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Osmium(II)--versus ruthenium(II)--arene carbohydrate-based anticancer compounds: similarities and differences.
PAPER www.rsc.org/dalton | DaltonTransactions
Osmium( )–versus ruthenium( )–arene carbohydrate-based anticancer
II II
compounds: similarities and differences†‡
MuhammadHanif,a AlexeyA.Nazarov,*a,b ChristianG.Hartinger,*a,c WolfgangKandioller,a
MichaelA.Jakupec,a,c VladimirB.Arion,a PaulJ.Dysonb andBernhardK.Kepplera,c
Received15thFebruary2010,Accepted19thMay2010
FirstpublishedasanAdvanceArticleontheweb2ndJuly2010
DOI:10.1039/c003085f
ThesynthesisandinvitroanticanceractivityofOsII–arenecomplexeswithcarbohydrate-derived
phosphiteco-ligandsarereported.Thecompoundswerecharacterizedbystandardmethodsandthe
molecularstructureofdichlorido(h6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropylidene-a-D-
glucofuranoside)osmium(II)wasdeterminedbyX-raydiffractionanalysis.Complexeswithchlorido
leavinggroupsundergohydrolysisbyconsecutiveformationofaquacompounds,followedbycleavage
ofaP–Obondofsugarphosphiteligands,asdemonstratedbyNMRstudies.Theseobservationsare
similartothoseofanalogousRuII–arenecomplexes;howevertherateofhydrolysisisveryslowfor
osmiumcompounds.Thecomplexeswithoxalatoleavinggroupsresisthydrolysis;nohydrolyticspecies
weredetectedby31P{1H}NMRspectroscopyoverseveraldays.WithinthisseriesofOscompounds,in
vitroanticanceractivityishighestforthemostlipophilicchloridocomplexdichlorido(h6-p-
cymene)(3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-a-D-glucofuranoside)osmium(II).
Introduction
The limitations of current metal-based chemotherapeutics have
prompted a search for novel antitumor drugs with improved
effectivenessandfewersideeffects.1–3Thousandsofnovelplatinum
andnon-platinumcompoundshavebeensynthesizedandevalu-
atedfortheirantitumorproperties.4 Amongthetestedtransition
metal compounds, ruthenium compounds have shown consider-
ablepotentialasanticancerdrugs.5-7 Rutheniumcompoundsare
usuallylesstoxicthancisplatinandarethereforebettertoleratedin
vivo.Inanimalmodels,rutheniumcompoundsareeffectiveinthe
treatment of cancer types which cannot be treated by platinum-
based compounds, most probably due to a different mode of
action. Two ruthenium(III)-based drugs, KP1019 and NAMI-
A (Fig. 1), have successfully completed phase I clinical trials.6,8
Bothruthenium(III)compoundsdifferconsiderablyfromcisplatin
in their in vivo behaviour; NAMI-A strongly inhibits metastasis
withouteffectsontheprimarytumour,whereasKP1019effectively
reducescolorectaltumoursinwhichcisplatinisinactive.
Recently,half-sandwichorganometallicRu(II)–arenecomplexes
of the general formula [(h6-arene)Ru(X)(Y)(Z)] have attracted
great attention as putative anticancer drugs, where X is a
monodentatelyboundmoiety,oftenaleavinggroup(e.g.chloride),
andYandZarebidentatechelatingortwomonodentateligands,
resultinginneutralorpositivelychargedcomplexes(thelatterof-
Fig. 1 Ruthenium compounds evaluated in clinical trials (KP1019,
NAMI-A), and Ru–arene complexes and their Os analogues with bio-
aUniversity of Vienna, Institute of Inorganic Chemistry, Waehringer Str.
logicalactivity(M=Ru,Os).
42, A-1090, Vienna, Austria. E-mail: christian.hartinger@univie.ac.at,
alex.nazarov@univie.ac.at;Fax:+43-1-4277-52680;Tel:+43-1-4277-52609
bInstitutdesSciencesetInge´nierieChimiques,EcolePolytechniqueFe´de´rale tenwithCl-,BF-,PF-,etc.ascounterions).Variousapproaches
4 6
deLausanne(EPFL),CH-1015,Lausanne,Switzerland have been investigated in this regard including coordination
cUniversityofVienna,ResearchPlatform“TranslationalCancerTherapy
of ethylenediamine (en),9,10 paullone derivatives,11 1,3,5-triaza-7-
Research”,WaehringerStr.42,A-1090,Vienna,Austria
phosphatricyclo[3.3.1.1]decane (pta),12 pyr(id)ones13–15 or per se
†CCDCreferencenumber766305.ForcrystallographicdatainCIFor
otherelectronicformatseeDOI:10.1039/c003085f bioactivegroups,suchasstaurosporinederivatives,16tothemetal
‡DedicatedtoProf.Tama´sKissontheoccasionofhis60thbirthday. centre. These water-soluble ruthenium–arene complexes offer a
This journal is © The Royal Society of Chemistry 2010 Dalton Trans., 2010,39, 7345–7352 | 7345
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potentialtofine-tunethelipophilicityofthemolecule,whichisan Table1 Crystaldataanddetailsofdatacollectionfor3†
importantparameterfordrugdevelopment.Forexample,Ru(II)
complexesofthetype[(h6-arene)Ru(en)X]+ (X=halide)exhibit Compound 3
highcytotoxicityinvitrocomparableor,insomecases,superior
Chemicalformula C H ClO OsP
19 23.70 2 6.35
to that of cisplatin.17,18 On the other hand, RAPTA complexes M/gmol-1 645.75
of the type [(h6-arene)Ru(pta)Cl] exhibit selective cytotoxicity T/K 100(2)
Crystalsize/mm 0.30¥0.30¥0.20
towardstheTS/AtumourigeniccelllineasopposedtotheHBL-
Crystalcolour,habit Orange,block
100 non-tumourigenic cell line, and notably in vivo some of the Crystalsystem Orthorhombic
compounds reduce significantly the growth of lung metastases Spacegroup P222
1 1 1
