Chloro half-sandwich osmium(II) complexes: influence of chelated N,N-ligands on hydrolysis, guanine binding, and cytotoxicity.

Inorg.Chem.2007, 46, 4049- 4059 Chloro Half-Sandwich Osmium(II) Complexes: Influence of Chelated N,N-Ligands on Hydrolysis, Guanine Binding, and Cytotoxicity Anna F. A. Peacock, Abraha Habtemariam, Stephen A. Moggach, Alessandro Prescimone, Simon Parsons, and Peter J. Sadler* School of Chemistry, UniVersity of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, U.K. ReceivedDecember8,2006 Relativelylittleisknownaboutthekineticsorthepharmacologicalpotentialoforganometalliccomplexesofosmium compared to its lighter congeners, iron and ruthenium. We report the synthesis of seven new complexes, [(Ł6- arene)Os(NN)Cl]+, containing different bidentate nitrogen (N,N) chelators, and a dichlorido complex, [(Ł6-arene)- Os(N)Cl]. The X-ray crystal structures of seven complexes are reported: [(Ł6-bip)Os(en)Cl]PF (1PF), 2 6 6 [(Ł6-THA)Os(en)Cl]BF (2BF),[(Ł6-p-cym)Os(phen)Cl]PF (5PF),[(Ł6-bip)Os(dppz)Cl]PF (6PF),[(Ł6-bip)Os(azpy- 4 4 6 6 6 6 NMe)Cl]PF (7PF), [(Ł6-p-cym)Os(azpy-NMe)Cl]PF (8PF), and [(Ł6-bip)Os(NCCH-N)Cl] (9), where THA ) 2 6 6 2 6 6 3 2 tetrahydroanthracene, en ) ethylenediamine,p-cym )p-cymene, phen ) phenanthroline, bip ) biphenyl, dppz )[3,2-a: 2¢,3¢-c]phenazineandazpy-NMe )4-(2-pyridylazo)-N,N-dimethylaniline.Thechelatingligandwasfound 2 to play a crucial role in enhancing aqueous stability. The rates of hydrolysis at acidic pH* decreased when the primaryamineN-donors(NN)en,t 1/2 )0.6hat318K)arereplacedwith(cid:240)-acceptingpyridinegroups(e.g.,NN )phen,t 1/2 )9.5hat318K).TheOsIIcomplexeshydrolyzeupto100timesmoreslowlythantheirRuIIanalogues. The pK* of the aqua adducts decreased with a similar trend (pK* ) 6.3 and 5.8 for en and phen adducts, a a respectively).[(Ł6-bip)Os(en)Cl]PF/BF (1PF/BF)and[(Ł6-THA)Os(en)Cl]BF (2BF)werecytotoxictowardboth 6 4 6 4 4 4 the human A549 lung and A2780 ovarian cancer cell lines, with IC values of 6- 10 (cid:237)M, comparable to the 50 anticancer drug carboplatin. 1BF binds to both the N7 and phosphate of 5¢-GMP (ratio of 2:1). The formation 4 constant for the 9-ethylguanine (9EtG) adduct [(Ł6-bip)M(en)(9EtG)]2+ was lower for OsII (logK) 3.13) than RuII (log K) 4.78), although the OsII adduct showed some kinetic stability. DNA intercalation of the dppz ligand in 6PF mayplayaroleinitscytotoxicity.Thisworkdemonstratesthatthenatureofthechelatingligandcanplaya 6 crucial role in tuning the chemical and biological properties of [(Ł6-arene)Os(NN)Cl]+ complexes. Introduction complexes has been little explored. In general, not much is known about the chemistry of osmium arene complexes Thedesignanddevelopmentoforganometallicdrugsisa comparedtothoseofironandruthenium.ASciFindersearch relativelynewbutrapidlydevelopingfield.1,2Organometallic revealed (cid:24)8 times fewer publications on osmium arene complexes of the group 8 elements ruthenium and iron are receivingincreasingattentionaspotentialanticanceragents,3-6 complexes. Rutheniumandplatinumcomplexescontainingchelating yetthepharmacologicalpotentialoforganometallicosmium N,N-heterocyclicligands,forexample,phenanthroline(phen), *To whom correspondence should be addressed. E-mail P.J.Sadler@ bipyridine(bipy),andphenylazo-pyridine(azpy),havebeen ed.ac.uk. (1) Fish,R.H.;Jaouen,G.Organometallics2003,22,2166-2177. extensively studied, and some have been reported to show (2) Melchart, M.; Sadler, P. J. In Bioorganometallics; Jaouen, G., Ed.; anticancer activity.7-9 Romeo et al. investigated the ligand Wiley-VCH: Weinheim,Germany,2006;Vol.1,pp39-64. exchangekineticsofplatinumcomplexescontainingdifferent (3) Allardyce, C. S.; Dorcier, A.; Scolaro, C.; Dyson, P. J. Appl. Organomet.Chem.2005,19,1-10. (4) Yan,Y.K.;Melchart,M.;Habtemariam,A.;Sadler,P.J.Chem.Comm. (7) Novakova,O.;Kasparkova,J.;Vrana,O.;vanVliet,P.M.;Reedijk, 2005,4764-4776. J.;Brabec,V.Biochemistry1995,34,12369-78. (5) Hillard,E.;Vessieres,A.;Le,Bideau,F.;Plazuk,D.;Spera,D.;Huche, (8) Bloemink, M. J.; Engelking, H.; Karentzopoulos, S.; Krebs, B.; M.;Jaouen,G.ChemMedChem.2006,1,551-559. Reedijk,J.Inorg.Chem.1996,35,619-627. (6) Hartinger,C.G.;Nazarov,A.A.;Arion,V.B.;Giester,G.;Jakupec, (9) Velders,A.H.;Kooijman,H.;Spek,A.L.;Haasnoot,J.G.;DeVos, M.;Galanski,M.;Keppler,B.K.NewJ.Chem.2002,26,671-673. D.;Reedijk,J.Inorg.Chem.2000,39,2966-2967. 10.1021/ic062350d CCC: $37.00 © 2007 American Chemical Society Inorganic Chemistry, Vol. 46, No. 10, 2007 4049 PublishedonWeb04/19/2007 Peacock et al. bidentate nitrogen-chelating ligands,10 and the DNA inter- [(Ł6-bip)Os(en)Cl]BF (1BF). A solution of [(Ł6-bip)OsCl] 4 4 22 calation ability of platinum and ruthenium complexes (187mg,0.22mmol)in12mLofmethanolwasheatedunderreflux containingthisclassofligandshasalsoreceivedattention.11-14 for80minunderargon;ethylenediamine(32(cid:237)L,0.48mmol)was Osmium complexes containing such heterocyclic bidentate added,andthereactionmixturewasheatedunderrefluxforafurther 40min.Themixturewasfilteredthrougha0.45(cid:237)mporesizefilter nitrogen chelates have been investigated as DNA photo- reagents;15-17 however these complexes were coordinately whilestillhot.NH 4 BF 4 (390mg,ca.8molequiv)wasadded;the mixture was stirred, and the solvent was removed on a rotary saturated, containing no exchangeable ligand, and so pro- evaporator. Soxlett extraction using dichloromethane was carried vided no information on ligand exchange rates on osmium outfor5.5h.Thesolventvolumewasreducedto(cid:24)5mL,andthe complexescontainingdifferentnitrogenchelators.However, samplewasstoredat253Kovernight.Theyellowmicrocrystalline itisclearthatbidentatenitrogenchelatorssuchasbipyform productwasrecoveredbyfiltration,washedwithdichloromethane characteristicallystableosmiumcomplexes.18Thisispoten- (10mL)anddiethylether(10mL),andair-dried.Yield: 129mg tiallyimportantforpossibleanticanceractivity,sincewehave (54%). Anal. Calcd for C ClH NOsBF (526.79): C, 31.92; H 14 18 2 4 found that dissociation of oxygen-chelating ligands from 3.44; N 5.32%. Found: C, 32.05; H, 3.20; N, 5.07%. 1H NMR osmium at micromolar concentrations can deactivate com- (DMSO-d): (cid:228)7.69(d,2H,J)7.2Hz),7.49(t,2H,J)7.6Hz), 6 plexes under biological testing conditions.19,20 7.44(t,1H,J)7.3Hz),7.07(b,2H),6.42(d,2H,J)5.7Hz), Here, we explore the solid-state structures, kinetics, and 6.13 (t, 1H, J ) 5.0 Hz), 6.03 (t, 2H, J ) 5.3 Hz), 4.79 (b, 2H), solutionbehaviorofOsIIarenecomplexescontainingaseries 2.44(m,2H),2.20(m,2H). ofdifferentbidentatenitrogenchelators,includingdiamines [(Ł6-bip)Os(en)Cl]PF 6 (1PF 6 ).Synthesiswasperformedinthe andazopyridines,andattempttorelatethistotheiractivity same manner as for 1BF 4 , using NH 4 PF 6 instead of NH 4 BF 4 . Yield: 139mg(67%).ESI-MSCalcdforC ClH NOs: m/z442.1. towardcancercelllines.Thisworkmayalsoberelevantto 14 18 2 Found: m/z441.4.1HNMR(DMSO-d): (cid:228)7.69(d,2H,J)7.2 the design of new catalysts. 6 Hz), 7.49 (t, 2H, J ) 7.4 Hz), 7.44 (t, 1H, J ) 7.3 Hz), 7.07 (b, 2H), 6.42 (d, 2H, J ) 5.5 Hz), 6.13 (t, 1H, J ) 5.1 Hz), 6.03 (t, Experimental Section 2H,J)5.4Hz),4.80(b,2H),2.45(m,2H),2.20(m,2H).Crystals Materials. OsCl 3 (cid:226)nH 2 O was purchased from Alfa Aesaer; of 1PF 6 suitable for X-ray diffraction were obtained by slow ethylenediamineand4-(2-pyridylazo)-N,N-dimethylaniline(azpy- evaporationofadichloromethanesolutionatambienttemperature. NMe 2 ) were obtained from Sigma-Aldrich. 