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Organometallic osmium(II) arene anticancer complexes containing picolinate derivatives.
Inorg. Chem. 2009, 48, 1753-1762
Organometallic Osmium(II) Arene Anticancer Complexes Containing
Picolinate Derivatives
Sabine H. van Rijt,† Anna F. A. Peacock,‡ Russell D. L. Johnstone,‡ Simon Parsons,‡
and Peter J. Sadler*,†
Department of Chemistry, UniVersity of Warwick, Gibbet Hill Road, CoVentry CV4 7AL, U.K., and
School of Chemistry, UniVersity of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, U.K.
ReceivedOctober22,2008
Chlorido osmium(II) arene [(η6-biphenyl)OsII(X-pico)Cl] complexes containing X ) Br (1), OH (2), and Me (3) as
ortho,orX)Cl(4),COH(5),andMe(6)asparasubstituentsonthepicolinate(pico)ringhavebeensynthesized
2
andcharacterized.TheX-raycrystalstructuresof1and6showtypical“piano-stool”geometrywithintermolecular
π-π stacking of the biphenyl outer rings of 6. At 288 K the hydrolysis rates follow the order 2 . 6 > 4 > 3 > 5
. 1 with half-lives ranging from minutes to 4.4 h illustrating the influence of both electronic and steric effects of
the substituents. The pK values of the aqua adducts 3A, 4A, 5A, and 6A were all in the range of 6.3-6.6. The
a
para-substitutedpicocomplexes4-6readilyformedadductswithboth9-ethylguanine(9EtG)and9-ethyladenine
(9EtA), but these were less favored for the ortho-substituted complexes 1 and 3 showing little reaction with 9EtG
and 9EtA, respectively. Density-functional theory calculations confirmed the observed preferences for nucleobase
bindingforcomplex1.IncytotoxicityassayswithA2780,cisplatin-resistantA2780cishumanovarian,A549human
lung, and HCT116 colon cancer cells, only complexes 4 (p-Cl) and 6 (p-Me) exhibited significant activity (IC
50
values<25µM).BothofthesecomplexeswereasactiveascisplatininA2780(ovarian)andHCT116(colon)cell
lines,andevenovercomecisplatinresistanceintheA2780cis(ovarian)cellline.Theinactivityof5isattributedto
the negative charge on its para carboxylate substituent. These data illustrate how the chemical reactivity and
cancer cell cytotoxicity of osmium arene complexes can be controlled and “fine-tuned” by the use of steric and
electroniceffectsofsubstituentsonachelatingligandtogiveosmium(II)arenecomplexeswhichareasactiveas
cisplatin but have a different mechanism of action.
Introduction A lack of understanding of the aqueous chemistry of
organometalliccomplexes,inparticularofRuIIandOsIIarene
Currently, there is much interest in ruthenium(II) arene
complexes, under biologically relevant conditions provides
complexesofthetype[(η6-arene)Ru(X)(YZ)](whereYZis
an obstacle in current attempts to design anticancer drugs.
a bidentate chelating ligand, and X a good leaving group,
Knowledge of the aqueous chemistry of these types of
e.g.Cl-)whichexhibitbothinvitroandinvivoanticancer
complexes may eventually lead to the control of their
activity,insomecasesevencomparabletothatofcisplatin.1
pharmacological properties, including cell uptake, distribu-
Yet the pharmacological potential of the heavier congener
tion, DNA binding, metabolism, and toxic side-effects.
osmium has been little explored. Previous work in our
For the OsII arene ethylenediamine (en) complex, [(η6-
laboratoryonOsIIarenecomplexes,[(η6-arene)Os(YZ)X]n+,
biphenyl)Os(en)Cl]+, hydrolysis occurs with a half-life of
hasshownthattheiraqueousreactivityishighlydependent
6.4 h at 298 K, which is about 40 times slower than that
on the nature of the chelating ligand YZ.2,3
(2) Peacock, A. F. A.; Habtemariam, A.; Fernandez, R.; Walland, V.;
* To whom correspondence should be addressed. E-mail: p.j.sadler@ Fabbiani,F.P.A.;Parsons,S.;Aird,R.E.;Jodrell,D.I.;Sadler,P.J.
warwick.ac.uk. J.Am.Chem.Soc.2006,128,1739.
†UniversityofWarwick. (3) (a)Peacock,A.F.A.;Parsons,S.;Sadler,P.J.J.Am.Chem.Soc.
‡UniversityofEdinburgh. 2007, 129, 3348. (b) Krostrhunova, H.; Florian, J.; Novakova, O.;
(1) Aird,R.E.;Cummings,J.;Ritchie,A.A.;Muir,M.;Morris,R.E.; Peacock,A.F.A.;Sadler,P.J.;Brabec,V.J.Med.Chem.2008,51,
Chen,H.;Sadler,P.J.;Jodrell,D.I.Br.J.Cancer2002,86,1652. 3635.
10.1021/ic8020222 CCC: $40.75 2009 American Chemical Society Inorganic Chemistry, Vol. 48, No. 4, 2009 1753
PublishedonWeb01/15/2009
van Rijt et al.
Chart1.OsmiumAreneComplexesStudiedinThisWork (IC ) 6 µM). These studies demonstrate that the kinetics
50
and thermodynamics of these types of complexes are
significantfortheirbiologicalactivityandimportantly,that
thesefactorscanbecontrolledbyappropriateliganddesign.
Hereweinvestigatetheoptimizationofthebiologicalactivity
of osmium(II) arene complexes where YZ is a picolinate
derivative (Chart 1). This has been approached by making
systematic changes to their design by placing different
substituentsintheortho-andpara-positionsofthepyridine
ringofthepicolinatechelatingligand.Solid-statestructures,
cytotoxicitydata,andinvestigationsoftheiraqueouschem-
istry using 1H NMR spectroscopy, including their rates of
hydrolysis,acidityoftheresultingaquaadducts,andnucleo-
base binding studies, are discussed. This work shows that
substituentsonthepicolinatebackbonecanhavesignificant
effectsontheaqueouschemistryinosmium(II)compounds
of the type [(η6-bip)Os(YZ)(Cl)] providing great scope for
design for this class of compounds.
Experimental Section
Materials.1,4-Dihydrobiphenylandthedimer,[(η6-bip)OsCl]
22
werepreparedbypreviouslyreportedprocedures.2,49-Ethylguanine
and 9-ethyl adenine were purchased from Sigma-Aldrich.
OsCl ·nHOand6-hydroxopicolinicacid(>97%)werepurchased
3 2
from Alfa Aesar. 2-Cyano-4-methyl pyridine (98%) and thionyl
chloridewereobtainedfromRiedeldeHae¨nandFluka,respectively.
6-Bromopicolinicacid(98%),6-methylpicolinicacid(95%),2,4
pyridinedicarboxylicacidmonohydrate(98%),dicyclohexylcarbo-
diimide, and all deuterated solvents were obtained from Aldrich.
Ethanolandmethanolweredistilledovermagnesium/iodineprior
oftheRuIIanalogue.Thishighlightsthelowerreactivityof touse.