in CBA mice bearing MCa mammary carcinoma.12 Other Ru– a/A˚ 8.7180(4)
arene complexes target kinases or other proteins, some of them b/A˚ 14.8768(7)
c/A˚ 17.0012(8)
beingveryinert.11,16,19 Osmiumanaloguesofmanyofthesehalf-
V/A˚3 2204.99(18)
sandwich organometallic complexes have been synthesized and
Z 4
shown to inhibit cancer cell growth in vitro, in some cases D/gcm-3 1.945
c withpotenciescomparabletotheclinicaldrugscarboplatinand m/cm-1 6.133
F(000) 1254
cisplatin.11,13,16,20–23 There are no clear-cut structure–activity rela- Hrangefordatacollection/◦ 2.71to30.09
tionshipsallowingtopredicttheeffectofexchangingruthenium hrange -12/12
forosmiuminanticancercompounds:asobservedbySadleretal. krange -20/20
theOscompoundscanbemoreactivethantheirRuanalogues,21 lrange -23/23
No.refls.usedinrefinement 6470
whereas in other cases no difference was observed,11,16 probably
No.parameters 269
duetoanexclusivelystructuralfunctionofthemetalcentre.16 R 0.075 int
In this paper we describe the influence of the metal cen- R 1 (obs.) 0.0240
wR (alldata) 0.0505
tre (Ru vs. Os) on hydrolysis and cytotoxicity of a series of 2 Flackparameter -0.010(4)
carbohydrate–metalcomplexes.WiththeintentiontotargetRu– S 1.011
arenecompoundsselectivelytothetumourmicro-environment,24
we have linked such metal moieties to glucose-derived ligands
Refine(cid:2)ment was by f(cid:2)ull-matrix least-squ(cid:2)ares (F
o
2) for(cid:2)all reflections,
R = (cid:2)F |-|F (cid:2)/ w|F |,wR =[ (F 2 -F 2)2/ wF 4]1/2,w=
via a phosphorus atom, resulting in RAPTA-C analogues with 1/ 1 [(s2(F
o
2)(cid:2) o +(0.017 c 3P)2+5. o 64P,w 2 ithP={[ o F
o
2+ c 2F
c
2)/3}. o Goodness
increasedinvitroactivity.25Glucoseuptakeisincreasedincancer offit,S= [(F 2-F 2)2]/(n-p)1/2.
o c
cellsduetoupregulationofglycolysisandglucosetransporters.26
Thispropertyisbeingexploitedforthevisualizationoftumoursby
usingtheglucoseanaloguetracer18fluorodeoxyglucoseinpositron the Laboratory for Elemental Analysis, Faculty of Chemistry,
emissiontomography(FdG-PET).26Asfarasweareaware,these University of Vienna, on a Perkin-Elmer 2400 CHN Elemental
arethefirstexamplesofosmiumcompoundswithcarbohydrate- Analyzer.Electrosprayionizationmassspectrawererecordedon
basedligands,andinadditiontotheirsynthesis,theirsolidstate aBrukeresquire .
3000
structures, behaviour in aqueous solution and cytotoxicity in X-Ray diffraction measurements of 3 were performed on a
cancercellsarediscussed. BrukerX8APEXIICCDdiffractometerat100K.Thecrystalwas
positioned at 35 mm from the detector and 1522 frames for 5 s
over1◦weremeasured.ThedatawereprocessedusingtheSAINT
Experimental
softwarepackage.30Crystaldata,datacollectionparameters,and
structurerefinementdetailsaregiveninTable1.
Materials
The structure was solved by direct methods and refined by
All reactions were carried out in dry solvents under an inert full-matrix least-squares techniques. Non-hydrogen atoms were
atmosphere. All chemicals were obtained from commercial refinedwithanisotropicdisplacementparameters.Hatomswere
suppliers and used as received and were of analytical grade. insertedatcalculatedpositionsandrefinedwitharidingmodel.
OsO (99.8%) and NH·2HCl were purchased from Johnson Thefollowingcomputerprogramswereused:structuresolution,
4 2 4
Matthey and Fluka, respectively. Methanol and CHCl were SHELXS-97;31 refinement, SHELXL-97;32 molecular diagrams,
2 2
dried using standard procedures. The dimer bis[dichlorido(h6- ORTEP-3;33computer,PentiumIV;scatteringfactors.34
p-cymene)osmium(II)] [(h6-p-cymene)OsCl(m-Cl)]
2
a,27 3,5,6-
bicyclophosphite-1,2-O-isopropylidene-a-D-glucofuranoside 1 Dichlorido(g6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopro-
and 3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-a-D-gluco- pylidene-a-D-glucofuranoside)osmium(II) (3). A solution of
furanoside 228 and disilver oxalate29 were synthesized using bis[dichlorido(h6-p-cymene)osmium(II)] a (79 mg, 0.1 mmol)
literature procedures. 1H, 13C{1H} and 31P{1H} NMR spectra in dry CHCl (5 mL) was added to a solution of 3,
2 2
were recorded at 25 ◦C on a Bruker FT NMR spectrometer 5,6-bicyclophosphite-1,2-O-isopropylidene-a-D-glucofuranoside
Avance III 500 MHz at 500.10 (1H), 125.75 (13C{1H}) and (50mg,0.2mmol)indryCHCl (15mL).Thereactionmixture
2 2
202.44 MHz (31P{1H}). 2D NMR spectra were collected in wasstirredat40◦Cfor2h.Thesolventwasreducedtoca.5mL
a gradient-enhanced mode. Specific optical rotations were andtheproductwasprecipitatedbytheadditionofpentane(ca.
determinedonaPerkin-Elmer341polarimeterina10cmcellat 20mL).Thesolidwasfiltered,washedwithpentane(3¥2mL)
20◦C.MeltingpointsweremeasuredonaBu¨chiB-540apparatus and dried under vacuum. Crystals suitable for X-ray diffraction
and are uncorrected. Elemental analysis was determined by studiesweregrownfromDOintheNMRtube.
2
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Yield: 126 mg (98%), m.p. 186–187 ◦C (decomp.), Elemental precipitation.Theyellowcrystallineproductwasfiltered,washed
analysis, found% C, 35.02; H, 4.14; calcd. for C H ClOPOs· withdiethylether(2¥5mL)anddriedundervacuum.