9-Ethylguanine and [(Ł6-THA)Os(en)Cl]BF 4 (2BF 4 ). A solution of [(Ł6-THA)- guanosine 5¢-monophosphate were purchased from Sigma, and OsCl] (27.1 mg, 0.03 mmol) in methanol (5 mL) was refluxed 22 ammonium tetrafluoroborate, ammonium hexafluorophosphate, for1.5hunderanargonatmosphere;ethylenediamine(5(cid:237)L,0.075 2-picolylamine(ampy),2,2¢-bipyridine(bipy),1,10-phenanthroline mmol) was added, and the reaction mixture was refluxed for a (phen), and deuterated solvents came from Aldrich. The dimers further40min.Themixturewasfilteredthroughaglasswoolplug [(Ł6-bip)OsCl 2 ] 2 , [(Ł6-p-cym)OsCl 2 ] 2 , [(Ł6-THA)OsCl 2 ] 2 , ligand whilestillhot.AfilteredsolutionofNH 4 BF 4 (29mg,(cid:24)9molequiv) [3,2-a: 2¢,3¢-c]phenazine(dppz),andtherutheniumcomplex,[(Ł6- in methanol (2 mL) was added; the mixture was stirred, and the bip)Ru(en)Cl]PF 6 , were prepared by previously reported pro- solvent was removed in vacuo. Soxlett extraction using dichlo- cedures.19,21-24Ethylenediamineandmethanol/ethanolweredistilled romethanewascarriedoutonthesolidresiduefor6.5h.Thesolvent oversodiumandmagnesium/iodine,respectively,priortouse. wasallowedtoevaporateslowlyovernightatambienttemperature Preparation of Complexes. Complexes 1PF 6 /BF 4 -9 were ((cid:24)0.5 mL). The crystalline product was recovered by filtration, synthesizedfromthedimerprecursor[(Ł6-arene)OsCl 2 ] 2 . washed with diethyl ether (10 mL), and air-dried. Yield: 6.4 mg (19%). Anal. Calcd for C ClH NOsBF (554.82): C, 34.63; H 16 22 2 4 (10) Romeo,R.;Scolaro,L.M.;Nastasi,N.;Arena,G.Inorg.Chem.1996, 4.00; N 5.05%. Found: C, 34.33; H, 3.44; N, 4.87%. 1H NMR (11) 3 L 5 ip , p 5 a 0 r 8 d 7 , - S. 50 J. 9 A 6. cc.Chem.Res.1978,11,211-217. (DMSO-d 6 ): (cid:228) 6.84 (b, 2H), 5.85 (dd, 2H, J ) 4.0 and 1.8 Hz), (12) Blasius,R.;Moucheron,C.;Kirsch-DeMesmaeker,A.Eur.J.Inorg. 5.78(s,2H),5.73(dd,2H,J)3.9and1.9Hz),4.72(b,2H),3.34 Chem.2004,20,3971-3979. (m,4H,J)175.1and16.4Hz),2.65(s,4H),2.45(m,2H),2.19 (13) Patel,K.K.;Plummer,E.A.;Darwish,M.;Rodger,A.;Hannon,M. (m, 2H). Crystals suitable for X-ray diffraction were obtained by J.J.Inorg.Biochem.2002,91,220-229. (14) vanderSchilden,K.;Garcia,F.;Kooijman,H.;Spek,A.L.;Haasnoot, slowevaporationofadichloromethanesolutionof2BF 4 atambient J.G;ReedijkJ.Angew.Chem.,Int.Ed.2004,43,5668-70. temperature. (15) Holmlin,R.E.;Barton,J.K.Inorg.Chem.1995,34,7-8. [(Ł6-p-cym)Os(ampy)Cl]PF (3PF). 2-Picolylamine (14 (cid:237)L, (16) Content,S.;Mesmaeker,A.K.-D.J.Chem.Soc.,FaradayTrans.1997, 6 6 93,1089-1094. 0.13 mmol) was added to a solution of [(Ł6-p-cym)OsCl 2 ] 2 (49.7 (17) Mishima, Y.; Motonaka, J.; Ikeda, S. Anal. Chim. Acta 1997, 345, mg, 0.06 mmol) in 4 mL of methanol. The reaction mixture was 45-50. shieldedfromlightandstirredatambienttemperaturefor20h.A (18) Dwyer,F.P.;Goodwin,A.;Gyarfas,E.C.Aust.J.Chem.1962,16, 42-50. filtered solution of NH 4 PF 6 (100 mg, (cid:24)5 mol equiv) in 2 mL of (19) Peacock, A. F. A.; Habtemariam, A.; Ferna´ndez, R.; Walland, V.; MeOHwasadded,andthesolventvolumewasreducedonarotary Fabbiani,F.P.A.;Parsons,S.;Aird,R.E.;Jodrell,D.I.;Sadler,P. evaporatoruntilayellowprecipitatebegantoform((cid:24)2mL).The J.J.Am.Chem.Soc.2006,128,1739-1748. (20) Peacock, A. F. A.; Melchart, M.; Deeth, R. J.; Habtemariam, A.; reaction vessel was stored at 253 K for 2 h. The product was Parsons,S.;Sadler,P.J.Chem.sEur.J.2007,13,2601-2613. recoveredbyfiltration,washedwithmethanol(4mL)anddiethyl (21) Stahl,S.;Werner,H.Organometallics1990,9,1876-1881. ether(10mL),andair-dried.Yield: 60.6mg(79%).Anal.Calcd (22) Chen,H.;Parkinson,J.A.;Parsons,S.;Coxall,R.A.;Gould,R.O.; Sadler,P.J.J.Am.Chem.Soc2002,124,3064-3082. for C 16 ClH 22 N 2 OsPF 6 (614.07): C, 31.35; H 3.62; N 4.57%. (23) Yamada,M.;Tanaka,Y.;Yoshimoto,Y.;Kuroda,S.;Shimao,I.Bull. Found: C,30.91;H,3.33;N,4.60%.1HNMR(MeOD-d): (cid:228)9.06 4 Chem.Soc.Jpn.1992,65,1006-1011. (d,1H,J)5.7Hz),7.97(dd,1H, J)8.5and7.6Hz),7.65(d, (24) Morris,R.E.;Aird,R.E.;Murdoch,P.d.S.;Chen,H.;Cummings, 1H,J)7.9Hz),7.48(dd,1H,J)7.6and6.6Hz),6.09(d,1H, J.;Hughes,N.D.;Parsons,S.;Parkin,A.;Boyd,G.;Jodrell,D.I.; Sadler,P.J.J.Med.Chem.2001,44,3616-3621. J)5.5Hz),6.06(d,1H,J)5.5Hz),5.91(d,1H,J)5.5Hz), 4050 InorganicChemistry,Vol.46,No.10,2007 ChloroHalf-SandwichOsmium(II)Complexes 5.88 (d, 1H, J ) 5.5 Hz), 4.41 (m, 2H), 2.71 (sep, 1H, J ) 6.9 precipitate. A solution of azpy-NMe (4-(2-pyridylazo)-N,N-dim- 2 Hz),2.17(s,3H),1.25(d,3H,J)6.8Hz),1.19(d,3H,J)6.6 ethylaniline)(35.3mg,0.16mmol)in5mLofMeOHwasadded Hz). dropwisetotheresultingpaleyellowfiltrate,whichresultedinan [(Ł6-p-cym)Os(bipy)Cl]PF (4PF).Asolutionof2,2¢-bipyridine instant color change to blue. The resulting mixture was stirred at 6 6 (21.1 mg, 0.14 mmol) and [(Ł6-p-cym)OsCl] (51.1 mg, 0.07 ambienttemperaturefor1hunderargon.Thesolventvolumewas 22 mmol) in 4 mL methanol was stirred for 19 h at ambient reducedto(cid:24)10mL;NH 4 PF 6 (114mg,>10molequiv)added,and temperature.Itwasthenfilteredthroughaglasswoolplug,anda themixturewasleftat253Kovernight.Theblueprecipitatewas filtered solution of NH PF (100 mg, (cid:24)5 mol equiv) in 2 mL of recoveredbyfiltration,washedwithethanol(20mL)anddiethyl 4 6 MeOH was added. The solvent volume was reduced on a rotary ether(10mL),andair-dried.Theproductwasrecrystallizedfrom evaporatorto(cid:24)3mL.Ayellowprecipitateformedasthesolution dichloromethane. Yield: 66.4 mg (73%). ESI-MS Calcd for C 25 - cooled to ambient temperature. The product was recovered by ClH 24 N 4 Os: m/z 607.1. Found: m/z 607.4. 1H NMR (CDCl 3 ): (cid:228) filtration,washedwithmethanol(4mL)anddiethylether(10mL), 8.57(d,1H,J)5.7Hz),8.31(d,1H,J)7.6Hz),8.00(d,2H, andair-dried.Yield: 63.2mg(74%).Anal.CalcdforC ClH N- J)8.7Hz),7.94(t,1H,J)7.9Hz),7.50(m,5H),7.32(t,1H, 20 22 2 OsPF (661.05): C,36.34;H3.35;N4.24%.Found: C,36.19;H, J)7.9Hz),6.64(d,2H,J)8.7Hz),6.50(t,2H,J)6.8Hz), 6 3.36; N, 4.08%. 1H NMR (CDCl ): (cid:228) 9.24 (d, 2H, J ) 5.6 Hz), 6.23 (t, 2H, J ) 5.7 Hz), 6.15 (t, 1H, J ) 5.7 Hz), 3.30 (s, 6H). 3 8.26(d,2H,J)8.0Hz),8.09(dd,2H,J)8.7and7.8Hz),7.67 CrystalssuitableforX-raydiffractionwereobtainedbydiffusion (dd,2H,J)7.8and6.6Hz),6.11(d,2H, J)5.8Hz),5.86(d, of diethyl ether into a solution of 7PF 6 in methanol at ambient 2H,J)5.7Hz),2.57(sep,1H,J)6.9Hz),2.32(s,3H),1.05(d, temperature. 6H,J)6.9Hz). [(Ł6-p-cym)Os(azpy-NMe)Cl]PF (8PF).Thedropwiseaddi- 2 6 6 [(Ł6-p-cym)Os(phen)Cl]PF (5PF).Asolutionof1,10-phenan- tionofasolutionofazpy-NMe (31.6mg,0.14mmol)in4mLof 6 6 2 throline(23.6mg,0.13mmol)in1mLofmethanolwasaddedto MeOHtoasolutionof[(Ł6-p-cym)OsCl] (55.5mg,0.07mmol) 22 asolutionof[(Ł6-p-cym)OsCl] (49.1mg,0.06mmol)in3mLof in 15 mL of MeOH, resulted in an instant color change to blue. 22 methanol,andthemixturewasstirredat323Kfor1hunderargon. Theresultingmixturewasstirredatambienttemperaturefor6.5h Thesolutionwasfilteredthroughaglasswoolplug,andthesolvent underargon,andthesolventvolumewasthenreducedto(cid:24)5mL volumewasreducedto(cid:24)2mLonarotaryevaporator.Afiltered on a rotary evaporator. Blue crystals formed overnight at 253 K solution of NHPF (100 mg, (cid:24)5 mol equiv) in 2 mL of MeOH after the addition of NHPF (100 mg, (cid:24)5 mol equiv) to the 4 6 4 6 was added, and the solvent was removed in vacuo. The residue solution. The product was recovered by filtration, washed with was sonicated in ethanol to give a bright yellow suspension. The ethanol(20mL)anddiethylether(10mL),air-dried,andrecrys- yellow powder was recovered by filtration, washed with ethanol talizedfromdichloromethane.Yield: 92.1mg(90%).Anal.Calcd (10mL)anddiethylether(10mL),andair-dried.Yield: 67.4mg for C ClH NOsPF (731.12): C, 37.78; H 3.86; N 7.66%. 23 28 4 6 (79%). Anal. Calcd for C ClH NOsPF (685.04): C, 38.57; H Found: C, 37.81; H, 3.74; N, 7.67%. 