OsII.2 Interestingly, despite its slow hydrolysis rate, [(η6-
PreparationofComplexes.Complexes1-6weresynthesized
biphenyl)Os(en)Cl]+stillexhibitspromisingactivityagainst from the dimeric precursor, [(η6-bip)OsCl 2 ] 2 , using procedures
similar to those reported previously for other half-sandwich OsII
thehumanovariancancerA2780cellline(IC )9µM).3b
50 arene complexes.5,6 Complex 7 was synthesized in EtOH using
ChangingtheligandfromtheneutralN,N-chelatorentothe
dicyclohexylcarbodiimide(DCC)ascouplingreagent.
anionic O,O-chelator acetylacetonate (acac) has a marked
4-Chloro Picolinic Acid. A suspension of picolinic acid (0.79
effect on the extent and rate of hydrolysis. The hydrolysis g,6.35mmol)andsodiumbromide(1.30g,12.7mmol)in10mL
rateof[(η6-arene)Os(acac)Cl],istoofasttomonitorbyNMR ofthionylchloridewasrefluxedmildlyfor20h.Theinitiallydark
at 298 K. However, hydrolysis of the acac compounds is green mixture changed to a dark red color. Excess SOCl was
2
complicatedbytheformationofthehydroxobridgeddimer, removedbyrotaryevaporation,andtheorangeresiduewastaken
[(η6-arene)Os(µ2-OH) 3 Os(η6-arene)]+,withlossoftheacac up in about 15 mL of CH 2 Cl 2 and was filtered through celite to
ligand. This hydroxo-bridged dimer is the only observed removeanyinsolublematerial.Theorangesolutionwascooledto
271K,and20mLofHO(doublydistilled)wasaddeddropwise
speciesatthemicromolarconcentrationsinsolutionssimilar 2
whilestirringvigorously,keepingthetemperaturebetween271and
tothoseusedinbiologicalcellculturetests.2Onaccountof
275K.Thecolorofthesolutionchangedtoalighterorange,and
formation of these hydroxo-bridged adducts, compounds
a white precipitate formed. The mixture was further stirred at
containing O,O-chelators are generally inactive toward the
ambienttemperaturefor20h.TheCHCl andHOwereremoved
2 2 2
humanovarian(A2780)andhumanlung(A549)cancercell
byrotaryevaporation.Thesolidwasrecrystallizedfromaminimum
lines. amountofEtOHtogiveayieldof0.28g(28%).1HNMR(DMSO-
Intermediatebehaviortothatofthecomplexescontaining d):δ)8.72(1H,d,J)4.9Hz),8.09(1H,d,J)1.6Hz),7.84
6
N,N-andO,O-chelatorsisobservedinaqueoussolutionfor (1H,dd,J)4.9,1.6Hz).
some complexes containing anionic N,O-chelators. Com- 4-Methyl Picolinic Acid. A solution of 2-cyano-4-methyl
plexescontainingapyridinederivativeastheN-donoratom pyridine (0.15 g, 1.27 mmol) in about 10 mL of 6 M HCl was
refluxedfor24h.Duringthistime,theinitiallylightyellowsolution
hydrolyze at an intermediate rate, are stable in aqueous
solutionatmicromolarconcentrations,andareactivetoward
(4) Stahl,S.;Werner,H.Organometallics1990,9,1876.
both A549 and A2780 cell lines.3 Notably, complexes
(5) Morris,R.E.;Aird,R.E.;Murdoch,P.D.;Chen,H.M.;Cummings,
containing picolinate (pico) as the N,O-chelate, display J.;Hughes,N.D.;Parsons,S.;Parkin,A.;Boyd,G.;Jodrell,D.I.;
Sadler,P.J.J.Med.Chem.2001,44,3616.
promisingactivitytowardthehumanovariancancercellline
(6) Fernandez,R.;Melchart,M.;Habtemariam,A.;Parsons,S.;Sadler,
with IC
50
) 4.5 µM, a value similar to that of carboplatin P.L.Chem.sEur.J.2004,10,5173.
1754 InorganicChemistry,Vol.48,No.4,2009
OrganometallicOsmium(II)AreneAnticancerComplexes
changed to a clear solution. The solution was taken to dryness, (534.03):C,42.73;H,3.40;N,2.62%.Found:C,42.57;H,2.99;
and a white solid remained. The solid was recrystallized from a N,2.58%.1HNMR(DMSO-d):δ)8.94(1H,d,J)5.66Hz),
6
minimalamountofdistilledwatertogiveayieldof148mg(85%). 7.74(1H,br),7.63(2H,m),7.52(1H,d,J)4.53Hz),7.45(3H,
1HNMR(DMSO-d):δ)8.65(1H,s),8.06(1H,s),7.66(1H,s), m), 6.73 (1H, d, J ) 5.29 Hz), 6.69 (1H, d, J ) 4.91 Hz), 6.40
6
2.05(3H,s). (1H,m),6.37(2H,m).CrystalssuitableforX-raydiffractionwere
[(η6-bip)Os(6-Br-pico)Cl] (1). A solution of [(η6-bip)OsCl] obtainedbyslowevaporationfromCHCl as6·CHCl atambient
22 2 2 2 2
(51.9 mg, 0.06 mmol) in dry and degassed MeOH (10 mL) was temperature.
refluxed under argon for 1 h before adding a solution of sodium [(η6-bip)Os(4-COEt-pico)Cl](7).Asolutionof[(η6-bip)OsCl]
2 22
methoxide(2.2molequiv,7.2mg)and6-bromopicolinicacid(2.2 (47.1mg,0.056mmol)anddimethylaminopyridine(3mg)indry
mol equiv, 27.2 mg) in 5 mL of dry and degassed MeOH. The anddegassedEtOHwasplacedundernitrogenandwasstirredfor
resultingmixturewasrefluxedmildlyfor3h,filtered,andsolvent 10 min after which it was cooled down to 273 K before adding
reducedonarotaryevaporatoruntilprecipitatebegantoformand dicyclohexylcarbodiimide (1 mol equiv, 19 mg). The reaction
wasleftstandingat278K.Theyellowpowderwasrecoveredby mixture was stirred at 273 K for a further 15 min, after which it
filtrationandwasair-driedtogiveafinalyieldof45.6mg(63%). waslefttostiratambienttemperatureforanother2.5h.Thiswas
Anal.CalcdforC H BrClNOOs(580.94):C,37.22;H,2.26;N, thenfiltered,andthesolventwasremovedontherotaryevaporator.
18 13 2
2.41%.Found:C,36.91;H,2.12;N,2.25%.1HNMR(CDCl):δ About3mLofdryDCMwasaddedtothebrownsolid,andthis
3
)8.09(1H,d,J)7.0Hz),7.83(1H,d,J)7.0Hz),7.70(1H, was filtered to remove the white precipitated urea side product.
t,8Hz),7.52(2H,d,J)7.9Hz),7.38(3H,m),6.71(1H,d,J) TheDCMfiltratewasdriedonarotaryevaporator,andthebrown
5Hz),6.63(1H,d,J)6Hz),6.46(1H,t,J)5.29Hz),6.35(1H, solid was washed with ether to remove the remaining dimethy-
t, J ) 6.0 Hz), 6.32 (1H, t, J ) 5.29 Hz). Crystals suitable for laminopyridinewhichresultedinabrowncrystallinesolid.Yield
X-raydiffractionwereobtainedbyslowevaporationfromCHCl 2.2mg(3.4%).1HNMR(MeOD-d):δ)9.10(1H,d,J)5.52
3 4
as1·CHCl atambienttemperature. Hz),8.40(1H,d,J)1.76Hz),8.05(1H,dd,J)3.76,2.01Hz),
3
[(η6-bip)Os(6-OH-pico)Cl] (2). Synthesis as for 1 using [(η6- 7.64(2H,m),7.44(3H,m),6.70(1H,d,J)5.02Hz),6.66(1H,
bip)OsCl] (51.7mg,0.06mmol),sodiummethoxide(2.2equiv, d, J ) 5.77 Hz), 6.39 (2H, q, J ) 4.76 Hz), 6.34 (1H, m), 4.48
22
6.71mg),and6-hydroxopicolinicacid(2.2equiv,19mg).Yield: (2H,q,J)7.03Hz),1.2(3H,t,J)7.03Hz).