19 27 2 6
0.15CHCl,C,35.05;H,4.19.MS(ESI+):m/z:667.0[M+Na]+, Yield: 195 mg (74%), m.p. 190–192 ◦C (decomp.), Elemental
2 2
1309.7[2M+Na]+;[a]20=6(c0.25,CHCl). analysis, found% C, 37.14; H, 3.98; calcd. for C H O POs·
D 2 2 21 27 10
1HNMR(500.10MHz,CDCl ,25◦C):d=6.13(d,J=3.5Hz, 0.75HO, C, 37.41; H, 4.26., MS (ESI+): m/z: 683 [M + Na]+;
3 2
1H,H-1),5.75(d,J =6.0Hz,1H,H-Ar),5.60(d,J =8.2Hz, [a]20=-2(c0.25,CHCl).
D 2 2
2H,H-Ar),5.59(d,J =8.2Hz,2H,H-Ar),5.04(m,1H,H-5), 1HNMR(500.10MHz,CDCl,25◦C):d=6.06(d,J=3.5Hz,
3
4.78(d,J=2.0Hz,1H,H-3),4.67(d,J=3.5Hz,1H,H-2),4.45 1H;H-1),5.97(d,J=4.7Hz,1H;H-Ar),5.94(d,J=5.0Hz,1H;
(dd,J=9.4,11.8Hz,1H,H-6),4.32(m,2H,H-6¢,H-4),2.86(m, H-Ar),5.80(d,J =4.7Hz,2H;H-Ar),5.10(m,1H;H-5),4.71
1H,CH(CH)),2.33(s,3H,CH),1.51(s,3H,C(CH)),1.36 (brs,1H;H-3),4.63(d,J =3.2Hz,1H;H-2),4.52(m,1H;H-6),
3 2 3 3 2
(s,3H,C(CH)),1.28(d,J =6.9Hz,6H,CH(CH )).13C{1H} 4.40(s,1H;H-4),4.35(m,1H;H-6¢),2.76(m,1H;CH(CH)),2.29
3 2 3 2 3 2
NMR(125.75MHz,CDCl ,25◦C):d =112.6(C(CH)),105.7 (s,3H;CH),1.48(s,3H;C(CH)),1.31(s,3H;C(CH)),1.29(d,
3 3 2 3 3 2 3 2
(C-1), 102.5 (C-Ar), 100.6 (C-Ar), 83.6 (J = 7.3 Hz, C-2), 82.0 J=6.6Hz,3H;CH(CH )),1.28(d,J=6.7Hz,3H;CH(CH ))
3 2 3 2
(C-Ar),81.8(C-Ar),81.4(C-Ar),78.6(J=8.2Hz,C-3),76.7(J= ppm; 13C{1H} NMR (125.75 MHz, CDCl , 25 ◦C): d = 165.1
3 5.5Hz,C-4),74.5(J=4.5Hz,C-5),69.6(J=8.2Hz,C-6),30.8 (J =6.0Hz;C=O),112.7(C(CH)),105.7(C-1),102.7(C-Ar),
3 2
(CH(CH)), 26.9 (C(CH)), 26.2 (C(CH)), 22.4 (CH(CH)), 99.1 (C-Ar), 83.6 (J = 6.1 Hz; C-2), 81.5 (C-Ar), 80.9 (C-Ar),
3 2 3 2 3 2 3 2
22.4(CH(CH)),18.4(CH)ppm.31P{1H}NMR(202.44MHz, 80.1(C-Ar),78.5(J=7.8Hz;C-3),76.7(J=10.1Hz;C-4),74.6
3 2 3
CDCl,25◦C):d=87.3ppm. (J =3.8Hz;C-5),69.5(J =7.1Hz;C-6),31.2(CH(CH)),26.9
3 3 2
(C(CH)), 26.2 (C(CH)), 22.7 (CH(CH)), 22.6 (CH(CH)),
Dichlorido(g6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclo- 3 2 3 2 3 2 3 2
18.2 (CH) ppm; 31P{1H} NMR (202.44 MHz, CDCl , 25 ◦C):
hexylidene-a-D-glucofuranoside)osmium(II) (4). A solution of
d=94.7p
3
pm.
3
bis[dichlorido(h6-p-cymene)osmium(II)] a (79 mg, 0.1 mmol) in
dry CHCl (5 mL) was added to a solution of 3,5,6-bicyclo- Oxalato(g6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclohexy-
2 2
phosphite-1,2-O-cyclohexylidene-a-D-glucofuranoside (58 mg, lidene-a-D-glucofuranoside)osmium(II)(6). [(h6-Cymene)OsCl(m-
0.2 mmol) in dry CHCl (15 mL). The mixture was stirred at Cl)] (158mg,0.2mmol)andsilveroxalate(133mg,0.44mmol)
2 2 2
40◦Cfor2h.Thesolventwasthenreducedtoca.5mLandthe were stirred in distilled water (25 mL) for 12 h. The mixture
compoundwasprecipitatedasyellowpowderbytheadditionof was filtered through a bed of Celite to remove the AgCl pre-
pentane(ca.20mL).Thesolidwasfiltered,washedwithpentane cipitate.Thesolventwasremovedundervacuum,theresiduewas
(3¥2mL)anddriedundervacuum. redissolvedinmethanol(25mL)and3,5,6-bicyclophosphite-1,2-
Yield: 135 mg (98%), m.p. 159–161 ◦C (decomp.), Elemental O-cyclohexylidene-a-D-glucofuranose (115 mg, 0.4 mmol) was
analysis, found% C, 37.48; H, 4.42; calcd. for C H ClOPOs· added. The reaction mixture was stirred for 2 h, the volume of
22 31 2 6
0.15CHCl, C, 37.22; H, 4.44. MS (ESI+): m/z: 649.0 [M - 2 the solvent was reduced to ca. 5 mL and diethyl ether (25 mL)
2 2
HO+H]+,705.0[M+Na]+;[a]20=12(c0.25,CHCl). was added. The slurry was cooled to 4 ◦C for 12 h to complete
2 D 2 2
1HNMR(500.10MHz,CDCl ,25◦C):d=6.13(d,J=3.8Hz, theprecipitation.Theyellowcrystallineproductwasfilteredoff,
3
1H,H-1),5.75(d,J =6.0Hz,1H,H-Ar),5.60(d,J =8.2Hz, washedwithdiethylether(2¥5mL)anddriedundervacuum.