1H NMR (CDCl): (cid:228) 9.18 22 22 2 6 3 3.24; N 4.09%. Found: C, 38.61; H, 3.15; N, 3.96%. 1H NMR (d,1H,J)5.8Hz),8.32(d,1H,J)7.9Hz),8.06(d,2H,J)9.0 (CDCl): (cid:228) 9.59 (d, 2H, J ) 5.3 Hz), 8.57 (d, 2H, J ) 8.2 Hz), Hz), 8.00 (t, 1H, J ) 7.7 Hz), 7.62 (t, 1H, J ) 6.6 Hz), 6.77 (d, 3 8.07(s,2H),8.03(dd,2H,J)8.2and5.3Hz),6.27(d,2H,J) 2H,J)9.2Hz),6.25(d,1H,J)5.7Hz),6.03(d,1H,J)5.8 5.9Hz),6.03(d,2H,J)5.8Hz),2.57(sep,1H,J)6.9Hz),2.29 Hz),5.93(d,1H,J)5.8Hz),5.82(d,1H,J)5.6Hz),3.32(s, (s, 3H), 1.00 (d, 6H, J ) 6.9 Hz). Crystals suitable for X-ray 6H), 2.34 (s, 3H), 2.33 (sept, 1H, J ) 7.0 Hz), 0.96 (d, 3H, J ) diffractionwereobtainedbydiffusionofhexaneintoasolutionof 7.0 Hz), 0.87 (d, 3H, J ) 6.9 Hz). Crystals suitable for X-ray 5PF inchloroformatambienttemperature. diffraction were obtained by slow evaporation of a methanol 6 [(Ł6-bip)Os(dppz)Cl]PF 6 (6PF 6 ).Asolutionof[(Ł6-bip)OsCl 2 ] 2 solutionof8PF 6 atambienttemperature. (22.3 mg, 0.03 mmol) was refluxed in MeOH (5 mL)/ H O (2.5 [(Ł6-bip)Os(NCCH-N)Cl](9).Acetonitrile(0.5mL)wasadded 2 3 2 mL) for 2 h under argon and then filtered through a cotton wool to a suspension of [(Ł6-bip)OsCl] (98 mg, 0.12 mmol) in 22 plugtoremoveadarkblackprecipitate.Asolutionofdppz(15.8 dichloromethane(6mL),andthereactionmixturewasheated(320 mg,0.06mmol)in5mLofMeOHwasaddedtotheresultingpale K) with stirring under argon, shielded from light, for 3 h. The yellow filtrate. The reaction mixture was stirred at 323 K for 80 reactionmixturewasfiltered;thevolumeofthefiltratewasreduced min under argon. The mixture was filtered through a glass wool (to4mL)onarotaryevaporator,anddiethyletherwasaddeduntil plug,andthesolventvolumewasreducedonarotaryevaporator yellow crystals began to form. After the mixture stood at 253 K (to(cid:24)5mL).TheadditionofafilteredsolutionofNH PF (90mg, overnight,theyellowcrystalswererecoveredbyfiltration,washed 4 6 (cid:24)10molequiv)in1mLofMeOHgaverisetoayellowprecipitate withdiethylether(5mL)andhexane(5mL),andair-dried.Yield: whichwasrecoveredbyfiltration,washedwithmethanol(5mL) 80.7mg(76%).Anal.CalcdforC ClH NOs(456.40): C,36.84; 14 2 13 and diethyl ether (10 mL), and air-dried. Yield: 32.5 mg (75%). H2.87;N3.07%.Found: C,36.24;H,2.69;N,2.80%.1HNMR Anal. Calcd for (6 + CHOH) C ClH NOOsPF (839.20): C, (DMSO-d): (cid:228) 7.70 (d, 2H, J ) 7.9 Hz), 7.45 (m, 3H), 6.66 (d, 3 31 24 4 6 6 44.37;H2.88;N6.68%.Found: C,43.61;H,2.43;N,7.15%.1H 2H, J ) 5.5 Hz), 6.36 (t, 1H, J ) 5.1 Hz), 6.32 (t, 2H, J ) 5.1 NMR(DMSO-d): (cid:228)9.80(d,2H,J)5.6Hz),9.73(d,2H,J) Hz),2.07(s,3H).Crystalsof9suitableforX-raydiffractionwere 6 8.4Hz),8.56(dd,2H,J)6.6and3.5Hz),8.24(dd,2H,J)6.7 obtainedbydiffusionofdiethyletherintoanaliquotoftheoriginal and3.4Hz),8.21(dd,2H,J)8.3and5.6Hz),7.60(d,2H,J) reactionmixture,atambienttemperatureinthedark. 7.9Hz),7.56(t,1H,J)7.1Hz),7.40(t,2H,J)7.7Hz),7.02 Instrumentation.X-rayCrystallography.Alldiffractiondata (d,2H,J)6.0Hz),6.86(t,2H,J)5.8Hz),6.49(t,1H,J)5.6 were collected using a Bruker (Siemens) Smart Apex CCD Hz). Crystals suitable for X-ray diffraction were obtained by diffractometerequippedwithanOxfordCryosystemslow-temper- diffusion of diethyl ether into a solution of 6PF 6 in methanol at aturedeviceoperatingat150K.Absorptioncorrectionsforalldata ambienttemperature. sets were performed with the multiscan procedure SADABS;25 [(Ł6-bip)Os(azpy-NMe)Cl]PF (7PF).Asolutionof[(Ł6-bip)- 2 6 6 OsCl 2 ] 2 (50.7 mg, 0.06 mmol) in 10 mL of MeOH was refluxed (25) Sheldrick,G.M.SADABS;UniversityofGo¨ttingen: Germany,2001- for 3 h under argon and hot-filtered to remove a dark black 2004. Inorganic Chemistry, Vol. 46, No. 10, 2007 4051 Peacock et al. structuresweresolvedusingdirectmethods(SHELXL,26SIR92,27 NMRspectrawererecorded.Thechemicalshiftsofthearenering or DIRDIF28). The complexes were refined against F2 using protons were plotted against pH*. The pH* titration curves were SHELXTL,andtheH-atomswereplacedingeometricallycalcu- fittedtotheHenderson-Hasselbalchequation,withtheassumption lated positions. X-ray crystallographic data for complexes 1PF, thattheobservedchemicalshiftsareweightedaveragesaccording 6 2BF, and 5PF-9 are available as Supporting Information and tothepopulationsoftheprotonatedanddeprotonatedspecies.These 4 6 havebeendepositedintheCambridgeCrystallographicDataCentre pK*valuescanbeconvertedtopK valuesbyuseoftheequation a a under the accession numbers CCDC 630298, 630299, 630300, pK ) 0.929pK*+ 0.42 as suggested by Krezel and Bal,30 for a a 630301,630303,630302,and630304,respectively. comparisonwithrelatedvaluesintheliterature. NMRSpectroscopy.1HNMRspectrawereacquiredonaBruker InteractionswithNucleobases.Thereactionofcomplex3PF 6 AVA600(1H)600MHz)spectrometer.1HNMRspectrainDO with9-ethylguanine(9EtG)atpH*7.4and310Kwasmonitored 2 were typically acquired with water suppression by Shaka29 or overtimeusing 1HNMRspectroscopybymixingequalamounts presaturation methods. 1H NMR chemical shifts were internally ofequilibriumD 2 Osolutions(1mM)of3PF 6 and9EtG.ThepH* referenced to 1,4-dioxane (3.75 ppm) for aqueous solutions, valuesofthesesolutionswereadjustedwithdiluteNaOHtoremain (CHD)(CD)SO(2.50ppm)forDMSO-d,CDHOD(3.34ppm) closetophysiologicalpH*7.4,andthesolutionswereallowedto 2 3 6 2 formethanol-d,andCHCl (7.26ppm)forchloroform-d solutions. equilibrate at 310 K before mixing. Their 1H NMR spectra were 4 3 1 All data processing was carried out using XWIN-NMR, version recordedat310Kaftervarioustimeintervals. 3.6(BrukerU.K.Ltd.). Similarly,equalvolumesofequilibriumsolutionsof1BF 4 and MassSpectrometry.Electrosprayionizationmassspectra(ESI- 5¢-GMPinD 2 Oweremixedat298K(finalconcentration1.6mM, MS)wereobtainedonaMicromassPlatformIIMassSpectrometer pH*6.9),and1HNMRspectrarecordedatvarioustimeintervals. andDO/HOsolutionswereinfuseddirectly.Thecapillaryvoltage To determine the OsII binding sites on 5¢-GMP, a pH* titration 2 2 was3.5V,andtheconevoltagewasvariedbetween20and45V over the range 2-12 was monitored by 1H NMR spectroscopy, dependingonsensitivity.Thesourcetemperaturewas353K.Mass andthechemicalshiftsoftheH8signalswereplottedagainstpH*. spectra were recorded with a scan range of m/z 200-1000 for Solutionscontaining1BF 4 ortherutheniumanalogue,[(Ł6-bip)- positiveions. Ru(en)Cl]PF 6 (1R),and1molequiv9EtGwerepreparedinD 2 O pH* Measurement. The pH* (pH meter reading without atvariousconcentrations(1mM,500,250,100,and50(cid:237)M),and correction for effects of D on glass electrode) values of NMR afterincubationat310Kfor24h(toreachequilibrium),their1H samples in D O were measured at (cid:24)298 K directly in the NMR NMR spectra were recorded at 298 K. Binding constants (log K) 2 tube, before and after the NMR spectra were recorded, using a for9EtGwereobtainedfromtheslopesofplotsof[bound9EtG]/ Corning240pHmeterequippedwithamicrocombinationelectrode [free9EtG]versus[freemetalcomplex],basedonpeakintegrals. calibratedwithAldrichbuffersolutionsatpH4,7,and10. Inaddition,a1:1stocksolutionof1BF 4 and9EtG(1mM),which had already been allowed to reach equilibrium (24 h at 310 K), Methods.KineticsofHydrolysis.Thekineticsofthehydrolysis ofcomplexes1BF-5PF weremonitoredatvarioustemperatures wasdilutedtogivesolutionsof500,250,100,and50(cid:237)M.These 4 6 wereincubatedat310Kforafurther24hbeforetheir 1HNMR bypreparationof0.8mMsolutions,preparedinmethanol-d (5% 4 spectrawererecordedat298K. final),followedbysubsequentdilution(1:20v/v)withDO(pH* 2 (cid:25)2,sothatdeprotonationoftheaqualigandisprevented)atthe CytotoxicityTowardA2780andA549HumanCancerCells. After