37.3mg(60%).CalcdforC H ClNOOs(517.99):C,41.74;H, Instrumentation.X-rayCrystallography.Diffractiondatafor
18 14 3
2.72; N, 2.70%. Found: C, 41.22; H, 2.62; N, 2.65%. 1H NMR compound1werecollectedat150KusingaBrukerSmartApex
(DMSO-d):δ)13.12(1H,br),7.86(1H,t,J)8Hz),7.65(2H, CCDdiffractometer.Diffractiondatafor6wascollectedat120K
6
d,J)7.2Hz),7.44(3H,m),7.33(1H,d,J)7.0Hz),7.11(1H, by the EPSRC National Crystallography Service (Southampton)
d,J)8.31Hz),6.74(1H,d,J)5.29Hz),6.66(1H,d,J)5.28 usingaBruker-NoniusAPEXIICCDcameraonkappa-goniostat.
Hz),6.42(2H,m),6.38(1H,t,J)4.53Hz). Absorption corrections for all data sets were performed with the
[(η6-bip)Os(6-Me-pico)Cl] (3). Synthesis as for 1 using [(η6- multiscanprocedureSADABS.7Thestructureof1wassolvedby
bip)OsCl] (52.2 mg, 0.06 mmol), sodium methoxide (2.2 mol direct methods (SIR92),8 and that of 6 by Patterson methods
2 2
equiv,7.0mg),and6-methylpicolinicacid(2.2molequiv,18mg). (DIRDIF).9 Refinement was against F2 using all data (SHELX-
Yield:50mg(77%)Anal.CalcdforC H ClNOOs(517.05):C, TL).10H-atomswereplacedingeometricallycalculatedpositions.
19 16 2
44.22;H,3.13;N,2.71%.Found:C,43.89;H,2.65;N,2.73%.1H In6amoleculeofdichloromethaneisdisorderedaboutacrystal-
NMR (CDCl): δ ) 7.95 (1H, d, J ) 7.93 Hz), 7.72 (1H, t, J ) lographic inversion center. The C-Cl bond distances were re-
3
7.56),7.43(3H,m),7.36(3H,m),6.51(1H,d,J)5.29Hz),6.47 strained to 1.73(1) Å during refinement. The programs Diamond
(1H,d,J)5.67Hz),6.30(1H,t,J)5.29Hz),6.27(1H,t,J) 3.020,11Mercury1.4.1,12andORTEP3213wereusedforanalysis
4.91Hz),6.22(1H,t,J)5.29Hz). ofdataandproductionofgraphics.X-raycrystallographicdatafor
[(η6-bip)Os(4-Cl-pico)Cl] (4). Synthesis as for 1 using [(η6- compounds 1 and 6 have been deposited in the Cambridge
bip)OsCl] (51.5 mg, 0.06 mmol), sodium methoxide (2.2 mol CrystallographicDataCentreundertheaccessionnumbersCCDC
2 2
equiv, 7.4 mg), and 4-chloro picolinic acid (2.2 mol equiv, 21.5 706164and706165,respectively.
mg).Yield:37.1mg(54%)Anal.CalcdforC H ClNOOs·HO NMRSpectroscopy.1HNMRspectrawereacquiredin5mm
18 13 2 2 2
(554.45):C,38.99;H,2.73;N,2.53%.Found:C,38.66;H,1.95; NMRtubesat298K(unlessstatedotherwise)oneitheraBruker
N,2.60%.1HNMR(DMSO-d):δ)9.11(1H,d,J)5.67Hz), DMX500(1H)500.13MHz)oranAVA600(1H)599.81MHz)
6
7.93(1H,d,J)1.89Hz),7.92(1H,dd,J)6.04,2.26Hz),7.65 spectrometer.1HNMRchemicalshiftswereinternallyreferenced
(2H,m),7.47(3H,m),6.76(1H,d,J)5.29Hz),6.72(1H,d,J to(CHD)(CD)SO(2.50ppm)forDMSO-d,CHCl (7.26ppm)
2 3 6 3
)5.66),6.46(1H,t,J)5.29Hz),6.43(1H,t,J)5.29Hz),6.40 for chloroform-d, and to 1,4-dioxane (3.75 ppm) for aqueous
1
(1H,t,J)4.91Hz). solutions.ForNMRspectraofaqueoussolutions,watersuppression
[(η6-bip)Os(4-COH-pico)Cl](5).Synthesisasfor1using[(η6-
2
bip)OsCl 2 ] 2 (53.6 mg, 0.06 mmol), sodium methoxide (2.2 mol (7) Sheldrick,G.M.SADABS,Version2006-1;UniversityofGo¨ttingen:
equiv,7.3mg),and2,4pyridinedicarboxylicacid(2.2molequiv, Go¨ttingen,Germany,2006.
23.3mg).Yield:29.7mg(41%).Anal.CalcdforC H ClNOOs (8) Altomare,A.;Cascarano,G.;Giacovazzo,C.;Guagliardi,A.J.Appl.
19 14 4 Crystallogr.1994,27,1045.
(546.00):C,41.80;H,2.58;N,2.57%.Found:C,41.19;H,2.12;
(9) Beurskens,P.T.,Beurskens,G.;Bosman,W.P.;deGelder,R.;Garcia-
N,2.55%.1HNMR(DMSO-d 6 ):δ)9.31(1H,m),8.14(1H,m), Granda;S.;Gould,R.O.;Israel,R.;Smits,J.M.M.Crystallography
8.01(1H,m),7.65(2H,m),7.47(3H,m),6.76(1H,d,J)5.67 Laboratory;UniversityofNijmegen:TheNetherlands,1996.
Hz), 6.72 (1H, d, J ) 6.04 Hz), 6.46 (1H, t, J ) 5.29 Hz), 6.44 (10) Sheldrick,G.M.SHELXL-97.Programfortherefinementofcrystal
structures;UniversityofGo¨ttingen:Germany,1997.
(1H,t,J)5.29Hz),6.41(1H,t,J)4.91Hz). (11) CrystalImpact DIAMOND, Version 3.0, Visual crystal structure
[(η6-bip)Os(4-Me-pico)Cl] (6). Synthesis as for 1 using [(η6- informationsystem.;CrystalImpactGbR:Bonn,Germany,2004.
bip)OsCl] (53.1 mg, 0.06 mmol), sodium methoxide (2.2 mol (12) Macrae,C.F.;Edgington,P.R.;McCabe,P.;Pidcock,E.;Shields,
2 2 G.P.;Taylor,R.;Towler,M.;vandeStreek,J.J.Appl.Crystallogr.
equiv, 7.4 mg), and 4-methyl picolinic acid (2.2 mol equiv, 19.2
2006,39,453.
mg).Yield:35.6mg(57%)Anal.CalcdforC 19 H 16 ClNO 2 Os·H 2 O (13) Farrugia,L.J.J.Appl.Crystallogr.1997,30,565.
