543. (2005)1526–1531. [38] F.M.Muggia,T.Fojo,J.Chemother.16(Suppl.4)(2004)77–82. [4] Z.Afrasiabi,E.Sinn,S.Padhye,S.Dutta,C.Newton,C.E.Anson,A.K.Powell,J.Inorg. [39] C.A.Lipinski,F.Lombardo,B.W.Dominy,P.J.Feeney,Adv.DrugDeliv.Rev.46 Biochem.95(2003)306–314. (2001)3–26. [5] R.W.Byrnes,M.Mohan,W.E.Antholine,R.X.Xu,D.H.Petering,Biochemistry29 [40] D.Kanamori,A.Furukawa,T.Okamura,H.Yamamoto,N.Ueyama,Org.Biomol. (1990)7046–7053. Chem.3(2005)1453–1459. [6] I.H.Hall,S.Y.Chen,B.J.Barners,D.X.West,Metal-BasedDrugs6(1999)143–147. [41] Z.Tao,J.Goodisman,H.S.Penefsky,A.K.Souid,Mol.Pharm.4(2007)583–595. [7] D.L.Klayman,J.F.Bartosovich,T.S.Griffin,C.J.Mason,J.P.Scovill,J.Med.Chem.22 [42] N.A.Thornberry,Y.Lazebnik,Science281(1998)1312–1316. (1979)855–862. [8] R.C.Condit,R.Easterly,R.F.Pacha,Z.Fathi,R.J.Meis,Virology185(1991)857–861.