they were plated, human ovarian A2780 cancer cells were requiredtemperature.SampleswerefilteredandplacedintheNMR treatedwithOsIIcomplexesonday3,andhumanlungA549cancer spectrometer preset at the desired temperature, and spectra were cellsweretreatedonday2,atconcentrationsrangingfrom0.1to recorded at various time intervals. Data, based on peak integrals, 100(cid:237)M.SolutionsoftheOsIIcomplexesweremadeupin0.125% werefittedtofirst-orderkineticstogivetherateconstant(k).The Arrheniusactivationenergies(E),activationenthalpies(¢Hq),and DMSOtoassistdissolution.Cellswereexposedtothecomplexes a activationentropies(¢Sq)weredeterminedfromtheslopesofthe for24h,washed,suppliedwithfreshmedium,andallowedtogrow for three doubling times (72 h), and then the protein content Arrheniusand(interceptsof)Eyringplots.Thekineticsofhydrolysis measured(proportionaltocellsurvival)usingthesulforhodamine and pK*values of coordinated water of complexes 6PF, 7PF, a 6 6 B(SRB)assay.31 and 8PF were not determined because of the poor aqueous 6 solubilityofthesecomplexes. StabilityStudies.Thestabilitiesofcomplexes1PF 6 ,1BF 4 ,and 2BF were investigated by preparation of 1 mM solutions in 5% To study the aqueous stability of complex 9, a 2 mM solution 4 MeOD-d/95% isotonic saline solution (150 mM NaCl) by dis- was prepared in DO, and its 1H NMR spectrum was recorded 4 2 directlyaftersamplepreparation((cid:24)15min). solution of the complex in MeOD-d 4 , followed by rapid dilution with the isotonic saline solution. 1H NMR spectra were recorded DeterminationofpK*Values.FordeterminationofpK*values a a aftervarioustimeintervals(10min,1week,2weeks,and2months) (pK valuesforD Osolutions),thepH*valuesofsolutionsofthe a 2 during which time the sample was stored at ambient temperature aquacomplexes2A-5AinDO(formedinsitubyhydrolysisof 2 inthedark. the parent chloro complexes 2BF-5PF) were varied from pH* (cid:24)2 to 10 by the addition of dilute 4 NaOD 6 and HNO , and the 1H The stability of 1BF 4 in DMSO under various conditions was 3 alsoinvestigated.Twosolutions((cid:24)4mM)of1BF wereprepared 4 in DMSO-d, and their 1H NMR spectra were recorded directly (26) Sheldrick,G.M..SHELXL-97,Programfortherefinementofcrystal after sample 6 preparation (<10 min). One sample was stored at structures;UniversityofGo¨ttingen: Go¨ttingen,Germany,1997. (27) Altomare,A.;Cascarano,G.;Giacovazzo,G.;Guagliardi,A.;Burla, ambienttemperature,stopperedinthedarkfor24h,andonewas M.C.;Polidori,G.;Camalli,M.J.Appl.Crystallogr.1994,27,435- exposed to heat, sonication, sunlight, and air for 24 h, before its 435. 1HNMRspectrumwasrecorded. (28) Beurskens,P.T.;Beurskens,G.;Bosman,W.P.;deGelder,R.;Garcia- Granda, S.; Gould, R. O.; Israel, R.; Smits, J. M. M. DIRDIF; CrystallographyLaboratory,UniversityofNijmegen: Nijmegen,The (30) Krezel,A.;Bal,W.J.Inorg.Biochem.2004,98,161-166. Netherlands,1996. (31) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; (29) Hwang,T.L.;Shaka,A.J.J.Magn.Reson.,Ser.A1995,112,275- Vistica,D.;Warren,J.T.;Bokesch,H.;Kenney,S.;Boyd,M.R.J. 279. Natl.CancerInst.1990,82,1107-1112. 4052 InorganicChemistry,Vol.46,No.10,2007 ChloroHalf-SandwichOsmium(II)Complexes Figure1. Osmium(II)arenecomplexescontainingN-donorligandsstudied inthiswork. Results Figure2. X-raystructuresandatomnumberingschemesforcomplexes Monofunctional (chlorido) osmium(II) arene complexes (A) [(Ł6-bip)Os(en)Cl]PF6 1PF6, (B) [(Ł6-THA)Os(en)Cl]BF4 2BF4, (C) [(Ł6-p-cym)Os(phen)Cl]PF65PF6,(D)[(Ł6-bip)Os(dppz)Cl]PF66PF6,(E) containing neutral diamine or azopyridine N,N-chelating [(Ł6-bip)Os(azpy-NMe2)Cl]PF67PF6,(F)[(Ł6-p-cym)Os(azpy-NMe2)Cl]- ligands, as well as the bifunctional complex [(Ł6-bip)Os- PF6 8PF6, and (G) [(Ł6-bip)Os(NCCH3-N)Cl2] 9 (50% probability el- lipsoids).TheHatomsandcounterionshavebeenomittedforclarity. (NCCH -N)Cl ] (9), Figure 1, were synthesized in good 3 2 yields via the Cl-bridged dimers, [(Ł6-arene)OsCl ] where molecules crystallize with their arenes parallel, see Figure 2 2 arene ) biphenyl (bip), tetrahydroanthracene (THA), or S1.ThedistancebetweentheTHAligandsis3.3Å,andthe p-cymene (p-cym). They were characterized by elemental rings are separated by 4.13, 4.10, and 4.13 Å at dihedral analysis or ESI-MS, NMR spectroscopy, and for seven anglesof32,27,and32(cid:176) ,respectively.Furtherintermolecular complexes, X-ray crystallography. arene ring stacking is observed in the crystal structures of complex5PF andthetwinnedcomplex6PF ,betweenone X-ray Crystal Structures. The X-ray crystal structures 6 6 of the bound pyridine rings and the related pyridine of a of [(Ł6-bip)Os(en)Cl]PF (1PF ), [(Ł6-THA)Os(en)Cl]BF 6 6 4 secondmolecule(3.72and4.75Åatdihedralanglesof37.0 (2BF ),[(Ł6-p-cym)Os(phen)Cl]PF (5PF ),[(Ł6-p-cym)Os- 4 6 6 and40.0(cid:176) ,respectively),FigureS2.Thebiphenylligandsin (dppz)Cl]PF (6PF ),[(Ł6-bip)Os(azpy-NMe )Cl]PF (7PF ), 6 6 2 6 6 the crystal structures of 6PF and 7PF are twisted by 33 [(Ł6-p-cym)Os(azpy-NMe )Cl]PF (8PF ),and[(Ł6-bip)Os- 6 6 2 6 6 and32(cid:176) ,respectively.Areneringstackingwasalsoobserved (NCCH -N)Cl ] (9) were determined. Their structures and 3 2 in the crystal structure of 7PF , between the coordinated atom numbering schemes are shown in Figure 2. The 6 pyridineandthephenylringattachedtotheazogroupofan complexesadopttheexpectedpseudo-octahedral“three-leg adjacent molecule (4.7 Å at a dihedral angle of 50(cid:176) ), but piano-stool” geometry with the osmium (cid:240)-bonded to the not in that of complex 8PF . Complex 9, in which the areneligand(1.65-1.71Åtocentroidofring),(cid:243)-bondedto 6 biphenylistwistedby42(cid:176) ,crystallizessuchthatdimerslink achloride(2.41-2.37Å)andthetwonitrogenatomsofthe four independent molecules through chloride ligand (Cl1) chelate (2.02-2.15 Å), which constitute the three legs of of one molecule and the arene protons C8H81 and C5H51 the piano stool. Crystallographic data and selected bond of two others (C8(cid:226)(cid:226)(cid:226)Cl1 ) 3.513(5) Å and C5(cid:226)(cid:226)(cid:226)Cl1 3.680- lengths and angles are shown in Tables 1 and 2. (6) Å, respectively), Figure S3. The PF - and BF - coun- 6 4 Complex1PF 6 crystallizeswith2independentmolecules terions in these crystal structures are extensively involved intheunitcell.Thebiphenylunitistwisted(by27or43(cid:176) ), in H-bonding to the chelated ligand or arene protons. andintermolecularareneringstackingisobservedbetween KineticsofHydrolysis.Therateofhydrolysisofcomplex the coordinated arene in two independent molecules (4.34 3PF at298Kinaqueoussolution(D O)wasmonitoredat 6 2 Åatadihedralangleof32.4(cid:176) ).Incontrast,thecoordinated pH* values of 8, 6 and 2. The rate of hydrolysis, based on THAligandincomplex2BF isalmostplanar,beingslightly peak integrals for p-cymene peaks, of the intact chloro 4 bent upward (away from the legs) by 6.5(cid:176) . Adjacent complex 3 (5.93 and 5.91 ppm) and the aqua complex 3A Inorganic Chemistry, Vol. 46, No. 10, 2007 4053 Peacock et al. Table1. CrystallographicDatafor[(Ł6-bip)Os(en)Cl]PF6,1PF6,[(Ł6-THA)Os(en)Cl]BF4,2BF4,[(Ł6-p-cym)Os(phen)Cl]PF6,5PF6, [(Ł6-p-cym)Os(dppz)Cl]PF6,6PF6,[(Ł6-bip)Os(azpy-NMe2)Cl]PF6,7PF6,[(Ł6-p-cym)Os(azpy-NMe2)Cl]PF6,8PF6,and[(Ł6-bip)Os(NCCH3-N)Cl2],9 1PF 2BF 5PF 6PF 7PF 8PF 9 6 4 6 6 6 6 formula C H ClF- C H BClF- C H ClF- C H ClF- C H ClF - C H ClF - C H ClNOs 14 18 6 16 22 4 22 22 6 30 21 6 50 48 2 12 46 56 2 12 14 13 2 NOsP NOs NOsP NOsP NOsP NOsP 2 2 2 4 8 2 2 8 2 2 MW 584.92 554.82 685.04 807.13 751.11 731.12 456.37 cryst colorlessblock yellowplate yellowblock yellowlath