Inorganic Chemistry, Vol. 48, No. 4, 2009 1755
van Rijt et al.
wascarriedoutusingShakaorpresaturationmethods.14Alldata Computation.Density-functionaltheory(DFT)calculationswere
processingwascarriedoutusingXWIN-NMRversion3.6(Bruker carried out by using the Amsterdam density functional (ADF)17
U.K.Ltd.). program(version2007.01).Thecoordinatesofcomplexesusedfor
pH* Measurements. pH* values (pH meter reading without the calculations were obtained directly from the X-ray crystal
correctionforeffectsofDonglasselectrode)ofNMRsamplesin structures. Modifications to the structures were performed in
DO were measured at about 298 K directly in the NMR tube, Chemcraft (version 1.5). Geometries and energies were obtained
2
beforeandafterrecordingNMRspectra,usingaCorning240pH byusingthePerdew-Wanggradient-correctedfunctional(GGA)
meterequippedwithamicrocombinationelectrodecalibratedwith withscalarZORArelativisticcorrection.18-22Thegeneralnumer-
AldrichbuffersolutionsofpH4,7,and10. ical intergration was 4.0. The frozen core approximation23 was
Elemental Analysis. Carbon, hydrogen, and nitrogen (CHN) applied using triple-(cid:4) polarization (TZP). Default convergence
elementalanalysiswerecarriedoutatTheUniversityofSt.Andrews criteriawereappliedforself-consistentfields(SCF)andgeometry
intheSchoolofChemistryonaCarloErbaCHNSanalyzer. optimization.TheCOSMO,asimplementedinADFprogram,was
Methods. Kinetics of Hydrolysis. The kinetics of hydrolysis used to simulate the aqueous environment with ε ) 78.4 and a
for complexes 1-6 were followed by 1H NMR at different proberadius)1.9Å.TheatomicradiiusedwereOs)1.958,F
temperatures. For this, solutions of the complexes with a final ) 1.425, Cl ) 1.725, Br ) 1.850, O ) 1.517, N ) 1.608, C )
concentration of 0.8 mM in 5% MeOD-d/95% DO (v/v) were 1.700,andH)1.350Å.
4 2
prepared by dissolution of the complexes in MeOD-d followed
4
Results
byrapiddilutionusingDOwithapH*ofabout2(acidifiedwith
2
HNO 3 ), so that the aqua ligand was not deprotonated. 1H NMR SynthesisandCharacterization.Theosmiumcompounds
spectra were taken after various intervals using the presaturation
weresynthesizedviatheCl-bridgeddimer,[(η6-bip)OsCl ] ,
methodforwatersurpression.Therateofhydrolysiswasdetermined wherebip)biphenyl.Compound7,however,wasobtain 2 e 2 d
byfittingplotsofconcentrations(determinedfrom1HNMRpeak
in low yield from compound 5 as starting material in dry
integrals)versustimetoapseudofirst-orderequationusingORIGIN
ethanol with DCC as coupling reagent. The X-ray crystal
version 5.0 (Microcal Software Ltd.). The Arrhenius activation
energy (E a ), activation enthalpies (∆Hq), and activation entropy structures of compounds [(η6-bip)Os(6-Br-pico)Cl]·CHCl 3
(∆Sq) for compound 6 were determined from the slopes of the (1·CHCl 3 )and[(η6-bip)Os(4-Me-pico)Cl]·CH 2 Cl 2 (6·CH 2 Cl 2 )
ArrheniusandinterceptsofEyringplots. weredetermined.Bothstructuresadoptthefamiliarpseudo-
pK* Calculations. For determinations of pK* values (pK octahedral“three-legpianostool”geometry(Figure1A,B).
a a a
values determined from solutions in D 2 O), the pH* values of the The osmium is π-bonded to the arene ligand (“the seat”)
aqua complexes of 3-6 in D 2 O (formed in situ by dissolution of withacentroidOs-ringdistanceof1.654Åfor1and1.660
theparentchlorocomplexes)werevariedfromaboutpH*2to10 Å for 6, σ-bonded to the chloride (2.4065(15) Å (1),
bytheadditionofdiluteNaODandHNO,and1HNMRspectra
3 2.4095(19) Å (6)) and σ-bonded to the chelating picolinate
were recorded. The chemical shifts of the arene ring protons
ligand(pyridinenitrogen-Os;2.154(5)Å(1),2.088(5)Å(6),
were plotted against pH*. The pH* titration curves were fitted
oxygen-Os2.077(4)Å(1),2.082(4)Å(6)),whichconstitute
to the Henderson-Hasselbalch equation, with the assumption
the three legs of the piano stool. Crystallographic data,
thattheobservedchemicalshiftsareweightedaveragesaccording
selectedbondlengths,andanglesaregiveninTables1and
to the populations of the protonated and deprotonated species.
ThesepK*valuescanbeconvertedtopK valuesbyuseofthe 2.Complex1hasapropellertwistofthebiphenylligandof
a a
equation pK ) 0.929pK* + 0.42 as suggested by Krezel and about 38°, while for complex 6 a propeller twist of 42° is
a a
Bal15 for comparison with related values in the literature. observed.Thereisanintermolecularinteractionbetweenthe
Interactions with Nucleobases. The reaction of 1-6 with outer rings of the biphenyl arene of adjacent molecules in
nucleobasestypicallyinvolvedadditionofasolutioncontaining1 compound 6, with a centroid-centroid distance of 3.7 Å
molequivofnucleobaseinD 2 O,toanequilibriumsolutionof1-6 (Figure 2). This value indicates intermolecular ring stack-
inD 2 O(>90%aquacomplex).ThepH*valueofthesamplewas ing.24
adjustedifnecessarysoastoremaincloseto7.4(physiological).
Kinetics of Hydrolysis. The rates of hydrolysis of
1H NMR spectra of these solutions were recorded at 298 K after
compounds 1-6 in a 5% MeOD-d /95% D O were moni-
varioustimeintervals. 4 2
tored by 1H NMR at 288 and 298 K by the observation of
CancerCellCytotoxicity.Afterplating,humanovarianA2780
andcisplatinresistantA2780ciscancercellsweretreatedwithOsII new peaks over time due to aqua adduct formation. Five
complexes on day 3, and human lung A549 and human colon percent MeOD was used to improve solubility, and acidic
HCT116cancercellsonday2,atconcentrationsrangingfrom0.5
µMto100µM.SolutionsoftheOsIIcomplexesweremadeupin (17) TeVelde,G.;Bickelhaupt,F.M.;Baerends,E.J.;FonsecaGuerra,
C.;VanGisbergen,S.J.A.;Snijders,J.G.;Ziegler,T.J.Comput.
0.125% (v/v) dimethylsulfoxide (DMSO) to assist dissolution
Chem.2001,22,931.
(0.03%finalconcentrationofDMSOperwellinthe96-wellplate). (18) vanLenthe,E.;Baerends,E.J.;Snijders,J.G.J.Chem.Phys.1993,
Cells were exposed to the complexes for 24 h, washed, supplied 99,4597.
(19) vanLenthe,E.;Baerends,E.J.;Snijders,J.G.J.Chem.Phys.1994,
withfreshmedium,allowedtogrowforthreedoublingtimes(72
101,9783.
h), and then the protein content measured (proportional to cell (20) vanLenthe,E.;Baerends,E.J.;Snijders,J.G.J.Chem.Phys.1996,
survival)usingthesulforhodamineB(SRB)assay.16 105,2373.
(21) vanLenthe,E.;Ehlers,A.;Baerends,E.-J.J.Chem.Phys.1999,110,
8943.
(14) Hwang,T.L.;Shaka,A.J.J.Magn.Reson.Ser.A1995,112,275. (22) vanLenthe,E.;vanLeeuwen,R.;Baerends,E.J.;Snijders,J.G.Int.
(15) Krezel,A.;Bal,W.J.Inorg.Biochem.2004,98,161. J.QuantumChem.1996,57,281.
(16) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; (23) Baerends, E. J.; Ellis, D. E.; Ros, P. Theor. Chim. Acta 1972, 27,
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. 339.
J.Natl.CancerInst.1990,82,1107. (24) Janiak,C.J.Chem.Soc.,DaltonTrans.2000,3885.
1756 InorganicChemistry,Vol.48,No.4,2009
OrganometallicOsmium(II)AreneAnticancerComplexes
Figure1.X-raycrystalstructureandatomnumberingschemefor(A)[(η6-bip)Os(6-Br-pico)Cl]·CHCl (1·CHCl),and(B)[(η6-bip)Os(4-Me-pico)Cl]·CHCl
3 3 2 2
(6·CHCl).Hatomsandsolventmoleculeshavebeenomittedforclarity.