1H,H-Ar),5.58(d,J =8.2Hz,2H,H-Ar),5.03(m,1H,H-5), Yield: 213 mg, (76%), m.p. 183–184 ◦C (decomp.), El-
4.79(d,J=2.5Hz,1H,H-3),4.67(d,J=3.5Hz,1H,H-2),4.45 emental analysis, found% C, 40.12; H, 4.27; calcd. for
(dd,J =7.0,11.8Hz,1H,H-6),4.32(m,2H,H-6¢,H-4),2.86 C H O POs·0.75HO, C, 40.36; H, 4.59; MS (ESI+): m/z: 725
24 31 10 2
(m,1H,CH(CH)),2.33(s,3H,CH),1.68(m,4H,CH ),1.56 [M + Na]+; [a]20 = 4 (c 0.25, CHCl). 1H NMR (500.10 MHz,
3 2 3 6 10 D 2 2
(m, 6 H, CH ), 1.28 (d, J = 6.9 Hz, 6H, CH(CH )). 13C{1H} CDCl,25◦C):d =6.07(d,J =3.2Hz,1H,H-1),5.94(brs,1
6 10 3 2 3
NMR(125.75MHz,CDCl ,25◦C):d =113.3(C(CH)),105.3 H,H-Ar),5.78(brs,1H,H-Ar),5.58(brs,2H,H-Ar),5.08(m,
3 3 2
(C-1), 102.5 (C-Ar), 100.7 (C-Ar), 83.2 (J = 6.4 Hz, C-2), 82.0 1H,H-5),4.71(brs,1H,H-3),4.62(d,J =3.2Hz,1H,H-2),
(C-Ar), 81.6 (C-Ar), 81.4 (C-Ar), 78.8 (J = 8.2 Hz, C-3), 76.6 4.49(m,1H,H-6),4.38(m,1H,H-4),4.32(m,1H,H-6¢),2.76
(J=5.5Hz,C-4),74.5(J=4.5Hz,C-5),69.4(J=9.1Hz,C-6), (m,1H,CH(CH)),2.29(s,3H,CH),1.65(m,4H,CH ),1.53
3 2 3 6 10
36.5 (CH ), 35.7 (CH ), 30.8 (CH(CH)), 24.8 (CH ), 23.8 (m, 6 H, CH ), 1.29 (d, J = 6.4 Hz, 3 H, CH(CH )), 1.28 (d,
6 10 6 10 3 2 6 10 6 10 3 2
(CH ), 23.5 (CH ), 22.4 (CH(CH)), 22.4 (CH(CH)), 18.4 J =6.5Hz,3H,CH(CH ))ppm;13C{1H}NMR(125.75MHz,
6 10 6 10 3 2 3 2 3 2
(CH) ppm. 31P{1H} NMR (202.44 MHz, CDCl , 25 ◦C): d = CDCl,25◦C):d =165.0(J =6.0Hz;C=O),113.4(C(CH)),
3 3 3 3 2
87.3ppm. 105.3 (C-1), 102.7 (C-Ar), 99.1 (C-Ar), 83.1 (J = 6.1 Hz; C-2),
81.5(C-Ar),80.9(C-Ar),80.1(C-Ar),78.7(J=7.8Hz;C-3),76.7
Oxalato(g6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropy-
(J=10.1Hz;C-4),74.7(J=3.8Hz;C-5),69.6(J=7.1Hz;C-6),
lidene-a-D-glucofuranoside)osmium(II)(5). [(h6-Cymene)OsCl(m-
36.5 (CH ), 35.6 (CH ), 31.2 (CH(CH)), 24.7 (CH ), 23.8
Cl)] (158mg,0.2mmol)andsilveroxalate(133mg,0.44mmol) 6 10 6 10 3 2 6 10
2 (CH ),23.5(CH ),22.7(CH(CH)-Ar),22.6(CH(CH)-Ar),
werestirredindistilledwater(25mL)for12h.Themixturewas 18. 6 2( 1 C 0 H-Ar)p 6 pm 1 ; 0 31P{1H}NMR( 3 2 2 02.44MHz,CDCl, 3 25 2 ◦C):
filtered through a bed of Celite to remove the AgCl precipitate. 3 3
d=94.6ppm.
The solvent was removed under vacuum, and the residue was
redissolved in methanol (25 mL) and 3,5,6-bicyclophosphite-
Hydrolysisandreactivitywith5¢-GMP
1,2-O-isopropylidene-a-D-glucofuranose(100mg,0.4mmol)was
added.Thereactionmixturewasstirredfor2h,thevolumeofthe For hydrolysis studies, the compounds were dissolved in DO
2
solvent was reduced to ca. 5 mL and diethyl ether (25 mL) was and the samples were analyzed by 31P{1H} NMR spectroscopy
added. The slurry was cooled to 4 ◦C for 12 h to complete the onceadayfor5days.The31P{1H}NMRspectraforthekinetic
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experimentswererecordedonaBrukerAvance400instrumentat exploitthealteredmetabolismassociatedwithcancercells,suchas
161.98MHz.ForGMPbindingexperiments,thecomplexeswere increasedglucoseuptakeascomparedtohealthytissues.Inprevi-
mixedatmolarratiosof1:2(complex:GMP)inDOandkeptat ouswork,welinkedcarbohydrate-derivedligandstoorganometal-
2
roomtemperaturefor2days. lic RuII–arene moieties to afford RAPTA-C analogues
such as dichlorido(h6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-
Cytotoxicityincancercelllines cyclohexylidene-a-D-glucofuranoside)ruthenium(II) 4Ru, which is
more cytotoxic than RAPTA-C, probably due to its higher
Celllinesandcultureconditions. CH1cellsoriginatefroman
lipophilicity and, hence, increased uptake. The complexes were
ascitessampleofapatientwithapapillarycystadenocarcinoma
showntoundergoaquationofahalidoligandinaqueoussolution,
of the ovary and were a generous gift from Lloyd R. Kel-
followedbyhydrolysisofaP–Obondofthephosphiteligand,and
land, CRC Centre for Cancer Therapeutics, Institute of Cancer
finally formation of dinuclear species.25 In order to investigate
Research, Sutton, UK. SW480 (adenocarcinoma of the colon,
the influence of the metal centre on hydrolysis and cytotoxicity,
human), and A549 (non-small cell lung cancer, human) cells
we prepared similar compounds based on osmium, the heavier
were kindly provided by Brigitte Marian (Institute of Cancer
homologueofruthenium.