darkgreenprism blackblock yellowplate description crystsize 0.08(cid:2)0.08(cid:2)0.34 0.09(cid:2)0.15(cid:2)0.44 0.14(cid:2)0.16(cid:2)0.36 0.13(cid:2)0.16(cid:2)0.61 0.17(cid:2)0.22(cid:2)0.26 0.33(cid:2)0.52(cid:2)0.55 0.17(cid:2)0.42(cid:2)0.81 (mm) (cid:236)(Å) 0.71073 0.71073 0.71073 0.71073 0.71073 0.71073 0.71073 temp(K) 150 150 150 150 150 150 150 cryst monoclinic monoclinic orthorhombic monoclinic, triclinic triclinic monoclinic syst twinned space P121/c1 P121/n1 Pbca P121/c1 P1h P1h P121/c1 group a(Å) 19.5853(8) 12.722(3) 12.7769(3) 11.8153(12) 8.1226(3) 8.2549(3) 10.0020(15) b(Å) 9.0300(4) 7.9298(16) 13.3958(2) 8.7125(9) 10.0165(4) 12.2787(4) 7.009(1) c(Å) 23.2542(8) 18.142(4) 26.6395(6) 26.175(3) 17.3516(6) 12.8556(5) 19.717(3) R(deg) 90 90 90 90 101.105(2) 84.792(2) 90 (cid:226)(deg) 112.608(2) 105.28(3) 90 102.952(4) 96.365(2) 82.980(2) 102.447(2) (cid:231)(deg) 90 90 90 90 109.271(2) 80.680(2) 90 vol(Å3) 3796.6(3) 1765.6(7) 4559.53(16) 2626.0(5) 1284.28(9) 1272.84(8) 1349.8(3) Z 8 4 8 4 1 1 4 R(F2) 0.0913 0.0307 0.048 0.043 0.039 0.031 0.0285 o R (F2) 0.0972 0.0907 0.066 0.045 0.077 0.065 0.0802 w o GOF 0.6814 0.9498 0.599 1.056 0.941 0.665 1.0434 Table2. SelectedBondLengths(Å)andAngles(deg)for[(Ł6-bip)Os(en)Cl]PF6,1PF6,[(Ł6-THA)Os(en)Cl]BF4,2BF4,[(Ł6-p-cym)Os(phen)Cl]PF6, 5PF6,[(Ł6-p-cym)Os(dppz)Cl]PF6,6PF6,[(Ł6-bip)Os(azpy-NMe2)Cl]PF6,7PF6,[(Ł6-p-cym)Os(azpy-NMe2)Cl]PF6,8PF6,and [(Ł6-bip)Os(NCCH3-N)Cl2],9a 1PF6 2BF4 5PF6 6PF6 7PF6 8PF6 9 Os-C(arene) 2.232(7) 2.156(5) 2.196(3) 2.215(8) 2.220(4) 2.278(4) 2.178(4) 2.208(7) 2.180(5) 2.208(3) 2.192(7) 2.207(5) 2.209(4) 2.193(5) 2.195(7) 2.224(4) 2.187(3) 2.195(5) 2.189(5) 2.214(3) 2.181(4) 2.176(8) 2.171(5) 2.224(3) 2.139(7) 2.219(5) 2.220(3) 2.170(4) 2.169(7) 2.218(5) 2.194(4) 2.173(8) 2.211(5) 2.175(3) 2.183(5) 2.194(7) 2.200(5) 2.178(3) 2.257(5) 2.201(5) 2.231(4) 2.186(4) Os-N1 2.136(6) 2.127(4) 2.091(3) 2.117(5) 2.055(3) 2.055(3) 2.057(4) Os-X2 2.152(6) 2.140(4) 2.094(3) 2.073(5) 2.071(4) 2.021(3) 2.4139(12) Os-Cl 2.3918(19) 2.4107(12) 2.4062(9) 2.3958(14) 2.3962(11) 2.3711(8) 2.3976(11) N1 -Os-X2 78.7(2) 78.48(16) 77.22(11) 76.98(17) 74.89(14) 74.99(12) 85.01(11) N1 -Os-Cl 83.47(17) 82.88(12) 83.17(8) 85.14(17) 85.10(10) 83.26(8) 84.00(11) Cl-Os-X2 83.65(18) 81.87(11) 83.06(8) 84.69(17) 83.94(10) 87.17(8) 86.14(4) aX2 )secondnitrogendonorgroupandN1 )pyridine(andX2 )azo)in7PF6and8PF6.Forcomplex9,X2 )Cl2. (5.76and5.74ppm)wasrapidatpH*8,withahalf-lifeof pK* Determination. Peaks for coordinated arene-ring a (cid:24)0.11h.AtpH*(cid:24)6,therateofhydrolysiswasslower(t protonsoftheaquacomplexes,[(Ł6-arene)Os(NN)(OD )]2+ 1/2 2 ) 0.66 h), with less hydrolysis of the complex and at pH* 2A-5A, shifted to high field with increase in pH*. The (cid:24)2,evenslower.Afittopseudo-first-orderkineticsyielded chemical shifts were plotted against pH* (Figure S5), and a rate constant of k ) 0.13 h-1 (t ) 5.3 h, Figure 3A) at thedatawerefittedtotheHenderson-Hasselbalchequation, 1/2 pH* 2. from which the pK*values of the coordinated water were a A comparison of the rates of hydrolysis of complexes determined. This yielded a value of 5.8 for complexes 1BF , 2BF , 3PF , 4PF , and 5PF under acidic aqueous containingtwo(cid:240)-acceptorpyridinegroupsasN-donors(4A/ 4 4 6 6 6 conditions (D O, pH* (cid:24)2), to prevent deprotonation of the 5A) and 6.3 for complexes containing a primary amine as 2 coordinatedwater(Figure3BandTable3),showedthatthe N-donor (2A/3A), see Table 4. ratesdecreaseintheorder3PF >2BF >1BF .4PF (cid:25) Guanine Adducts. New peaks, assignable to the adduct 6 4 4 6 5PF . From the data obtained at various temperatures, the [(Ł6-p-cym)Os(ampy)(9EtG)]2+3G,appearedinthe1HNMR 6 Arrhenius activation energies (E), activation enthalpies spectrum of 3PF and 9EtG (1:1, 0.5 mM) over time and a 6 (¢Hq), and activation entropies (¢Sq) were determined, under biologically relevant conditions (pH* (cid:24)7.4, 310 K), Figure S4 and Table 3. The large negative ¢Sq values for Figure 5A. On the basis of peak integration, the reaction complexes 1BF , 2BF , and 3PF are notable. half-life was (cid:24)3.2 h (Figure 5B). 4 4 6 The 1H NMR spectrum of a solution of complex 9, [(Ł6- Similarly,newpeaksappearedovertimeintheH8region bip)Os(NCCH )Cl ], in D O (2 mM) showed peaks for a in 1H NMR spectra of 1:1 solutions of 1BF and 5¢-GMP 3 2 2 4 numberofminorunidentifiedspeciesandmajorarenepeaks (1.6 mM, D O, pH* 6.9, 298 K). After 30 h, (cid:24)40% of the 2 ((cid:24)60% of species) at 6.46, 6.25, and 6.01 ppm assignable 5¢-GMP had reacted, Figure 6A. The H8 peak for free 5¢- to the trihydroxo-bridged dimer, [(Ł6-bip)Os((cid:237)-OD) Os(Ł6- GMP (8.15 ppm, b), decreased in intensity with an ap- 3 bip)]+, Figure 4.19 proximate half-life of (cid:24)8.5 h, and peaks for new species a 4054 InorganicChemistry,Vol.46,No.10,2007 ChloroHalf-SandwichOsmium(II)Complexes Figure6. (A)Low-fieldregionofthe1HNMRspectrumofanaqueous solutionof1BF4(1.6mM)containing1molequiv5¢-GMPafter30hat 298K.(B)Timedependence,basedon1HNMRpeakintegrals,ofdecay/ formationofpeaksa,b,andcfromtheabovereaction.(C)pH*titrations ofthereactionmixture.PlotsofH8chemicalshiftvspH*.Solidcurves arecomputerfitsgivingpKa*valuesfora(5.94and8.09),b(2.55,6.57, and 9.86), and c (0.95 and 9.66). Peak assignments (see structures): a, F o (a n i m g 1 u p H y re )C N 3 l M ] . P R T F6 im p ( e A e a ) d k e a p i t n e 2 t n e 9 d g 8 e r n a K l c s e ) i f n d o u r D r f 2 i o n O r g m a h a t t y i v o d a n r r o i o o ly f u s s t i h s p e H o a f q * u 3 v a P a c F l o u 6 m es [ p , (Ł l 8 e 6 x - ( p 9 3 - A c ), y ( m 6 b ) a ( O s b e s ) d - , O co s m -N p 7 le G x M O P s ; -O b ( , P f O re 3) e G 5 M ¢- P G N M 7 P -O ; s c . , Os-O(PO3)GMP; x, possible dinuclear and 2 (2), giving half-lifes of (cid:24)0.11, 0.66 and 5.3 h, respectively. (B) Hydrolysisof3PF6inacidicD2O(pH*(cid:24)2)at298K(9),304K(b),310 K(2),and316K(1). Figure 4. Formation of hydroxo-bridged dimer [(Ł6-bip)Os((cid:237)-OD)3Os- (Ł6-bip)]+ from [(Ł6-bip)Os(NCCH3-N)Cl2] 9 in aqueous solution. Low- fieldregionofthe1HNMRspectrum,showingthecoordinatedbiphenyl Figure 7. Determination of the apparent formation constants for the arene ring protons, of 9 in D2O within 15 min of sample preparation. osmiumandrutheniumanalogues,[(Ł6-bip)Os(en)(9EtG)]2+(1G)and[(Ł6- Assignments: xcorrespondstothehydroxo-bridgeddimer,whichaccounts for(cid:24)60%of{(Ł6-bip)Os}2+speciespresentinsolution.Otherpeakshave bip)Ru(en)(9EtG)]2+(1RG).Theextentofbindingwasdeterminedbythe integrationof1HNMRpeaks(seeFigureS5).Fromtheslopesoftheplots notbeenassigned. of [(Ł6-bip)M(en)(9EtG)]2+/[free 9EtG] vs [(Ł6-bip)M(en)(OD2)]2+, the bindingconstantslogK)3.13(0.03and4.78(0.09weredetermined forOsII(1G)andRuII(1RG),respectively. Figure8. Cytotoxicityof[(Ł6-bip)Os(en)Cl]PF6(1PF6,s),[(Ł6-bip)Os- (en)Cl]BF4(1BF4,--),and[(Ł6-THA)Os(en)Cl]BF4(2BF4,---)toward (A)humanA549lungcancercellsand(B)humanA2780ovariancancer cells. The IC50 values (concentrations that inhibit cell growth by 50%) obtainedfromthesecurvesaregiveninTable5. establishedbymonitoringapH*titrationofthemixtureby 1HNMRspectroscopy(Figure6C).Peakbforfree5¢-GMP Figure5. (A)Low-fieldregionofthe1HNMRspectrumofanaqueous was assigned by addition of excess 5¢-GMP to the mixture solutionof3PF6(0.5mM)afterreactiionwith1molequiv9EtGfor15h (pH*(cid:25)7.4and310K).Peakassignments: [(Ł6-p-cym)Os(ampy)(OD)]+ andshiftedwithpH*togivethreepK a *valuesof2.55,6.57, 3A,H8offree9EtGGandthe9EtGboundproduct[(Ł6-p-cym)Os(ampy)- and 9.86, corresponding to N7H, the phosphate