2 2
Table1.CrystallographicDatafor[(η6-bip)Os(2-Br-pico)Cl]·CHCl
3
(1·CHCl)and[(η6-bip)Os(4-Me-pico)Cl]·CHCl (6·CHCl)
3 2 2 2 2
1·CHCl 6·CHCl
3 2 2
formula C H BrCl NO Os C H ClNO Os
19 14 4 2 19 16 2
molecularweight 700.22 558.44
crystaldescription colorlessblock colorlesslath
size 0.34×0.17×0.14mm 0.16×0.06×0.02mm
λ(Å) 0.71073 0.71073
T/K 150(2) 120(2)
crystalsystem triclinic triclinic
j j
spacegroup P1 P1 Figure2.X-raystructureof[(η6-bip)Os(4-Me-pico)Cl]·CHCl (6·CHCl)
a(Å) 6.8555(8) 8.065(5) showingintermolecularringstackingoftheextendedbiph 2 eny 2 lring. 2 2
b(Å) 11.0638(11) 10.495(5)
c(Å) 13.9209(19)A 11.672(5)
R(deg) 99.783(7) 81.806(5) energy(E a ),activationenthalpy(∆Hq),andactivationentropy
(cid:8)(deg) 98.387(8) 78.860(5) (∆Sq)ofcompound6(Figure3B)arelistedinTable4.The
γ(deg) 91.757(7) 77.776(5) large negative ∆Sq value for compound 6 is notable. The
volume(Å3) 1027.7(2) 942.0(8)
Z 2 2 extentofhydrolysisatequilibriumishighforallcompounds,
R 0.0376 0.0399 with even 100% hydrolysis observed for compounds 4 and
R 0.0868 0.0837
w 5andbetween88%and96%for1,3,and6(Figure3A,B).
GOF 0.997 1.120
Thehydrolysisrateofcompound1(6-Brpico)wastooslow
Table2.SelectedBondLengths(Å)andAngles(deg)for to monitor at 288 K, but its half-life was successfully
[(η6-bip)Os(2-Br-pico)Cl]·CHCl (1·CHCl)and
[(η6-bip)Os(4-Me-pico)Cl]·CHC 3 l (6·CH 3 Cl) determined by increasing the temperature to 298 K (Table
2 2 2 2
bondlength/angle compound1·CHCl compound6·CHCl 3, Figure 3A). Compound 6 hydrolyzes the fastest with a
3 2 2 half-life of just under 1 h (288 K). Complexes 1, 3-5
Os-C(arene) 2.178(7) 2.164(6)
2.207(7) 2.208(7) hydrolyzedwithhalf-livesrangingfrom2.4to4.4hat288
2.175(6) 2.193(6) K (Table 3).
2.177(6) 2.199(6)
The effects of chloride concentrations typical of blood
2.159(6) 2.173(6)
2.208(6) 2.176(6) plasma (100 mM), cell cytoplasm (22.7 mM), and cell
Os-O 2.077(4) 2.082(4) nucleus (4 mM) on the aqueous chemistry of 3 and 6 were
Os-N 2.154(5) 2.088(5) investigated. 1H NMR spectra of 3 or 6 (1 mM) were
Os-Cl 2.4065(15) 2.4095(19)
O-Os-N 77.35(18) 77.10(18) recorded within 10 min of sample preparation and after
O-Os-Cl 84.82(12) 84.50(14) incubationat310Kfor24h(SupportingInformation,Figure
N-Os-Cl 81.74(13) 83.30(15)
S2). On the basis of 1H NMR peak integrals, 35% of
conditions (D O, pH* 2) were used to prevent the deproto- hydrolyzedcomplex3(3A)and10%ofhydrolyzedcomplex
2
nation of the aqua complex as a secondary reaction. The 6(6A)werefoundtobepresentin100mM[Cl](pH*6.22),
hydrolysis of compound 2 containing 6-hydroxy picolinate 60% of 3A, and 53% of 6A at 22.7 mM [Cl] (pH* 6.50)
was too rapid to be observed by 1H NMR at 288 K. The and 60% of 3A and 82% of 6A at 4 mM [Cl] (pH* 6.75)
percentage of aqua peak formation for 1, 3-6 was plotted after 10 min. For complex 6, no significant change was
against time and was fitted to pseudofirst order kinetics observed after 24 h, while compound 3 had not reached
(Figure3,SupportingInformation,FigureS1),andtheirhalf- equilibriumafter24h,seeTable5andSupportingInforma-
lifetimeswerecalculated(Table3).TheArrheniusactivation tion, Figure S2.
Inorganic Chemistry, Vol. 48, No. 4, 2009 1757
van Rijt et al.
pH.ThechemicalshiftswereplottedagainstpH*,Supporting
Information,FigureS4,andtheresultingpHtitrationcurves
were fitted to the modified Henderson-Hasselbalch equa-
tion.25,26 This gave rise to pK* values between 6.30 and
a
6.60(Table6).Forcompound5,thepK*valueofthepara-
a
substituentcarboxylicacidprotonwasdeterminedtobe2.50.
Reactions with Nucleobase Models, 9-Ethyl Guanine
(9EtG) and 9-Ethyl Adenine (9EtA). Since DNA is often
believed to be the main target for transition metal antican-
cer drugs,27 nucleobase binding reactions of compounds
1,[(η6-bip)Os(6-Br-pico)Cl],2,[(η6-bip)Os(6-OH-pico)Cl],
3,[(η6-bip)Os(6-Me-pico)Cl],4,[(η6-bip)Os(4-Cl-pico)Cl],
5, [(η6-bip)Os(4-CO --pico)Cl], and 6, [(η6-bip)Os(4-Me-
2
pico)Cl],withnucleobasemodels9-ethylguanine(9EtG)and
9-ethyladenine(9EtA),wereinvestigated.Solutionsof1-6
(1mM)(containinganequilibriummixtureof1-6andtheir
respectiveaquaadducts1A-6Aasthemajorspecies)with
1 mol equivalent of 9EtG or 9EtA in D O were prepared,
2
and 1H NMR spectra were recorded at different time
intervals.Thepercentagesofnucleobaseadductsformedby
all compounds based on 1H NMR peak integrals are
displayed in Table 7A,B and Figure 4. The addition of 1
Figure3.Timedependenceforformationoftheaquacomplexes1A,3A, mol equiv 9EtG or 1 mol equiv 9EtA to aqueous solutions
4A,5A,and6A(basedon1HNMRpeakintegrals)duringhydrolysisof1,
of all compounds resulted in no new peaks after about 5
3,4,and5inacidicDO(pH*2)at(A)298Kfor1A(9),3A(b),4A
2
(2),and5A(1)and(B)at298K(1),288K(2),285K(9),and278K min.However,afterincubationat310Kfor24h,newpeaks
(b)for6A.TheinsetshowstheArrheniusplot,theslopeofwhichgives for all compounds were observed.
theArrheniusactivationenergyE of90.6kJmol-1.
