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Synthesis, structure, spectroscopic properties, and antiproliferative activity in vitro of novel osmium(III) complexes with azole heterocycles.
Inorg. Chem. 2008, 47, 7338-7347
Synthesis, Structure, Spectroscopic Properties, and Antiproliferative
Activity In Vitro of Novel Osmium(III) Complexes with Azole
Heterocycles§
Iryna N. Stepanenko, Artem A. Krokhin, Roland O. John, Alexander Roller, Vladimir B. Arion,*
Michael A. Jakupec, and Bernhard K. Keppler*
UniVersity of Vienna, Institute of Inorganic Chemistry, Wa¨hringerstr. 42, A-1090 Vienna, Austria
ReceivedApril18,2008
Reactions of (Hazole)[OsCl], where Hazole ) pyrazole, Hpz, (1), indazole, Hind, (2), imidazole, Him, (3) and
2 2 6
benzimidazole,Hbzim,(4)withthecorrespondingazoleheterocyclein1:4molarratioinboilingisoamylalcoholor
hexanol-1 afforded novel water-soluble osmium(III) complexes of the typetrans-[OsCl(Hazole)]Cl, where Hazole
2 4
) Hpz (5a), Hind (6a), Him (7a), and Hbzim (9a) in 50-70% (5a, 7a, 9a) and 5% (6a) yields. The synthesis of
7a was accompanied by a concurrent reaction which led to minor formation (<4%) of cis-[OsCl(Him)]Cl (8). The
2 4
complexeswerecharacterizedbyelementalanalysis,IRspectroscopy,UV-visspectroscopy,ESImassspectrometry,
cyclic voltammetry, and X-ray crystallography. 5a, 7a, and 9a were found to possess remarkable antiproliferative
activityinvitroagainstA549(non-smallcelllungcarcinoma),CH1(ovariancarcinoma),andSW480(coloncarcinoma)
cells, which was compared with that of related ruthenium compounds trans-[RuCl(Hazole)]Cl, where Hazole )
2 4
Hpz (5b), Hind (6b), Him (7b), and Hbzim (9b).
Introduction calculatedaccordingtoLever’sparametrizationmethodand
measured by cyclic voltammetry, showed that a higher
Thecompounds(Him)[trans-RuIIICl(Him)(DMSO)](NAMI-
2 4 indazole-to-chloride ratio results in a higher reduction
A,Him)imidazole)and(Hind)[trans-RuIIICl(Hind)](KP1019,
2 4 2 potential.Theantiproliferativeactivityofthesecompounds
Hind ) indazole) were selected for clinical development as
incoloncancercells(SW480)andovariancancercells(CH1)
anticanceragentsfromalargenumberofrutheniumcomplexes.1–10
varied broadly, but largely correlated with their RuIII/RuII
WhereasKP1019isactiveagainstmalignanttumors(inparticular,
redox potentials in the following order: [RuIIICl ]3- <
colon carcinoma) and their metastases, NAMI-A was shown to 6
[RuIIICl (Hind) ]- < [RuIIICl (Hind)]2- < [RuIIICl (Hind) ]
belesscytotoxicincellculturesbutabletoinhibittheformation 4 2 5 3 3
< [RuIIICl (Hind) ]+ ≈ [RuIICl (Hind) ].17 A similar rela-
ofmetastases. 2 4 2 4
tionship between RuIII/RuII redox potentials and cytotoxic
Itisgenerallybelievedthattheactivityofruthenium(III)
potencyintheSW480celllinewasobservedfor(H azole)-
complexesdependsontheirreductioninvivotomorelabile 2
[trans-RuIIICl (Hazole) ] complexes, where Hazole ) imi-
ruthenium(II)complexes.Suchareductionhasbeenreported 4 2
to occur under the hypoxic conditions in solid tumors.11–14 dazole, 1,2,4-triazole and indazole.18,19
Inaddition,theinterestinosmiumcomplexesaspotential
Theresultingruthenium(II)speciesshowahigherpropensity
antitumoragentsisgrowing,16,20–25andarecentcomparative
for ligand exchange reactions and might therefore interact
studyonruthenium(III)NAMI-A-typecomplexesandtheir
withtargetmoleculesmorerapidly.Theircytotoxicityoften
correlates with the DNA binding ability.5,11,15,16 osmium(III) analogues revealed that the latter have reason-
Thecorrelationbetweencytotoxicityandelectrochemical able antiproliferative activity in vitro in two human cancer
properties for [RuIIICl (6-n) (Hind) n ](3-n)- (n ) 0-4) com- celllines,HT-29(coloncarcinoma)andSK-BR-3(mammary
poundswasreportedpreviously.17Reductionpotentials,both carcinoma),withIC
50
valuesmostlyinthe10-5Mrange.26
It is therefore worthwhile (i) to develop synthetic routes to
*Towhomcorrespondenceshouldbeaddressed.E-mail:vladimir.arion@ osmiumcongenersofthecomplexes[RuIIICl (Hazole) ]Cl17
2 4
univie.ac.at (V.B.A.), bernhard.keppler@univie.ac.at (B.K.K.). Fax: +43 (Hazole ) azole heterocycle) with 2:1 Hazole-to-chloride
1427752680.