backbone, (9EtG)]2+ 3G. (B) Time dependence, based on 1H NMR peak integrals, forformationof[(Ł6-p-cym)Os(ampy)(9EtG)]2+(3G)intheabovereaction. and N1H deprotonation, respectively. Peak a shifted with two associated pK*values of 5.94 and 8.09 (phosphate and a (8.45 ppm) and c (8.03 ppm) appeared with half-lives of N1Hdeprotonation),consistentwithN7-bound5¢-GMP,[(Ł6- (cid:24)8.5and9.0h,respectively,withcformingbeforea(Figure bip)Os(en)(5¢-GMP-N7)].Peakcshiftedwithtwoassociated 6B).Peakswereassignedandbindingsiteson5¢-GMPwere pK*values of 0.95 and 9.66 assignable to N7H and N1H a Inorganic Chemistry, Vol. 46, No. 10, 2007 4055 Peacock et al. Table3. RateDatafortheAquationofComplexes1BF4 -5PF6atVariousTemperatures T k t1/2 Ea ¢Hq ¢Sq (K) (h-1) (h) (kJmol-1) (kJmol-1) (JK-1mol-1) 1BF4 298 0.046(0.007 15.11(0.22 101.2(2.0 98.6(2.0 -60.6(6.3 310 0.250(0.001 2.80(0.08 318 0.615(0.014 1.13(0.03 333 3.439(0.151 0.20(0.01 2BF4 298 0.082(0.001 8.47(0.13 97.8(4.1 95.1(4.2 -54.2(13.3 310 0.423(0.009 1.64(0.03 318 1.205(0.052 0.58(0.03 333 5.105(0.232 0.14(0.01 3PF6 298 0.131(0.002 5.29(0.08 94.3(4.6 91.7(4.6 -46.2(15.2 304 0.308(0.007 2.25(0.05 310 0.545(0.009 1.27(0.02 316 1.203(0.037 0.58(0.02 4PF6 323 0.150(0.003 4.63(0.10 98.3(14.1 95.5(14.1 -35.1(42.5 328 0.266(0.007 2.61(0.07 333 0.643(0.016 1.08(0.03 343 1.217(0.039 0.57(0.02 5PF6 318 0.073(0.001 9.45(0.14 105.4(9.8 102.6(9.8 -56.9(29.6 328 0.343(0.006 2.02(0.04 333 0.609(0.012 1.14(0.02 347 2.105(0.125 0.33(0.02 Table4. pK*aandpKaValuesafortheDeprotonationofthe Table5. InVitroGrowthInhibitionofHumanA549LungandA2780 CoordinatedD2OinHydrolyzedComplexes1A-5A OvarianCancerCellLines complex pK*a pKa IC50((cid:237)M) [(Ł6-bip)Os(en)(OD2)]2+ 1A 6.37b 6.34b complex A549 A2780 [ [ [ [ ( ( ( ( Ł Ł Ł Ł 6 6 6 6 - - - - T p p p - - - H c c c y y y A m m m )O ) ) ) O O O s( s s s e ( ( ( n a b p ) m i h ( p e O p y n y ) D ) ( ) ( O 2 ( O O ) D ] D 2 D 2 + 2 ) 2 ) ] ) ] 2 ] 2 + 2 + + 2 3 4 5 A A A A 6 6 5 5 . . . . 3 3 8 8 6 2 1 0 6 6 5 5 . . . . 3 2 8 8 3 9 2 1 1 2 3 1 B B P P F F F F 6 6 4 4 >1 1 1 0 0 0 6 0 .4 7 7 9. . . 4 0 6 apKavaluescalculatedfrompK*aaccordingtoref30.bRef19. 4 5 P P F F 6 6 > > 1 1 0 0 0 0 deprotonation,andconsistentwithphosphatebound5¢-GMP, 6 7 P P F F 6 6 >10 a 0 b [(Ł6-bip)Os(en)(5¢-GMP-O)]. Peak x may be the result of a 8PF6 >100 dinuclear complex in which two {(Ł6-bip)Os(en)}2+ frag- 9 >100 mentsbindtoN7andphosphateofthesame5¢-GMPunit.32 a<40(cid:237)M,seetext.b <2(cid:237)M,seetext. Solutions of 1BF or the RuII analogue [(Ł6-bip)Ru(en)- 4 Anticancer Activity. The cytotoxicity of complexes Cl]PF 1R (1 mM to 50 (cid:237)M) containing 1 mol equiv 9EtG 6 1BF -9 toward the human lung A549 cancer cell line and were incubated at 310 K, and their 1H NMR spectra were 4 ofcomplexes1BF ,1PF ,2BF ,and6PF towardthehuman recorded. Peaks for free 9EtG (H8, 8.10 ppm) increased in 4 6 4 6 ovarianA2780cancercelllinewasinvestigated.33Complexes intensitywithadecreaseinmetalconcentration,FigureS6. 3PF -5PF and 7PF -9 were nontoxic up to the highest Speciesdistributionplotsbasedonpeakintegrals,Figure7, 6 6 6 testconcentration(50(cid:237)M).TheIC (concentrationinhibiting yieldedapparentformationconstantsoflogK)3.13(0.03 50 cellgrowthby50%)valuesarethereforelikelytobe>100 and 4.78 ( 0.09 for the formation of [(Ł6-bip)Os(en)- (cid:237)M, and the complexes are deemed inactive. However, (9EtG)]2+ (1G) and [(Ł6-bip)Ru(en)(9EtG)]2+ (1RG), re- complexes1BF ,1PF ,2BF ,and6PF wereactive(Figure spectively. 4 6 4 6 8 and Table 5). Complexes 1BF /PF and 2BF displayed 4 6 4 Similar solutions were prepared by dilution of an equi- IC valuesof6-10(cid:237)M.TheIC valuesforcomplex6PF 50 50 6 librium stock solution of 1BF 4 and 9EtG (1 mM) to give were estimated to be <40 (cid:237)M for A549 cells and <2 (cid:237)M 500-50 (cid:237)M solutions, which were incubated at 310 K for forA2780cells.Testson6PF (aswellas7PF and8PF ) 24hbeforetheir1HNMRspectrawererecorded.Basedon 6 6 6 were complicated by its poor solubility in water. It was 1HNMRpeakintegrationofH8peaksforbound9EtG(8.21 initially dissolved in DMSO and then rapidly diluted with ppm) and free 9EtG (8.10 ppm), no significant change in water but could only be administered to the cells as a ratio occurred with decrease in concentration (Figure S7). suspension34 to maintain a low exposure of the cells to Bindingof1to9EtGwasfurtherconfirmedbyESI-MS. DMSO (0.25%). AnequilibriumNMRsample(containingequalamountsof 1BF 4 and 9EtG), gave peaks at m/z 294.1 and 585.7 (33) TheIC50valuesforcisplatinwere2.6and0.5(cid:237)Mforthesehuman consistentwith{(Ł6-bip)Os(en)(9EtG)}2+(calcdm/z292.6) A549 lung and A2780 ovarian cancer cell lines, respectively, as reportedpreviously: S.M.Guichard,R.Else,E.Reid,B.Zeitlin,R. and {(Ł6-bip)Os(en)(9EtG)-H}+ (calcd m/z 584.2), respec- Aird,M.Muir,M.Dodds,H.Fiebig,P.J.Sadler,D.I.JodrellBiochem. tively. Pharmacol.2006,71,408-415. (34) Drugsaresometimesadministeredassuspensions,althoughusually in peanut oil and not as a suspension in water as used here. Cells (32) Chen,H.;Parkinson,J.A.;Morris,R.E.;Sadler,P.J.J.Am.Chem. were washed after removal of complex, in an attempt to remove Soc.2003,125,173-186. unreactedparticles. 4056 InorganicChemistry,Vol.46,No.10,2007 ChloroHalf-SandwichOsmium(II)Complexes Stability Studies. Since complex 1BF had previously the flexible extended arene ring system of bip may mean 4 been found to have a low cytotoxicity,19 its stability during that intercalation via both dppz and bip can contribute to thesamplepreparationprocesswasinvestigated.Thisinitially the cytotoxicity of 6PF . Finally, the crystal structures of 6 involveddissolutioninDMSOandsubsequentdilutionwith the highly colored complexes containing azopyridine type water. Solutions of 1BF in DMSO were prepared; one ligands in 7PF and 8PF , also show (cid:240)-(cid:240) stacking. The 4 6 6 sample was stored stoppered at ambient temperature in the crystal structure of the ruthenium analogue of 8PF has 6 dark, and the second sample was exposed to the air, heat, previouslybeendeterminedandagainappearstobestructur- sonication,andlight,allofwhichmayhavebeenencountered ally similar.36 These appear to be the first reported X-ray duringtheearliersamplepreparation.Directlyaftersample crystal structures of osmium arene complexes, containing preparation the solution was clear and pale yellow color. coordinated dppz or azopyridine ligands. After 24 h, the first sample appeared unchanged, and no RatesofHydrolysisandAcidityofCoordinatedWater. additional peaks were present in the 1H NMR spectrum. TheincorporationofachelatingligandintotheseOsIIarene However,thesecondsample(exposedtoextremeconditions) complexesiscrucialformaintainingtheiraqueousstability. wasnowdarkred,andnewpeakswereseeninthe1HNMR Complex 9, which contains only monodentate non-arene spectrum (Figure S8). ligands and a single donor nitrogen ligand, is largely 1H NMR spectra of 1 mM solutions of the active deactivatedinwatertoformpredominantlytheinerthydroxo- complexes, 1PF /BF and 2BF in isotonic saline solution bridged dimer, [(Ł6-bip)Os((cid:237)-OD) Os(Ł6-bip)]+ (Figure 4). 