Research, Department of Medicine I, Medical University of
TheorganometallicOsII-chloridocomplexes3and4weresyn-
Vienna, Austria). All cell culture reagents were obtained from
thesizedinverygoodyieldbyreactingthedimerbis[dichlorido(h6-
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2
Cl
2
at 40 ◦C with 3,5,6-
bicyclophosphite-1,2-O-isopropylidene-a-D-glucofuranoside 1
sentialMedium(MEM)supplementedwith10%heat-inactivated
and 3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-a-D-gluco-
foetalcalfserum,1mMsodiumpyruvateand2mML-glutamine.
furanoside 2, respectively (Scheme 1). In contrast, the
Cultures were maintained at 37 ◦C in a humidified atmosphere
oxalato analogues 5 and 6 were prepared via a two step
containing95%airand5%CO.
2 synthesis, following a literature procedure.29 In the first step,
MTT assay conditions. Cytotoxicity was determined by the [oxalato(h6-p-cymene)(H 2 O)osmium(II)] was formed by reacting
colorimetricMTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- bis[dichlorido(h6-p-cymene)osmium(II)] with disilver oxalate
tetrazoliumbromide,purchasedfromFluka)microcultureassay. (AgCO). In the second step the active aqua species was
2 2 4
For this purpose, cells were harvested from culture flasks by stirredfor2hinCHOHwiththerespectivecarbohydrate-based 3
trypsinizationandseededin100mLaliquotsMEMsupplemented phosphorus ligand and the compounds ultimately precipitated
with 10% heat-inactivated foetal calf serum, 1 mM sodium fromthereactionmixture.Thereasonforchoosingthisreaction
pyruvate, 4 mM L-glutamine and 1% non-essential amino acids sequencewastoavoidsidereactionsinvolvingthesilverionsand
(100¥)into96-wellmicrocultureplates(Iwaki).Celldensitiesof thephosphorusligand.
1.5¥103 cells/well(CH1),2.5¥103 cells/well(SW480)and4¥
103 cells/well(A549)werechoseninordertoensureexponential
growth of untreated controls throughout the experiment. Cells
were allowed to settle and resume exponential growth for 24 h.
The test compounds were dissolved and serially diluted in the
samemediumandaddedin100mLaliquotstothemicrocultures
(ifnecessaryduetolimitedsolubility,themaximumconcentration
testedwasaddedin200mLaliquotsafterremovalofthemedium),
and cells were exposed to the test compounds for 96 h. At
the end of the exposure period, all media were replaced by
100mL/wellRPMI1640culturemedium(supplementedwith10%
heat-inactivatedfoetalcalfserum)plus20mL/wellMTTsolution
inphosphate-bufferedsaline(5mgml-1).Afterincubationfor4h,
thesupernatantswereremoved,andtheformazancrystalsformed
by vital cells were dissolved in 150 mL DMSO per well. Optical
densitiesat550nmweremeasuredwithamicroplatereader(Tecan
SpectraClassic),usingareferencewavelengthof690nmtocorrect
forunspecificabsorption.Thequantityofvitalcellswasexpressed
intermsofT/Cvaluesbycomparisontountreatedcontrolmicro-
cultures,and50%inhibitoryconcentrations(IC 50 )werecalculated Scheme 1 Synthesis of the dichlorido– (3, 4) and oxalato–OsII (5, 6)
from concentration-effect curves by interpolation. Evaluation is compoundsofP-derivedsugarligands(1,2).
basedonmeansfromatleastthreeindependentexperiments,each
comprisingatleastthreereplicatesperconcentrationlevel. All the complexes were fully characterized by 1D and 2D
NMR spectroscopy, ESI-MS, elemental analysis, and the solid
Resultsanddiscussion state structure of complex 3 was determined by single crystal
X-raydiffractionanalysis.UponcoordinationoftheP-containing
In recent years, the tumour microenvironment has become one ligandstotheOscentre,achangeinchemicalshiftofthe31P{1H}
of the focuses of targeted cancer chemotherapy. Carbohydrate NMRsignalswasobservedfromapproximately117ppmtoabout
compoundshavebeenextensivelystudiedastheycouldpotentially 87 ppm in the case of the chlorido complexes and to 94 ppm
7348| Dalton Trans., 2010,39, 7345–7352 This journal is © The Royal Society of Chemistry 2010
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in the case of the oxalato complexes. For the analogous Ru to the RAPTA-C analogous Os compound, the Os–centroid
arene
compounds low-field shifts to about 130 ppm were observed.25 distanceisslightlylonger(1.698A˚),whereastheOs–ClandOs–P
Inthe13C{1H}NMRspectraof5and6,themostindicativesignal bondsareshorter[2.4344(15),2.4194(15);2.3324(16)A˚].37
forcomplexformationisfoundat165.0ppm,whichcorresponds Thehydrolysisof3wasstudiedby31P{1H}NMRspectroscopy.