§DedicatedtoProf.JanReedijkontheoccasionofhis65thbirthday. stoichiometry at the metal center, which were found to
7338 InorganicChemistry, Vol.47,No.16,2008 10.1021/ic8006958CCC:$40.75 2008AmericanChemicalSociety
PublishedonWeb07/03/2008
Osmium(III)ComplexeswithAzoleHeterocycles
Chart1.Anticancer[MIIICl2(Hazole)4]Clcomplexes;underlinedcomplexeshavebeencharacterizedinthisworkbyX-raycrystallography
exhibit the highest cytotoxicity within a series of indazole Experimental Section
complexes (vide supra); (ii) to study their structure, spec-
Materials. The starting compound [(DMSO) H] [OsCl ] was
troscopic properties, electrochemical behavior, aqueous 2 2 6
synthesized as previously reported in the literature.27,28 OsO
stability,andDNAbasebindingability;(iii)toevaluatetheir 4
(99.8%) and NH ·2HCl were purchased from Johnson Matthey
antiproliferative activity in vitro in human cancer cell lines 2 4
and Fluka, correspondingly. Pyrazole, indazole, imidazole, and
in comparison to relevant trans-[RuIIICl (Hazole) ]Cl com-
2 4 benzimidazole were from Aldrich and Fluka. The synthesis of
plexes;and(iv)toelucidaterelationshipsbetweenOsIII/OsII
[OsCl (Hazole) ]Clwasperformedunderanargonatmosphereby
2 4
redoxpotentialsandcytotoxicityofthepreparedcomplexes.
using standard Schlenk techniques. trans-[RuIIICl(Hazole) ]Cl
2 4
In this article, we show that a number of trans- (Hazole ) Hpz (5b), Him (7b), Hbzim (9b)) were synthesized
[OsIIICl 2 (Hazole) 4 ]Clcomplexes,whereHazole)pyrazole, accordingtotheliteratureprotocols.19
Hpz, (5a), indazole, Hind, (6a), imidazole, Him, (7a), and
benzimidazole, Hbzim, (9a), can be prepared as major
isomers (Chart 1) when starting from (H azole) [OsIVCl ] (13) ClarkeM.J.InMetalComplexesinCancerChemotherapy;Keppler
(1-4)andanexcessofazoleheterocycle.H
2
owev
2
er,forma
6
-
B.K.,Ed.;VCH:Weinheim,1993;129-157.
(14) Mestroni,G.;Alessio,E.;Sava,G.;Pacor,S.;Coluccia,M.InMetal
tion of cis isomers as minor products is also possible, as Complexes in Cancer Chemotherapy; Keppler B. K., Ed.; VCH:
found by the isolation and characterization of 8. Compre-
Weinheim,1993;157-185.
(15) Romerosa, A.; Saoud, M.; Campos-Malpartida, T.; Lidrissi, C.;
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biologicalevaluationaspotentialanticanceragentsprovides Eur.J.Inorg.Chem.2007,18,2803–2812.