a Compounds 4-6 formed 9EtG and 9EtA adducts to the
Table3.HydrolysisDataforCompounds1-6at288K/298K, extentof60%-100%completionafter24h.Onlycompound
Determinedby1HNMR 6showedincreasedbindingto9EtG,with20%morebinding
compound T/K k/h-1 t 1/2 /h compared to 9EtA. Compound 5 showed an exceptionally
1 298 0.345(0.0002 2.01(0.001 high nucleobase affinity with 100% nucleobase adduct
2 288 g5.9 e0.12 formation with both 9EtG and 9EtA after 24 h. The ortho-
3 288 0.219(0.005 3.17(0.07
298 0.676(0.039 1.03(0.04 substituted pico compounds 1-3 were less reactive toward
4 288 0.288(0.001 2.40(0.08 the nucleobases compared to the para-substituted com-
298 0.656(0.032 1.06(0.05 pounds 4-6 (Table 7, Figure 4). Compound 3 (o-Me)
5 288 0.158(0.011 4.42(0.32
298 0.384(0.004 1.81(0.02 showed a preference for 9EtG, while compounds 1 (o-Br)
6 288 0.710(0.012 0.98(0.02 and 2 (o-OH) formed no adducts with 9EtG, but did form
298 2.32(0.08 0.30(0.01 9EtAadducts.Incontrasttocompounds3-6,compounds1
pK a * Determination. The changes in the 1H NMR and 2 formed two adenine nucleobase adducts, most likely
chemicalshiftsfortheprotonsofthecoordinatedphenylring throughosmiumbindingtotheN1orN7ofadenineforming
incompounds3-6,presentinequilibriumsolutionsof3-6
9EtA-1/2aandb,Chart1.Itwasnotpossibletodetermine
withtheiraquaadductsasthemajorspecies(3A-6A),were
thepreciseadductratiofor9EtA-1adductsbecauseofpeak
followed with change in pH* over a range of 2-10 overlap. Compound 2 formed 9EtA adducts in a 14% and
(SupportingInformation,FigureS3A).WhenthepH*values 22% ratio.
ofthesolutionswereincreasedfromabout2to10,themajor Cytotoxicity Data. The cytotoxicity of complexes 1-7
set of NMR peaks assigned to 3A, 4A, and 6A gradually toward colon HCT116, human lung A549, ovarian A2780,
shiftedtohighfieldinthespectrum.Incontrast,thesignals andovariancisplatin-resistantA2780ciscancercelllineswas
assignable to parent chloro species 3, 4, and 6 remained investigated (Table 8). Complexes 1, 2, 3, and 5 were non-
unchangedwithchangingpH*,buttheirintensitiesincreased toxic up to the highest test concentration of 50 µM in all
at lower pH*. The protonation of the para-carboxylate on fourcelllines.TheIC values(concentrationatwhich50%
50
the picolinate ring of complex 5 caused the NMR peaks of the cell growth is inhibited) of these complexes are
assigned to both species (5 and 5A) to shift to high field at
low pH* (Supporting Information, Figure S3B), while at (25) Lee,S.A.;Eyeson,R.;Cheever,M.L.;Geng,J.M.;Verkhusha,V.V.;
higherpH*onlythepeaksassignableto5Ashiftedtohigher Burd, C.; Overduin, M.; Kutateladze, T. G. Proc. Natl. Acad. Sci.
U.S.A.2005,102,13052.
field.
(26) Sigel, R. K. O.; Sabat, M.; Freisinger, E.; Mower, A.; Lippert, B.
Becauseofthechiralityattheosmiummetalcenter,none Inorg.Chem.1999,38,1481.
(27) Zhang,C.X.;Lippard,S.J.Curr.Opin.Chem.Biol.2003,7,481.
of the protons of the coordinated arene are equivalent, and
(28) Peacock,A.F.A.;Habtemariam,A.;Moggach,S.A.;Prescimone,
each gave rise to separate 1H NMR peaks at high and low A.;Parsons,S.;Sadler,P.J.Inorg.Chem.2007,46,4049.
1758 InorganicChemistry,Vol.48,No.4,2009
OrganometallicOsmium(II)AreneAnticancerComplexes
Table4.RateDataandActivationParametersfortheAquationofActiveComplex6atVariousTemperatures
compound T/K k/h-1 t /h E /kJmol-1 ∆Hq/kJmol-1 ∆Sq/JK-1mol-1
1/2 a
6 278 0.17 4.06
285 0.44 1.57 90.6(1.0 87.7(2.1 -55.6(1.6
288 0.71 0.98
298 2.32 0.30
Table5.PercentageofAquaAdductFormationinaSolutionof1mM Table8.InVitroGrowthInhibitionofA2780,A2780cis,A549,and
3or6inDOatChlorideLevelsTypicalofBloodPlasma(100mM), HCT116CellLinesforCompounds1-7andCisplatin(CDDP)as
2
CellCytoplasm(22.7mM),andCellNucleus(4mM) Control
%aquaadduct A2780 A2780cis A549 HCT116
t)10min t)24h compound IC 50 (µM) IC 50 (µM) IC 50 (µM) IC 50 (µM)
1 >50 >50 >50 >50
[NaCl] 3A 6A 3A 6A
2 >50 >50 >50 >50
100mM 35 10 27 20 3 >50 >50 >50 >50
22.7mM 60 53 49 53 4 4.8 5.45 24 2.8
4mM 60 82 70 81 5 >50 >50 >50 >50
6 4.4 7.61 25.1 2.5
Table6.pK a *andpK a ValuesafortheDeprotonationofthe 7 44.4 61 NDa NDa
CoordinatedH 2 OinComplexes3A-6A CDDP 3 16.9 7.7 2.7
complex pK* pK aND)notdetermined.
a a
[(η6-bip)Os(6-Me-pico)OD]+ 3A 6.34 6.31
2
[(η6-bip)Os(4-Cl-pico)OD]+ 4A 6.30 6.27 6 overcome cisplatin resistance in the A2780cis cell line.
2
[(η6-bip)Os(4-CO 2 H-pico)OD 2 ]+ 5A 6.61 6.56 Compound7,theethylesterofcompound5,showsmoderate
[(η6-bip)Os(4-Me-pico)OD]+ 6A 6.42 6.38
2 activityinthehumanovarianA2780andA2780ciscelllines
apK valuescalculatedfrompK*accordingtoKrezelandBal.15
a a (Table 8).
Table7.Extentof9EtGand9EtAAdductFormationforCompounds Computation.Usingcomputationalmethodsweattempted
1-6atDifferentTimeIntervals
to gain insight into the steric effects caused by the ortho
compound t)10min t)24h t)72h substituentsuponnucleobasebindingandtheireffectonthe
rate of hydrolysis. The minimum energy structures of
(A)%9EtGNucleobaseAdduct
1 0 0 0 nucleobaseadductsofcompounds1[(η6-bip)Os(6-Br-pico)-
2 0 0 0 (9EtG/9EtA)]+,3[(η6-bip)Os(6-Me-pico)(9EtG/9EtA)]+,and
3 0 30 30 6[(η6-bip)Os(4-Me-pico)(9EtG/9EtA)]+obtainedfromDFT
4 0 59 76
5 0 100 100 calculationsareshowninSupportingInformation,FigureS5.
6 0 80 85 The 9EtG adduct of p-Me complex 6 is 6.6 kcal/mol lower
(B)%9EtANucleobaseAdduct in energy, and the 9EtA adduct is 5.8 kcal/mol lower in
1 0 ∼30a ∼35a energy compared to similar adducts of its isomer the o-Me
2 0 14:20b 14:20b
complex 3.
3 0 9 9
4 0 62 71 Geometry optimizations for the nucleobase adducts of
5 0 100 100 complex1revealedH-bondingbetweentheNH ofadenine
2
6 0 60 60 andthecaboxylateoxygenofthepicolinateligand(NH···OC,
aApproximatevalueduetopeakoverlap.bFormationoftwonucleobase
1.833Å).Incontrast,aclosecontactbetweenguanineC6O
adducts(9EtA-2aandb,Chart1).
and the ortho-bromide substituent (3.260 Å) was observed
in the guanine adduct (Supporting Information, Figure S5).
Discussion
Mixed N,O-chelating ligands can be used to “fine-tune”
theaqueousreactivityofOsIIarenecompoundsbythechoice
ofthetypesofN-andO-donorgroups.Theaimofthisstudy
wastooptimizethebiologicalactivityofosmium(II)arene
complexes by making systematic changes to their design
usingdifferentsubstituentsintheortho-andpara-positions
Figure4.Barchartshowingtheextentofbindingofcompounds1-6to
of the pyridine ring of the picolinate chelating ligand.
thenucleobases9EtG(darkgray)and9EtA(lightgray)after48h.