tothecarboxylatemoietiesofthecoordinatedoxalatoligand.1H Initially only a single peak is observed at 90.0 ppm which was
NMR spectra of oxalato complexes in CDCl gave two sets of assigned to unmodified 3 (Fig. 3). After 24 h, two additional
3
signals(twodoublets)fortheAr-CH(CH) protons,whichisin peaksofequalrelativeintensityat95.8and95.3ppmappeardueto
3 2
contrasttoNMRinDOwherethemethylgroupsareobserved formationofdiastereomersbyexchangeofasinglechloridoligand
2
as a single doublet, since the inversion of the metal centre only withanaquamolecule(Scheme2).Theseobservationsaresimilar
occursinproticsolvents.35 Asimilarobservationwaspreviously tothosemadeforrelatedruthenium(II)compounds,12,25,38asisthe
reportedforrelatedcomplexes.36 nextstepinthehydrolysismechanismwhichinvolvescleavageof
X-Ray diffraction analysis of 3 demonstrated the presence aP–Obond(afterapproximately4d;peakatd(31P)=62.1ppm,
of piano-stool geometry (Fig. 2), as usually observed for such Fig.3).IncontrasttotheRucompounds,therateofhydrolysisis
metal–arene organometallics.11,14,36 Complex 3 crystallizes in the veryslowfortheOscompounds,asalsoobservedinothercases.39
orthorhombic space group P222. The osmium–centroid Within24honly5%ofthecomplexundergohydrolysisinthecase
1 1 1 arene
distanceis1.713A˚ in3andtheOs–Cl1,Os–Cl2,andOs–Pbond ofOscomplex,whereasfortheRuanaloguemorethan50%ofthe
lengths are 2.4203(8), 2.4106(9) and 2.2411(9), respectively. The initialcomplexwasdegraded.Fortheruthenium(II)compounds
Os–ClbondsareslightlylongerthantheRu–Clbondsobserved theformationofmono-aquaspeciesandthehydrolysisoftheP–
in 3Ru, whereas the Os–P bond length is similar.25 As compared O bond of the phosphite was observed already after 12 h and
a dimeric species had formed within 24 h. The diaqua complex
was not observed in the NMR spectrum, probably because it
is converted immediately into the dimeric species. In contrast
to the Ru complexes, no dimeric species were detected for the
Os compound after 72 h. Since the dimer is believed to be not
cytotoxic,14,36 avoiding the formation of a dimeric species could
proveadvantageous(seebelow).Forcedhydrolysisof3byaddition
oftwoequivalentsofAgNO resultedinareleaseofbothchlorido
3
ligandsandformationofaquaspecieswithhydrolyzedP–Obonds
(d(31P)=99.1and97.9for3Ruand62.2ppmfor3).
Introduction of bidentate ligands has been demonstrated to
be an option to overcome the instability problem of titanocene
anticancer compounds and has also proven useful for platinum
drugs by leading to higher stability, reduced side-effects and
Fig.2 Molecularstructureof3.Selectedbondlengths(A˚)andangles extendingtherangeoftreatabletumors.7,40 Inordertostudythe
(◦)are:Os–Cl12.4203(8),Os–Cl22.4106(9),Os–P2.2411(9);P–Os–Cl2 influenceofchelatingoxalatoligandsonthehydrolysisbehaviour,
88.51(3),P–Os–Cl188.74(3),Cl2–Os–Cl186.29(3). 5and6wereprepared(Scheme1)andsimilarhydrolysisstudies
Fig.3 31P{1H}NMRspectraforthehydrolysisof3Ruand3inDOupto72h.Peakassignment:a,monoaquacomplex;b,productsobtainedbycleavage
2
oftheP–Obond;c,dimericspecies.
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Scheme2 Reactionschemeforthehydrolysisof3(charges/counterionswereomittedforclarity).
Table2 Invitroanticanceractivityinhumanovariancancer(CH1),colon Evaluationoftheinvitroanticanceractivity
cancer(SW480)andnon-smallcelllungcancer(A549)cells(exposuretime
96 h unless stated otherwise). Values are means ± standard deviations,
The antiproliferative potential of3–6 was determined in human
obtainedfromatleastthreeindependentexperiments
SW480 colon adenocarcinoma, CH1 ovarian cancer and A549
IC values/mM non-smallcelllungcancercellsbymeansoftheMTTassay,and
50
theobtainedresultsarecomparedtothoseoftheanalogousRu(II)
Compound CH1 SW480 A549
compounds(Table2).Structure–activityrelationshipscanbestbe
3 113±24 n.d.a n.d.a assessed in CH1 cells, the most sensitive of these cell lines, and
4 50±6 215±7 >640 concentration–effect curves of 3, 4, 3Ru and 4Ru are depicted in
5 436±72 >640 >640 Fig.4.
6 764±215 >640 >640
Among the series of Os derivatives, 4 is the most cytotoxic
3Ru 60±14b 361±122b 498±17bc
4Ru 29±4b 150±19b 223±14ac in CH1 cells, followed by the chlorido species 3 and the oxalate
complexes 5 and 6. As in the case of the Ru compounds
aNotdetermined.bFromref.25.cExposuretime72h.
with chlorido ligands, the Os analogue bearing the lipophilic
cyclohexylidene protection group, i.e., 4, is more cytotoxic than
asfor3wereconducted.Theexchangeofthechloridoligandsby the isopropylidene derivative. The Ru–chlorido compounds are
oxalato stabilized the compounds and there were no hydrolysis approximatelytwiceaspotentastheirOscounterparts3and4.
productsidentifiedduringaweekofincubation(aftertwoweeks TheRucomplexesexhibitedactivityincisplatin-resistantA2780
a very small peak at 59.1 ppm was assigned to a P–O bond cells,whichsuggestsamodeofactiondifferentfromthatofDNA-
hydrolysisproduct).AsimilarapproachwasfollowedforRAPTA- targetedagents,andfurthermoreselectivityfornon-tumourigenic
Candmostnotablysuchmodificationdidnotaltertheanticancer cellswasobserved.25 Anon-DNA-relatedmodeofactionisalso
activity of the compounds.29 In contrast, 5 and 6 are much less supportedbythefactthattheRuanaloguesforminactivedimers
activeininvitroanticancerassaysthantheirRucounterparts(see whicharenotdetectablefortheOscompounds.However,theOs
below;Table2). complexes still lack reactivity to 5¢-GMP and the cytotoxicity
Inanattempttostudythereactivityofthecompoundsto5¢- is low. In general, the cytotoxicities of the tested Os and Ru
GMP,31P{1H}NMRstudieswereconductedonmixtureswith5¢- organometallicscoverabroadrangeofIC valuesof30–760mM
50
GMPin1:2ratioinDO.However,noneofthecomplexesreacted inthemostsensitivecelllineCH1.