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the basis for a comparison with the previously reported
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2 4
7b, and 9b).17,19 (17) Jakupec,M.A.;Reisner,E.;Eichinger,A.;Pongratz,M.;Arion,V.B.;
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Inorganic Chemistry, Vol. 47, No. 16, 2008 7339
Stepanenko et al.
(Hpz)[OsCl](1).Toasolutionof[(DMSO)H][OsCl](997.6 trans-[OsCl(Hpz)]Cl(5a).Pyrazole(100mg,1.47mmol)was
2 2 6 2 2 6 2 4
mg,1.4mmol)indryethanol(20mL)anexcessofpyrazole(267.9 addedtoasuspensionof1(195mg,0.36mmol)inhexanol-1(10
mg, 3.9 mmol) was added. This suspension was stirred at room mL), and the mixture was heated at 170 °C for 24 h. After the
temperaturefor2handthenconcentratedunderreducedpressure reaction mixture was allowed to cool to room temperature, the
to a minimal volume. The solution above the precipitate was alcoholwasevaporatedunderreducedpressureat60°C.Addition
decanted, and the product was washed with diethyl ether (2 × 5 of diethyl ether (15-20 mL) to the oily residue and treatment of
mL) and dried in vacuo at 60-70 °C for 24 h. Yield: 693 mg, the mixture in an ultrasound bath for 1.5 h afforded a brown
92%.Anal.CalcdforCH ClNOs(M )541.09g/mol):C,13.32; precipitate. This was filtered off, dried in vacuo, and purified by
6 10 6 4 r
H,1.86;N,10.35.Found:C,13.17;H,2.05;N,10.12.IRspectrum column chromatography on silica, using as eluent a mixture of
in KBr, cm-1: 517, 608, 637, 795, 872, 919, 1052, 1102, 1165, CHCl 3 /CH 3 OH(5:1)andcollectingthesecondfraction(R f )0.68).
1233, 1318, 1404, 1461, 1519, 1546, 1596, and 1716 ν(C-C), Thecomplexcrystallizedasdark-redcrystalsbydiffusionofdiethyl
ν(C-N) and σ(C-H); 2746, 2851, 2926, 3136, 3249, 3499, and ether into a methanol solution of 5a and dried under nitrogen at
3562ν(C-H)andν(N-H).UV-vis(CH 3 OH),λ max ,nm(ε,M-1 140°Cfor1h.Yield:105mg,50%.Anal.CalcdforC 12 H 16 Cl 3 N 8 Os
cm-1):215(22374),253sh(2233),276sh(1161),302sh(1219), (M r )568.90g/mol):C,25.33;H,2.83;N,19.70.Found:C,25.05;
336(7614),347(7495),371(7060),422(869).1HNMR(400MHz, H, 2.80; N, 19.45. ESI-MS in MeOH (positive), m/z: 534.2
d 6 - ( D H M 2 i S n O d) ) 2 : [O δ s 7 C .6 l 6 8 ] ( ( s 2 , ). 2H Th , e H 3 co a m nd pl H ex 5 ) w ,6 a . s 31 pr ( e s p , a 1 r H ed 4 ) a p s p a m n . orange [ [ O O s s C C l l 2 2 ( ( H H p p z z ) ) 2 4 - ]+ 2 , H E ]- S . I- I M R S spe i c n tru M m e i O n H KB ( r n , e c g m at - iv 1: e) 6 , 00 m , / 7 z 6 : 0, 39 7 2 9 . 2 7 ,
875,907,1047,1115,1144,1260,1276,1350,1385,1406,1470,
powderbyfollowingaprocedureanalogoustothatreportedabove
1506, 1526, 1644 ν(C-C), ν(C-N), and σ(C-H); 2852, 2936,
for1,startingfrom[(DMSO) H] [OsCl ](1005.7mg,1.4mmol)
2 2 6 2970,3128,3293,and3445ν(C-H)andν(N-H).UV-vis(HO),
andindazole(484.7mg,4.1mmol)indryethanol(20mL).Yield: 2
709.2 mg, 79%. Anal. Calcd for C H Cl NOs (M ) 641.21 λ max , nm (ε, M-1 cm-1): 212 (9134), 247 (12600), 289 (4930),
14 14 6 4 r 373(1447).1HNMR(400MHz,d-DMSO):δ-6.4,-17.7ppm.
g/mol): C, 26.22; H, 2.20; N, 8.74. Found: C, 26.11; H, 2.50; N, 6
8.69. IR spectrum in KBr, cm-1: 429, 538, 611, 643, 756, 831, X-ray diffraction quality crystals of 5a were obtained by recrys-
tallizationinmethanol.