6 4 4 3 (150mMNaCl)wererecordeddirectlyaftersampleprepara- The rate of hydrolysis of complex 3, [(Ł6-p-cym)Os- tionandafterstorageatambienttemperatureinthedarkfor (ampy)Cl]+, was found to be markedly pH*-dependent, 1 week, 2 weeks, and 2 months. Small peaks for the aqua increasingwithincreaseinpH*.Twoequilibriaareinvolved complexes (<10%) were present. The only changes in the (eq 1), the first of which is hydrolysis and the second is 1H NMR spectra were loss of peaks assigned as the NH deprotonationofcoordinatedwater.Becauseoftherelatively protons of en because of the exchange with deuterium low pK*values of water coordinated to osmium in these a (Figure S9). complexes, the hydroxo species is formed even at neutral The 1H NMR spectrum of a 50 (cid:237)M solution of 3PF in pH*. This shifts the equilibrium to the right, effectively 6 D O, (cid:24)10 min after sample preparation, gave rise to increasingtherateofhydrolysis.Tomeasuretherateofthe 2 p-cymenearenepeakspredominantlyfortheaquacomplex hydrolysis step, alone, the pH* was lowered. 3A (78%) with minor peaks present for the intact chloro species 3 (12%). No new peaks appeared after incubation {Os-Cl}+y\ +H 2 O z{Os-OH }2+y\ -H+z{Os-OH}+ (1) of the sample at 310 K for 24 h (Figure S10). +Cl- 2 +H+ Discussion The strong dependence of the rate of hydrolysis of 3PF 6 on pH*, and the presence of exchangeable NH protons on Eight new osmium arene complexes with the general thechelatedligand,suggestthataconjugatebasemechanism formula [(Ł6-arene)Os(NN)Cl]+, where NN ) bidentate may be involved. nitrogen-chelating ligands, 1PF -8PF and [(Ł6-bip)Os- 6 6 The rate of hydrolysis of complexes 1BF and 2BF (NCCH )Cl ](9)weresynthesizedviatheCl-bridgeddimers, 4 4 3 2 containingachelatingligandwithtwoprimaryaminedonors, [(Ł6-arene)OsCl ] . Complex 1BF , [(Ł6-bip)Os(en)Cl]BF , 2 2 4 4 en, is faster than that for complexes containing two (cid:240)-ac- hadbeensynthesizedpreviously,19butanimprovedsynthesis ceptor pyridine groups, bipy and phen, ((cid:24)5.5 and 8 times, is reported here. respectively). This is attributable to the reduced electron X-ray Crystal Structures. We determined the X-ray densityonOsII.Intriguingly,complex3PF ,whichcontains crystalstructureofthePF-saltofcomplex1forcomparison 6 6 the unsymmetrical chelated ligand, 2-picolylamine, hydro- withthatoftheBPh -saltwhichwereportedpreviously.19 4 lyzed the most rapidly, despite replacement of one of the They are structurally very similar. The X-ray structure of aminegroupswithapyridine.Thisiscontrarytotheexpected complex2BF appearstobethefirstofanosmiumcomplex 4 trend and that observed for square-planar platinum com- bound to tetrahydroanthracene (THA). In 2BF , the arene, 4 plexes.10 The rate of hydrolysis of complex 3PF was also THA,isplanar,asitisintherutheniumanalogueof2,and 6 the most sensitive to changes in pH*. A direct comparison ithasbeenproposedthattheincreaseincytotoxicityof[(Ł6- betweencomplexes1BF -2BF and3PF -5PF cannotbe arene)Ru(en)Cl]+,onreplacingbipwithTHAmaybepartly 4 4 6 6 made because of the different nature of the arenes, with the result of the ability of coordinated THA to intercalate p-cymenebeingmoreelectron-richthaneitherbiphenyl(bip) intoDNA.22TheabilityoftheTHAligandto(cid:240)-(cid:240)stackis or tetrahydroanthracene (THA). Complex 2BF , containing evidentinthecrystalstructureoftheosmiumcomplex2BF , 4 4 THA as the arene, hydrolyzed almost twice as fast as Figure S1; (cid:240)-(cid:240) stacking is also evident in the crystal complex1BF ,inwhichtheareneisbip,inagreementwith structuresofcomplexes5PF and6PF ,especiallyfor6PF 4 6 6 6 observations made for the ruthenium analogues.37 involvingthedppzligand,whichhasbeenextensivelystudied asaDNAintercalator.15,35Thisfeatureincombinationwith (36) Dougan,S.J.;Melchart,M.;Habtemariam,A.;Parsons,S.;Sadler, P.J.Inorg.Chem.2006,45,10882-10894. (35) Frodl,A.;Herebian,D.;Sheldrick,W.S.J.Chem.Soc.,DaltonTrans. (37) Wang,F.;Chen,H.;Parsons,S.;Oswald,I.D.H.;Davidson,J.E.; 2002,19,3664-3673. Sadler,P.J.Chem.sEur.J.2003,9,5810-5820. Inorganic Chemistry, Vol. 46, No. 10, 2007 4057 Peacock et al. Osmium complexes are generally more inert than those nucleotide, one to the N7 and the other to the phosphate of their lighter congener ruthenium, and the data reported group,[(Ł6-bip)Os(en)(OP-5¢-GMP-N7)Os(en)(Ł6-bip)]2+;how- here show that complexes 1BF and 2BF hydrolyze 100 everitsconcentrationwastoolowtofollowby1HNMRin 4 4 timesmoreslowlythantheirrutheniumanalogues,[(Ł6-bip/ the pH* titration of the reaction mixture. The time depen- THA)Ru(en)Cl]+.37Hydrolysisoftheosmiumcomplex[(Ł6- denceofthespeciesdistribution(Figure6B)withinthefirst bz)Os(en)Cl]+ and anation of the aqua adduct [(Ł6-bz)Os- 5 h suggests that phosphate binding occurs first, followed (en)(OH )]2+ have also been reported to occur (cid:24)100 times byN7binding.Thiswouldbeconsistentwiththemigration 2 moreslowly(k )1.2(cid:2)10-5s-1andk )0.13(cid:2)10-2 mechanism proposed by Chen et al.32 for the ruthenium hydro Cl M-1s-1,298K)38thanfortherelatedrutheniumcomplexes, analogue 1R. The migration of OsII from the phosphate to [(Ł6-bip)Ru(en)(Cl/OH )]+/2+ (k ) 1.3 (cid:2) 10-3 s-1 and N7 of 5¢-GMP appears to be slower than that for the 2 hydro k )0.15M-1s-1,298K).37Theactivationparametersfor ruthenium analog. After 22 h at 298 K, all of the RuII was Cl hydrolysis of the complexes [(Ł6-bip)M(en)Cl]+, where M boundtotheN7of5¢-GMP,32comparedto66%ofthebound ) Os or Ru, can now be compared.37 As expected the GMP for OsII (Figure 6B). Arrheniusactivationenergy,E a ,forhydrolysisoftheosmium ThebindingconstantforformationoftheOsIIadductwith complexishigherthanthatforitsrutheniumanalogue(¢E a 9EtG[(Ł6-bip)Os(en)(9EtG)]+(1G)oflogK)3.13(0.03 (cid:25)26kJmol-1)asistheenthalpyofactivation,¢Hq,(¢¢Hq isanorderofmagnitudelowerthanthatfortheRuIIanalog (cid:25) 26 kJ mol-1) with similar entropies of activation, ¢Sq. (log K ) 4.78 ( 0.09), Figure 7. Intriguingly, the stronger The¢E of26kJmol-1isingoodagreementwithreported a bindingtoRu(II)isoppositetothatobservedforthemaltolate theoretical calculations, which give a ¢E a value of 20 kJ (mal)complexes[(Ł6-p-cym)M(mal)(9EtG)]+,forwhichlog mol-1forthehydrolysisoftheseOsIIandRuIIcomplexes.20,39 K)4.41(Os)and3.87(Ru).20Despitethelowerformation Thelargenegative¢Sqvaluesforthehydrolysisofosmium constant compared to RuII, the kinetic stability of the OsII complexes 1BF -3PF are indicative of an associative 4 6 complex 1G is high: 9EtG did not readily dissociate, even process(Table3).Theerrorsassociatedwiththe¢Sqvalues in50(cid:237)Msolutionsincubatedat310Kfor24h.Thissuggests forthehydrolysisofcomplexes4PF 6 and5PF 6 aretoolarge that once formed on DNA (or RNA), {(Ł6-bip)Os(en)}2+ to allow comparisons. adducts with G bases may persist long enough to interfere Anotherindicationoftheelectrondensityonosmiumand with downstream DNA processing (including protein rec- oftheOs-Obondstrength,istheacidityofthecoordinated ognition and excision/repair). water.WhenthepK*valuesoftheaquacomplexes1A,2A, a Cancer Cell Cytotoxicity. We reported previously that 3A, 4A, and 5A (Table 4) are compared, it is evident that complex1BF ,[(Ł6-bip)Os(en)Cl]BF ,wasinactiveagainst watercoordinatedtoosmiumboundtotwo(cid:240)-acceptorgroups 4 4 thehumanovarianA2780cancercellline(IC >100(cid:237)M).19 suchaspyridinesismoreacidic(pK*)5.8)thanwhenone 50 a Because the chemical properties of the complex (rate of ofthenitrogendonorsisaprimaryamine(pK*)6.3).Water a hydrolysisandbindingtoguanine)appearedtobecompatible bound to ruthenium analogues is less acidic but shows the withactivity,wereinvestigateditscytotoxicity.Apartfrom sametrend: pK valuesof7.71and7.2for[(Ł6-bip)Ru(en)- a thepotentialvariabilityofcellculturemedia(especiallyfetal (OH )]2+and[(Ł6-p-cym)Ru(bipy)(OH )]2+,respectively.37,40 2 2 calf serum) and the cell population, a possible explanation Binding to Guanine. The kinetics for the reactions of of the previous finding of inactivity (IC > 100 (cid:237)M) and 3PF and 1BF with 9EtG and 5¢-GMP, respectively, were 50 6 4 the present finding of activity can be related to partial investigated.Inaddition,thestabilityconstantsof[(Ł6-bip)- decomposition of the complex in stock solutions prepared Os(en)(9EtG)]2+ 1G and its ruthenium analogue [(Ł6-bip)- in DMSO. We have shown here that solutions of 1BF in Ru(en)(9EtG)]2+ 1RG were determined. 