2
with 5¢-GMP within 48 h, whereas the Ru organometallics are Such a low anticancer potency in vitro has often been ob-
relativelyreactiveandformadductsviatheN7atomsofguanine. served for osmium and ruthenium drug candidates, including
Hydrolysis prior to reaction with biomolecular targets can be theclinicallytestedNAMI-AandcertainorganometallicRAPTA
considered an essential step for Ru and Pt compounds in their compounds,especiallywhencomparedtoplatinum-baseddrugs.
modesofaction.1,5However,forthestudiedderivatives,although Nevertheless, the Ru compounds exhibit excellent in vivo ac-
hydrolysisisobserved,nobindingofOs(II)toN-donorligandsoc- tivities, in particular against tumour metastases.12,41–43 Ru and
curs.ThispropertyhasbeennotedforsimilarOs(II)complexes,18,21 Pt complexes bearing carbohydrate ligands were shown to be
butmightalsoberelatedtotheinsufficientdetectionlimitofNMR generallylesscytotoxicthanstructuralanalogues,24buttheyoften
spectroscopy,sinceonlyaminorfractionoftheOscompoundwas exhibitconsiderableactivityinvivo,whichmakesthempromising
foundtohydrolyzewithintheexperimentaltime. compoundsforfurtherdevelopment.12,44
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Fig.4 Concentration–effectcurvesofOsandRucarbohydratecomplexesinCH1cells.
Conclusions References
RAPTA-type complexes are among the best studied and most 1M.Galanski,M.A.JakupecandB.K.Keppler,Curr.Med.Chem.,
2005,12,2075–2094.
promising Ru–arene anticancer agents. In an attempt to confer
2P.Heffeter,U.Jungwirth,M.Jakupec,C.Hartinger,M.Galanski,L.
tothebasicscaffoldtumour-targetingproperties,sugarmoieties Elbling,M.Micksche,B.KepplerandW.Berger,DrugResist.Updates,
hadbeenlinkedtoRucomplexesofthiskind,whichyieldedcom- 2008,11,1–16.
poundswithselectivityfortumourigeniccelllines.Inthispaper,we 3P. J. Sadler, Dalton Transactions themed issue on Metal Anticancer
Compounds,DaltonTrans.,2009,10629–10936.
reportonthesynthesisofOsanalogueswithcarbohydrate-derived
4Z.GuoandP.J.Sadler,Angew.Chem.,Int.Ed.,1999,38,1512–1531.
phosphorus-containingligands.Thesecompoundsexhibitslightly 5C. G. Hartinger, S. Zorbas-Seifried, M. A. Jakupec, B. Kynast, H.
higherIC valuesthantheirRucounterparts.Incontrasttothe ZorbasandB.K.Keppler,J.Inorg.Biochem.,2006,100,891–904.
50 6C. G. Hartinger, M. A. Jakupec, S. Zorbas-Seifried,M. Groessl, A.
Rucompounds,studiesonthereactivityto5¢-GMPshowedthat
Egger,W.Berger,H.Zorbas,P.J.DysonandB.K.Keppler,Chem.
theOscomplexesdonotreactwiththisDNAmodelwithin48h, Biodiversity,2008,5,2140–2155.
whichmayaccountfortheirlowercytotoxicity. 7C.G.HartingerandP.J.Dyson,Chem.Soc.Rev.,2009,38,391–401.
In addition, the chlorido ligands were replaced by oxalate 8J.M.Rademaker-Lakhai,D.VanDenBongard,D.Pluim,J.H.Beijnen
andJ.H.M.Schellens,Clin.CancerRes.,2004,10,3717–3727.
groups, in order to increase the stability of the compounds. In
9R.E.Morris,R.E.Aird,P.d.S.Murdoch,H.Chen,J.Cummings,
aquation studies, both the Os–chlorido and –oxalato complexes N.D.Hughes,S.Parsons,A.Parkin,G.Boyd,D.I.JodrellandP.J.
were significantly more stable than the Ru counterparts, as Sadler,J.Med.Chem.,2001,44,3616–3621.
demonstratedby31P{1H}NMRspectroscopy,thoughstructurally 10R.E.Aird,J.Cummings,A.A.Ritchie,M.Muir,R.E.Morris,H.
Chen,P.J.SadlerandD.I.Jodrell,Br.J.Cancer,2002,86,1652–1657.
similar species are formed, including a species in which an
11W. F. Schmid, R. O. John, V. B. Arion, M. A. Jakupec and B. K.
intramolecular P–O bond break occurs upon replacement of a Keppler,Organometallics,2007,26,6643–6652.
chlorido ligand by an aqua moiety. These oxalato compounds 12C.Scolaro,A.Bergamo,L.Brescacin,R.Delfino,M.Cocchietto,G.
Laurenczy,T.J.Geldbach,G.SavaandP.J.Dyson,J.Med.Chem.,
inhibitcancercellgrowthwithamuchlowerpotencyinvitrothan
2005,48,4161–4171.
thechloridoanalogues. 13M.G.Mendoza-Ferri,C.G.Hartinger,A.A.Nazarov,R.E.Eichinger,
M.A.Jakupec,K.SeverinandB.K.Keppler,Organometallics,2009,
28,6260–6265.
14W.Kandioller,C.G.Hartinger,A.A.Nazarov,C.Bartel,M.Skocic,
Acknowledgements
M.A.Jakupec,V.B.ArionandB.K.Keppler,Chem.–Eur.J.,2009,
15,12283–12291.
WethanktheHigherEducationCommissionofPakistan,theAus- 15M.G.Mendoza-Ferri,C.G.Hartinger,M.A.Mendoza,M.Groessl,
trian Exchange Service (O¨AD), the Hochschuljubila¨umsstiftung A. E. Egger, R. E. Eichinger, J. B. Mangrum, N. P. Farrell, M.
Maruszak,P.J.Bednarski,F.Klein,M.A.Jakupec,A.A.Nazarov,K.