893, 928, 962, 997, 1093, 1239, 1263, 1312, 1369, 1385, 1451,
1526, 1549, 1605, 1638, 1722, 1818, 1955, and 1991 ν(C-C), [OsCl 2 (Hind) 4 ]Cl(6a).Indazole(160mg,1.35mmol)wasadded
ν(C-N)andσ(C-H);2676,2735,2842,2880,2953,3014,3142, toasuspensionof2(210mg,0.32mmol)in5mLofhexanol-1,
and 3472 ν(C-H) and ν(N-H). UV-vis (CH OH), λ , nm (ε,
andthemixturewasheatedfor24hat170°C,leadingtoformation
3 max of a red-brown suspension. Then, the alcohol was evaporated in
M-1cm-1):211(52780),251(10611),258sh(9610),285(9132),
vacuoat60°C.Additionofdiethylether(15-20mL)totheresidue
337(8551),349(8356),371(7909),421(994).1NMR(400MHz,
andtreatmentofthemixtureinanultrasoundbathfor1.5hresulted
d-DMSO):8.06(s,1H ),7.76(d,J)8.36Hz,1H or1H ),7.53
6 3 4 7 in a dirty-brown powder. The product 6a as a yellow-greenish
(d,J)8.6Hz,1H or1H ),7.34(t,J)7.7Hz,1H or1H ),7.10
4 7 5 6 powder was purified by column chromatography on silica, using
(t,J)7.58Hz,1H or1H )ppm.
5 6 aseluentamixtureofCHCl/CHOH5:1andcollectingthesecond
(H im) [OsCl ] (3). The complex was prepared as an orange 3 3
2 2 6 fraction (R ) 0.58). Yield: 12 mg, <5%. ESI-MS in MeOH
powderbyfollowingaprocedureanalogoustothatreportedabove f
(positive),m/z:497.9[OsCl (Hind) ]+,734[OsCl(Hind) ]+;ESI-
for1,startingfrom[(DMSO) 2 H] 2 [OsCl 6 ](225.7mg,0.32mmol) MS in MeOH (negative), m 2 /z: 495. 2 9 [OsCl (Hind 2 ) -2H] 4 -, 731.9
andimidazole(64.6mg,0.95mmol)indryethanol(10mL).Yield: [OsCl(Hind)-2H]-.1HNMR(400MHz,d 2 -DMSO 2 ):δ7.39,6.28,
153.4 mg, 90%. Anal. Calcd for C 6 H 10 Cl 6 N 4 Os (M r ) 541.09 5.83, 2 4.69, 2 4 .77, -24 ppm. Red crystals 6 of 6a·CH OH·HO
3 2
g/mol):C,13.32;H,1.86;N,10.35.Found:C,13.49;H,1.69;N,
suitableforX-raystructureanalysiswereobtainedfromaCHOH/
10.12.IRspectruminKBr,cm-1:620,690,773,884,922,1046, 3
hexanesystem.