Complexes 1-7 were synthesized and X-ray structures of
therefore likely to be >100 µM and then can be described compound1[(η6-bip)Os(6-Br-pico)Cl],asaCHCl solvate,and
3
asinactivecompounds.Compounds4(p-Cl)and6(p-Me), 6[(η6-bip)Os(4-Me-pico)Cl],asaCHCl solvate,weredeter-
2 2
however, exhibited promising toxicity with IC values mined.Bothcompoundsadoptthefamiliarpseudo-octahedral
50
similar to those found for cisplatin in the human ovarian “three-legpianostool”geometryasfoundpreviouslyforother
A2780 and colon HCT116 cancer cell lines and moderate osmium(II) biphenyl compounds (i.e., [(η6-bip)Os(YZ)(Cl)]),
cytotoxicity in the A549 cancer cell line (ca. 3 times less forwhichapropellertwistofthebiphenylligandofabout40°
active than cisplatin). Interestingly, both compounds 4 and andOs-Clbondlengthsof2.40Åareobserved.2
Inorganic Chemistry, Vol. 48, No. 4, 2009 1759
van Rijt et al.
Theπ-πstackingbytheflexibleextendedbiphenylarene and at the lowest chloride concentration of 4 mM close to
ringsystemobservedintheX-raystructureofcompound6 that of the cell nucleus, complex 6 is 81% present as the
(Figure2)couldallowintercalationintoDNAtocontribute reactive aqua species. These data indicate that in the cell
to the cytotoxicity of this class of compounds. nucleus, complex 6 might be selectively activated through
Aqueous Solution Chemistry. Previous studies on the hydrolysis as a mode of activation toward DNA binding,
hydrolysis rates of OsII arene compounds of the type [(η6- while outside the cell and in particular in blood plasma
arene)Os(YZ)Cl]n+ have shown that the aqueous reactivity complex6ispredominatelypresentasitslessreactiveintact
ofthesecomplexesishighlydependentonthenatureofthe chloro species.
chelatingligandYZ.2,3,28Compounds1-6(Chart1)hydro- Forcompound3,thedifferentchlorideconcentrationshad
lyzerelativelyfastwithhalf-livesrangingfrom0.98to4.4h little effect on the concentration of hydrolyzed complex 3
at288K,apartfromtheo-OHcompound2,[(η6-bip)Os(6- (3A) after 10 min, although after equilibration for 24 h the
OH-pico)Cl],whichhydrolyzestoofasttobemonitoredby extentofhydrolysiswassimilartothatofcompound6.This
1H NMR at 288 K (Table 3). In addition, the equilibrium can be explained by the steric bulk of the ortho-methyl
forhydrolysisliestoagreatextenttowardtheaquaadducts substituent in complex 3 slowing down hydrolysis. It is
forallcompoundswitheven100%hydrolysisobservedfor important to note that depending on the pH of the cell and
compounds2,4,and5,andbetween88%and96%for1,3, the pK of the aqua adduct, the complex can be present in
a
and 6. The rapid hydrolysis of compound 2 might be aided eitherthelessreactivehydroxoorinitsreactiveaquaform
by the hydrogen bonding ability of the ortho-hydroxo (Table 5, Supporting Information, Figure S2).
substituentwhichcanstabilizetheincomingwatermolecule Compounds3A,[(η6-bip)Os(6-Me-pico)D O]+,4A,[(η6-
2
inthetransitionstate,andinaddition,theelectron-donating bip)Os(4-Cl-pico)D O]+, 5A, [(η6-bip)Os(4-CO H-pico)-
2 2
properties of the ortho-hydroxo substituent can contribute D O]+,and6A,[(η6-bip)Os(4-Me-pico)D O]+,havesimilar
2 2
to the weakening of the Os-Cl bond. pK*values,rangingfrom6.30to6.61(Table6).Thisshows
a
Compound 6 also hydrolyzes rapidly with a half-life of thatsubstituentsonthepicolinatechelatingligandhavelittle
just under 1 h (288 K), indicating an electronic substituent effect on the acidity of the bound water. These values are
effect of the para-methyl group. The electron-donating significantlylower(ca.1.5units)thanthoseforstructurally
propertiesofthemethylgroupgiverisetoincreasedelectron similar RuII arene aqua complexes.2,6 The pK* values of
a
density around osmium which may facilitate chloride loss 3A-6A(<7,Table6)indicatethatatphysiologicalpH(7.4)
andthereforeresultinafasterhydrolysisrate.Thissubstitu- almostallof3A-6Awouldbepresentintheirlessreactive
ent effect is also observed for compound 4 which contains hydroxoforms.Furthermore,thecarboxylicacidsubstituent
theelectron-withdrawing(throughtheinductiveeffect)para- of compound 5 (pK* 2.5), would be present in its depro-
a
chloride group and for compound 5 with the electron- tonatedformatphysiologicalpHgivinganoverallnegative
withdrawing (through the resonance effect) carboxyl group charge on 5 (i.e., [(η6-bip)Os(4-CO -pico)Cl]-).
2
alsointhepara-positiononthepyridinering,withhydrolysis Interaction with Nucleobase Derivatives; 9-Ethyl
half-livesof2.4and4.4h(288K)for4and5,respectively. Guanine (9EtG) and 9-Ethyl Adenine (9EtA). Nuclear
A similar trend is observed for compounds 1 and 3 which DNA is believed to be a major target for transition metal-
containbromide(1)andmethyl(3)substituentsintheortho- based anticancer complexes.27 Osmium arene complexes,
positionofthepyridinering.Compound3,withanelectron- [(η6-arene)Os(XY)Cl]n+, containing a neutral N,N-chelate
donatingmethylgroup,hydrolyzestwiceasfastascompound (e.g., ethylenediamine) bind selectively to G nucleobases,
1, which contains an electron-withdrawing bromide group. andthosecontainingananionicO,O-chelate(e.g.,acetylac-
However, important to note is the slower hydrolysis for etonate) have similar affinities for both G and A nucleo-
compound 3 compared to compound 6 (Table 3), both of bases.2
whichcontainamethylsubstituentbutindifferentpositions Inthisstudy,theinteractionswithnucleobasederivatives,
on the ring (i.e., ortho for 3 and para for 6). This can be 9-ethyl guanine (9EtG) and 9-ethyl adenine (9EtA) with
explainedbythestericbulkoftheortho-methylgrouparound compounds1-6inD Owereinvestigated(Table7,Figure
2
themetalcenter,whichmayforcealessfavorabledissocia- 4). Compounds 4-6 bind significantly (60-100%) to both
tive pathway. A more favorable associative pathway is nucleobases. Compound 6 appears to bind more weakly to
allowedinthetransitionstateofcompound6sinceherethe 9EtA which indicates a slightly weaker binding of 6 to
methyl group does not interfere with the reactive site. The adenine compared to guanine. Compound 5 shows excep-
largenegativeactivationentropyofhydrolysis(-55.6JK-1 tionally high nucleobase affinity with 100% nucleobase
mol-1, Table 4, Figure 3B) for compound 6, suggests that adduct formation for both 9EtG and 9EtA after 24 h.
themechanismofhydrolysisforthiscompounddoesindeed Compound4reactedmoreslowlywithbothnucleobaseswith
involve an associative pathway. equilibrationreachedafter72h,whiletheothercompounds
At high chloride concentrations typical of blood plasma reacted with the nucleobases within 24 h. (Table 7).