Vienna, the Theodor-Ko¨rner-Fonds, the FFG – Austrian Re-
SeverinandB.K.Keppler,J.Med.Chem.,2009,52,916–925.
search Promotion Agency (811591), the Austrian Council for 16J. Maksomiska, D. S. Williams, G. E. Atilla-Gokcumen, K. S. M.
Research and Technology Development (IS526001), COST D39 Smalley, P. J. Carroll, R. D. Webster, P. Filippakopoulos, S. Knapp,
andCM0902andtheAustrianScienceFundforfinancialsupport. M.HerlynandE.Meggers,Chem.–Eur.J.,2008,14,4816–4822.
17Y. K. Yan, M. Melchart, A. Habtemariam and P. J. Sadler, Chem.
This research was supported by a Marie Curie Intra European
Commun.,2005,4764–4776.
Fellowship within the 7th European Community Framework 18A.F.A.Peacock,A.Habtemariam,R.Fernandez,V.Walland,F.P.A.
Programmeproject220890-SuRuCo(A.A.N.).Wegratefullyac- Fabbiani,S.Parsons,R.E.Aird,D.I.JodrellandP.J.Sadler,J.Am.
Chem.Soc.,2006,128,1739–1748.
knowledge Alexander Roller for collecting the X-ray diffraction
19C. A. Vock, W. H. Ang, C. Scolaro, A. D. Phillips, L. Lagopoulos,
data,MichaelaHejlforperformingtheinvitroanticancerassays L.Juillerat-Jeanneret,G.Sava,R.ScopellitiandP.J.Dyson,J.Med.
andProf.MarkusGalanskiforrecordingthe2DNMRspectra. Chem.,2007,50,2166–2175.
This journal is © The Royal Society of Chemistry 2010 Dalton Trans., 2010,39, 7345–7352 | 7351
2102
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F580300C/9301.01:iod
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no
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20A.Dorcier,W.H.Ang,S.Bolano,L.Gonsalvi,L.Juillerat-Jeannerat, 31G.M.Sheldrick,UniversityGo¨ttingen(Germany),Editonedn,1997.
G.Laurenczy,M.Peruzzini,A.D.Phillips,F.ZanobiniandP.J.Dyson, 32G.M.Sheldrick,UniversityGo¨ttingen(Germany),Editonedn,1997.
Organometallics,2006,25,4090–4096. 33L.J.Farrugia,J.Appl.Crystallogr.,1997,30,565.
21A.F.A.Peacock,S.ParsonsandP.J.Sadler,J.Am.Chem.Soc.,2007, 34InternationalTablesforX-rayCrystallography,KluwerAcademicPress,
129,3348–3357. Dordrecht,TheNetherlands,1992.
22S. H. van Rijt, A. J. Hebden, T. Amaresekera, R. J. Deeth, G. J. 35R.Lang,K.Polborn,T.SeverinandK.Severin,Inorg.Chim.Acta,
Clarkson,S.Parsons,P.C.McGowanandP.J.Sadler,J.Med.Chem., 1999,294,62–67.
2009,52,7753–7764. 36A. F. A. Peacock, M. Melchart, R. J. Deeth, A. Habtemariam, S.
23S.H.vanRijt,A.F.A.Peacock,R.D.L.Johnstone,S.Parsonsand ParsonsandP.J.Sadler,Chem.–Eur.J.,2007,13,2601–2613.
P.J.Sadler,Inorg.Chem.,2009,48,1753–1762. 37A.Dorcier,P.J.Dyson,C.Gossens,U.Rothlisberger,R.Scopellitiand
24C.G.Hartinger,A.A.Nazarov,S.M.Ashraf,P.J.DysonandB.K. I.Tavernelli,Organometallics,2005,24,2114–2123.
Keppler,Curr.Med.Chem.,2008,15,2574–2591. 38C.Scolaro,C.G.Hartinger,C.S.Allardyce,B.K.KepplerandP.J.
25I.Berger,M.Hanif,A.A.Nazarov,C.G.Hartinger,R.O.John,M.L. Dyson,J.Inorg.Biochem.,2008,102,1743–1748.
Kuznetsov,M.Groessl,F.Schmitt,O.Zava,F.Biba,V.B.Arion,M. 39J.Reedijk,PlatinumMet.Rev.,2008,52,2–11.
Galanski,M.A.Jakupec,L.Juillerat-Jeanneret,P.J.DysonandB.K. 40M.A.Jakupec,M.GalanskiandB.K.Keppler,Rev.Physiol.,Biochem.,
Keppler,Chem.–Eur.J.,2008,14,9046–9057. Pharmacol.,2003,146,1–53.
26R.A.GatenbyandR.J.Gillies,Nat.Rev.Cancer,2004,4,891–899. 41E. Alessio, G. Mestroni, A. Bergamo and G. Sava, Curr. Top. Med.
27S.StahlandH.Werner,Organometallics,1990,9,1876–1881. Chem.,2004,4,1525–1535.
28N.K.Kochetkov,E.E.Nifant’ev,M.P.Koroteev,Z.K.Zhaneand 42A.Bergamo,A.Masi,P.J.DysonandG.Sava,Int.J.Oncol.,2008,33,
A.A.Borisenko,Carbohydr.Res.,1976,47,221–231. 1281–1289.
29W.H.Ang,E.Daldini,C.Scolaro,R.Scopelliti,L.Juillerat-Jeannerat 43S.Chatterjee,S.Kundu,A.Bhattacharyya,C.G.HartingerandP.J.
andP.J.Dyson,Inorg.Chem.,2006,45,9006–9013. Dyson,JBIC,J.Biol.Inorg.Chem.,2008,13,1149–1155.
30M.R.PressprichandJ.Chambers,BrukerAnalyticalX-raysystems, 44I.Berger,A.A.Nazarov,C.G.Hartinger,M.Groessl,S.-M.Valiahdi,
Madison,Editonedn,2004. M.A.JakupecandB.K.Keppler,ChemMedChem,2007,2,505–514.
7352| Dalton Trans., 2010,39, 7345–7352 This journal is © The Royal Society of Chemistry 2010
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