4 DMSOcandecomposeaftersonication,heating,andalonger Thereactionofthebip/encomplex1BF with5¢-GMPat 4 termofstandinginairanddirectsunlight,asisevidentfrom 298KresultedintheformationofthreeOsII-GMPadducts color change from yellow to dark red (Figure S8A/B). (2 major and 1 very minor) as judged by the H8 1H NMR However,suchsolutionsarestablewhenstopperedandstored signals.Fromreportedworkonthebindingoftheruthenium at ambient temperature in the dark, and then exhibit good analogue [(Ł6-bip)Ru(en)Cl]PF 1R to 5¢-GMP32 and from 6 activity, as does the PF salt (1PF ), giving IC values of pH* titrations of the products, the major species can be 6 6 50 7-10(cid:237)MagainstthehumanovarianA2780andlungA549 assigned as N7-bound 5¢-GMP, [(Ł6-bip)Os(en)(5¢-GMP- cancer cell lines (Table 5). The change of the arene from N7)], with the phosphate-bound 5¢-GMP adduct [(Ł6-bip)- biphenylin1BF totheextendedtetrahydroanthracenegave Os(en)(5¢-GMP-OP)]alsobeingformedinsignificantamounts 4 risetoasimilarpotency(complex2BF ,IC valuesof6-9 (ratio of 2:1 for N7/phosphate-bound adducts, after 30 h at 4 50 (cid:237)M, Table 5), whereas for the ruthenium analogues this 298 K). The minor product (labeled x in Figure 6) may be change in arene increases the activity by 10-fold.41 Further a dinuclear adduct, with two osmium units bound to the work will be required to investigate whether Os-bound (38) Hung,Y.;Kung,W.;Taube,H.Inorg.Chem.1981,20,457-463. extendedarenescanintercalateintoDNAinamannersimilar (39) Wang, F.; Habtemariam, A.; Geer, E. P. L. v. d.; Fernandez, R.; to Ru-bound arenes.42 Melchart,M.;Deeth,R.J.;Aird,R.;Guichard,S.;Fabbiani,F.P.A.; Lozano-Casal,P.;Oswald,I.D.H.;Jodrell,D.I.;Parsons,S.;Sadler, P.J.Proc.Natl.Acad.Sci.U.S.A.2005,102,18269-18274. (41) Aird,R.E.;Cummings,J.;Ritchie,A.A.;Muir,M.;Morris,R.E.; (40) Dadci,L.;Elias,H.;Frey,U.;Hornig,A.;Koelle,U.;Merbach,A. Chen,H.;Sadler,P.J.;Jodrell,D.I.Br.J.Cancer2002,86,1652- E.;Paulus,H.;Schneider,J.S.Inorg.Chem.1995,34,306-315. 1657. 4058 InorganicChemistry,Vol.46,No.10,2007 ChloroHalf-SandwichOsmium(II)Complexes Thep-cym/ampycomplex3PF wasnon-cytotoxictoward chelator hydrolyze relatively rapidly (t ) 1.6 h at 310 K 6 1/2 A549 cells despite its similar rate of hydrolysis to the bip/ forcomplex2BF ),theintroductionof(cid:240)-acceptorpyridines 4 en and p-cym/en complexes 1PF /BF and 2BF , stability as the N-donors, considerably slows down hydrolysis (e.g., 6 4 4 atmicromolarconcentrations(50(cid:237)M),andtheabilitytoreact by an order of magnitude for complex 5PF ), and a large 6 with 9EtG under biologically relevant conditions with an window of reactivity can therefore be accessed. The intro- associated half-life of 3.2 h. The reasons for the inactivity ductionofan(cid:240)-acceptorN-donoralsoincreasestheacidity arethereforenotclear.Howevertheothercomplexestested of the aqua adduct. The en complexes [(Ł6-bip)Os(en)Cl]- which have pyridyl ligands (4PF and 5PF ) were also PF /BF and [(Ł6-THA)Os(en)Cl]BF are as cytotoxic to 6 6 6 4 4 inactive, as are some of the RuII arenes with unsubstituted human A549 lung and A2780 ovarian cancer cells as the pyridyl ligands.43 drug carboplatin, whereas those complexes containing a Theinactivityofcomplexes4PF ,5PF ,7PF ,and8PF pyridine N-donor were inactive. An exception is the dppz 6 6 6 6 canbecorrelatedwiththeirslowkineticsofhydrolysisand complex with biphenyl as the arene [(Ł6-bip)Os(dppz)Cl]- higher acidity of coordinated water. Complex 9, which PF (6PF ), which can also act as a DNA intercalator. 6 6 containsonlymonodentatenon-areneligands,isdeactivated Although{(Ł6-bip)Os(en)}2+adductswithGnucleobases in water by formation of the inert hydroxo-bridged dimer. appear to be less thermodynamically stable than similar The dppz complex 6PF is probably too substitution-inert adductsthoughttoberesponsiblefortheanticanceractivity 6 to bind effectively to G bases on DNA, but both the arene of the RuII analogues, once formed the OsII adducts have (bip) and chelating ligand (dppz) have the ability to somekineticstabilitywithrespecttodissociation.Itisunclear intercalateintoDNA15,35(videsupra),whichmaycontribute why the complex [(Ł6-p-cym)Os(ampy)Cl]PF (3PF ) ex- 6 6 to the mechanism of cytotoxicity of 6PF against both the hibitslowcytotoxicity,sinceithydrolyzesatasimilarrate, 6 A549 and A2780 cancer cell lines. has a similar pK*for its aqua adduct, and a similar rate of a Despite the instability of complex 1BF 4 in DMSO, bindingtoGnucleobasesastheactivecomplexes1PF 6 /BF 4 solutionsoftheactivecomplexes1PF 6 /BF 4 and2BF 4 were and 2BF 4 . foundtobestableinisotonicsalinesolutions(150mMNaCl) Thus we have shown that half-sandwich OsII arene storedatambienttemperatureinthedarkoveraperiodof2 complexescontainingN,N-chelatingligandscanbedesigned months. This suggests that drug formulation in aqueous whichhavecancercellcytotoxicitycomparabletotheirRuII solution would be feasible for these complexes. analogs,butthereactivitiesare100timesless.Sucharange of kinetic effects can be useful for balancing cytotoxicity Conclusions andunwantedside-effectsofanticancerdrugs,asillustrated bytheclinicalprofilesofcisplatinandtheless-labilesecond Although organometallic complexes offer a versatile generation drug carboplatin. Some of the OsII complexes platformforthedesignofnewmedicines,andofanticancer preparedhereappeartobestableunderconditionsrelevant agents in particular, their aqueous solution chemistry has todrugformulation(e.g.,isotonicsaline)andareinteresting been relatively little explored. An understanding of how candidates for further investigation. ligand design can be used to control their redox and ligand substitutionreactionsunderconditionsofbiologicalrelevance Acknowledgment. We thank the EPSRC and The Uni- is vital for the establishment of structure-activity relation- versityofEdinburgh(studentshipforA.F.A.P.),Dr.Michael ships and for understanding their mechanisms of action. Melchart(Edinburgh)forthegiftofthedppzligand,Rhona Although osmium complexes are often considered to be E. Aird and Professor Duncan Jodrell (Western General kinetically inert, we have shown that the reactivity of half- Hospital/Cancer Research U.K. Centre) for advice and sandwichOsIIarenecomplexesdependsstronglyontheother assistance with cell culture, and members of EC COST ligands in the complex, which can play both steric and groups D20 and D39 for stimulating discussions. electronic roles. Here we have shown that although [(Ł6- arene)Os(N,N)Cl]+ complexes containing en as the N,N- Supporting Information Available: Details of the crystal- lographicdata(FiguresS1-S3),aqueousandnucleobasebinding studies(FiguresS4-S7)andstabilitystudies(FigureS8-S10),and (42) Liu,H.-K.;Berners-Price,S.J.;Wang,F.;Parkinson,J.A.;Xu,J.; Bella,J.;Sadler,P.J.Angew.Chem.,Int.Ed.2006,45,8153-8156. X-raycrystallographicdatainCIFformat.Thismaterialisavailable (43) Habtemariam,A.;Melchart,M.;Ferna´ndez,R.;Parsons,S.;Oswald, freeofchargeviatheInternetathttp://pubs.acs.org. I.;Parkin,A.;Fabbiani,F.P.A.;Davidson,J.;Dawson,A.;Aird,R. E.;Jodrell,D.I.;Sadler,P.J.J.Med.Chem.2006,49,6858-6868. IC062350D Inorganic Chemistry, Vol. 46, No. 10, 2007 4059