(100 mM), complex 6 is 80% present as the intact chloro Compounds 1-3 containing ortho-substituents, reacted
species,alsoreferredtoasthe“prodrug”formsinceinthis significantly less with the model nucleobases (Figure 4),
formitisrelativelyunreactive.Atalowerchlorideconcen- indicating weaker binding to G and A bases for these
trationresemblingthatinthecellcytoplasm(22.7mM),53% complexes. This weaker binding is most likely due to the
of complex 6 is present in its active hydrolyzed form (6A) steric hindrance caused by the ortho-substituents on the
1760 InorganicChemistry,Vol.48,No.4,2009
OrganometallicOsmium(II)AreneAnticancerComplexes
picolinate ring. This is more evident for 3 compared to Conclusions
compound 6, which both contain a methyl substituent
In this study, we have shown how steric and electronic
displayingasimilarelectroniceffectbutwithonly3(ortho-
effects of substituents on the ortho- and para-position of
Me) causing steric hindrance at Os. Compound 3 is
picolinate chelated to [(η6-arene)OsIICl]+ fragments can be
significantly less reactive toward these nucleobases com-
usedtocontrolhydrolysisrates,extentofnucleobase(Gand
pared to compound 6 with about 50% less binding
A) binding, and cancer cell cytotoxicity. Faster hydrolysis
observedtoboth9-ethylguanineand9-ethyladenineafter
is observed with an electron-donating methyl group (com-
24h.ADFcalculationsconfirmthattheminimumenergies
pound6)andslowerhydrolysisfortheelectron-withdrawing
of geometry-optimized structures of the nucleobase ad-
chloro and carboxyl groups (compounds 4 and 5, respec-
ducts of 6 are significantly lower than those found for its
tively, see Table 3) in the para-position.
isomer complex 3; 9EtG-6 was found to be 6.6 kcal/mol
Of the 6 compounds tested, compounds 4 and 6 show
more stable than 9EtG-3, and 9EtA-6 was 5.8 kcal/mol
promisingactivityinthehumanovarianA2780andhuman
more stable than 9EtA-3, indicating that the nucleobase
colon HCT116 cancer cell lines with similar IC values to
adductsofcomplex6arethermodynamicallymorestable 50
thoseofcisplatin(seeTable8)andevenovercomecisplatin
than those of complex 3.
resistance in the A2780cis cell line, indicating that the
In contrast to compounds 3-6, compound 1 formed no
mechanism of action of these osmium arene complexes is
adduct with 9EtG but does, however, form adducts with
different from that of cisplatin.
9EtA. Minimum energy structures of the 9EtA adduct with
Thecytotoxicinactivityofcompounds1-3maybecaused
complex 1, ([(η6-bip)Os(6-Br-pico)(9EtA)], Supporting In-
by the presence of sterically demanding ortho-substituents
formation,FigureS5B),obtainedfromDFTcalculationsdo
onthepyridineringwhichhinderattackonthemetalcenter.
indeed show hydrogen bonding between the NH group of
2 The effect of the steric hindrance is further demonstrated
adenineandthecarboxylateofthepicolinateligand,stabiliz-
by their relatively slow hydrolysis rates and their reduced
ing this adduct. In contrast, the minimum energy structure
and weaker binding to guanine and adenine especially for
ofthe9EtGadduct,([(η6-bip)Os(6-Br-pico)(9EtG)]),shows
complex3incomparisontocomplex6(Table7,Figure4),
a close contact between the bromide substituent and the
which differ only by having the methyl substituent at the
guanine C6O which might explain the lack of reactivity of
ortho-andpara-positionsofthepicolinatering,respectively.
complex1with9EtG(SupportingInformation,FigureS5A).
The cytotoxic inactivity of compound 5 might be caused
Thisargumentcanalsobeusedtoexplainthesameobserved
bythedeprotonationofitspara-substituentcarboxylgroup
nucleobasespecificityofcomplex2.Also,bothcompounds
(pK* 2.5), resulting in an overall negative charge (in its
1 and 2 form two adducts with 9EtA, a behavior which is a
chloroform)inaqueousconditionshinderingcellularuptake.
not observed for any of the other osmium complexes 3-6
Also,evenifcomplex5istakenupintothecellandactivated
(Table 7).
byhydrolysistoitsneutralaquaform,thelackofapositive
Cytotoxicity. Compounds 1-3 and 5 were non-toxic
chargemighthinderitsattackonDNA(whichisnegatively
toward colon HCT116, human lung A549, human ovarian
charged). To investigate the influence of charge, the ethyl
A2780, and cisplatin resistant A2780cis cancer cell lines.
ester,compound7,wassynthesized,anditscytotoxicitywas
Compounds4and6,however,exhibitedpromisingtoxicity,
determined in the A2780 and A2780cis cell lines. Its
with IC values similar to those for cisplatin in the human
50 moderatecytotoxicactivity(Table8)suggeststhattheoverall
ovarian A2780 and colon HCT116 cancer cell lines, and
negative charge in complex 5 does indeed contribute to its
moderatecytotoxicityintheA549cancercelllinewithIC
50 inactivity.
valuesaboutthreetimesthatofcisplatin.Interestingly,both
This work shows that substituents on the picolinate
compounds 4 and 6 overcome cisplatin resistance in the
backbone can have significant effects on the aqueous
A2780cis cell line indicating that the mechanism of action
chemistry of osmium(II) compounds of the type [(η6-
of these osmium arene complexes is different from that of
bip)Os(YZ)(Cl)] allowing a great scope for design for this
cisplatin (Table 8).
class of compounds. The synthetic route for compound 7
Thestericbulkpresentaroundthemetalcentercausedby
illustratesaswellanewwayoffunctionalizingosmiumarene
the ortho-substituents in the picolinate compounds 1-3
compoundswhichcouldbeappliedtoawiderangeofother
accompaniedbytheirreluctancetoformnucleobaseadducts
osmium compounds. This study shows that we are able to
may account for their cytotoxic inactivity. This is further
introduce desirable features into these types of complexes
supported by their reduced 9EtG and 9EtA binding, espe-
to optimize their design as anticancer drugs.
ciallycomparingcomplex3withcomplex6andtheslower
hydrolysis rates observed for compounds 1 and 3. The Acknowledgment. We thank the EPSRC National Crys-
cytotoxic inactivity of compound 5 might arise from poor tallographyService(DrPeterHorton,UniversityofSouthamp-
cellular uptake as a result of the deprotonation of its para- ton) for collecting the diffraction data for complex 6. We
substituent carboxyl group (pK* 2.5, Table 6), and overall thankDr.R.DeethforadviceontheuseofADF.Wethank
a
negativecharge.Thishypothesisissupportedbytheobserved DrM.Khan(WarwickUniversity),DrA.M.Pizarro(War-
activityofcompound7whichhasanestergroupinthepara wickUniversity),andRhonaAird(GeneralHospital,Edin-
position in the human ovarian A2780 and A2780cis cancer burgh)forhelpandadviceoncellculture.Thisresearchwas
cell lines (Table 8). supported by Engineering and Physical Sciences Research
Inorganic Chemistry, Vol. 48, No. 4, 2009 1761
van Rijt et al.
Council(EPSRC)andWarwickUniversity.Theauthorsalso ofCl-iononthehydrolysisof3and6(FigureS2),1HNMRspectra
acknowledgethattheirparticipationintheEUCOSTAction ofthepHtitrationof6Aand5A(FigureS3),plotsofthe1HNMR
D39 enabled them to exchange regularly the most recent chemical shifts of the coordinated arene ring protons versus pH*
ideas in the field of anticancer metallodrugs with several (FigureS4),andoptimizedgeometriesforthenucleobaseadducts
European colleagues. of1,3,and6(FigureS5).Thismaterialisavailablefreeofcharge
viatheInternetathttp://pubs.acs.org.
Supporting Information Available: Time dependence for
hydrolysisof3,4,and5(FigureS1),effectofvariousconcentrations IC8020222
1762 InorganicChemistry,Vol.48,No.4,2009