1085,1107,1162,1189,1304,1421,1438,1529,1578,1636,and
trans-[OsCl(Him)]Cl(7a)andcis-[OsCl(Him)]Cl(8).Imi-
1757ν(C-C),ν(C-N),andσ(C-H);3035,3129,3163,3249,and 2 4 2 4
dazole(60.5mg,0.89mmol)wasaddedtoasuspensionof3(108.1
3316ν(C-H)andν(N-H).UV-vis(CH OH),λ ,nm(ε,M-1
3 max mg,0.19mmol)inisoamylalcohol(4mL).Themixturewasheated
cm-1): 216 (19 809), 256 sh (1621), 279 sh (662), 303 sh (838), at130°Cfor47h.Then,theisoamylalcoholwasevaporatedunder
336(8277),346(8194),371(7668),423(879).1HNMR(400MHz, reduced pressure at 60 °C, the residue was washed with diethyl
d -DMSO): δ 14.22 (s, H ), 9.07 (s, 1H ), 7.69 (s, 2H, H and
6 NH 2 4 ether, and dried in vacuo. The solid was dissolved in a CHCl /
3
H 5 )ppm. CH 3 OH mixture (2:1) and passed through a silica column, using
(H 2 bzim) 2 [OsCl 6 ](4).Thepreparationof4asanorangepowder CHCl 3 /CH 3 OH2:1aseluent.Thesecond(R f )0.7)andthethird
wascarriedoutfollowingtheprocedureusedforthesynthesisof (R )0.5)fractionwerecollected.Removingthevolatilecompo-
f
1,startingfrom[(DMSO) 2 H] 2 [OsCl 6 ](711.5mg,0.99mmol)and nents of the second fraction under reduced pressure followed by
benzimidazole(328.9mg,2.8mmol)indryethanol(25mL).Yield: vacuumsublimationofimidazoleascontaminantat90°Cafforded
618.4 mg, 97%. Anal. Calcd for C 14 H 14 Cl 6 N 4 Os (M r ) 641.21 theredsolidof7a.Yield:68.2mg,60%.Dark-red8wasisolated
g/mol): C, 26.22; H, 2.20; N, 8.74. Found: C, 25.96; H, 2.48; N, from the third fraction after removing the solvent under reduced
8.49. IR spectrum in KBr, cm-1: 418, 507, 542, 598, 622, 702, pressure.Yield:3-5mg,<4%.X-raydiffractionqualitycrystals
757,861,866,939,966,1005,1110,1137,1238,1266,1384,1447, of7aand8wereobtainedfrommethanolsolutionsofcomplexes
1498, 1507, 1596, 1621, 1734, 1845, 1931, and 1968 ν(C-C), saturatedwithdiethylether.
ν(C-N)andσ(C-H);2752,2811,2958,3041,3157,3260,3516, Complex 7a. Anal. Calcd for C H ClN Os (M ) 568.9
12 16 3 8 r
and 3581 ν(C-H) and ν(N-H). UV-vis (CH 3 OH), λ max , nm (ε, g/mol):C,25.33;H,2.83;N,19.69.Found:C,25.27;H,2.59;N,
M-1cm-1):211(22098),222sh(13896),267(8796),274(8830), 19.41. ESI-MS in MeOH (positive), m/z: 398.2 [OsCl(Him) ]+,
2 2
336(5786),346(5691),372(5326),423(655).1HNMR(400MHz, 534.3 [OsCl (Him) ]+, ESI-MS in MeOH (negative), m/z: 465.4
2 4
d -DMSO): δ 9.43 (s, 1H), 7.84 (d, J ) 6.08 Hz, 1H or 1H), [OsCl (Him) -2H]-, 532.0 [OsCl(Him) -2H]-. IR spectrum in
6 2 4 7 2 3 2 4
7.83(d,J)6.32Hz1H or1H),7.57(d,J)6.28Hz,1H,or KBr,cm-1:614,658,729,743,769,840,1074,1100,1144,1182,
4 7 5
1H),7.56(d,J)6.32Hz,1H or1H )ppm. 1264,1330,1444,1492,1508,1548,1560,1578,and1686ν(C-C),
6 5 6
7340 InorganicChemistry,Vol.47,No.16,2008
Osmium(III)ComplexeswithAzoleHeterocycles
Table1.CrystalDataandDetailsofDataCollectionfor5a,6a,7a,8,and9a
complex 5a 6a·CH3OH·H2O 7a 8 9a·CH3OH·(C2H5)2O
empiricalformula C12H16Cl3N8Os C29H30Cl3N8O2Os C12H16Cl3N8Os C12H16Cl3N8Os C33H38Cl3N8O2Os
fw 568.88 819.16 568.88 568.88 875.26
j
spacegroup P1 C2/c C2/c Cm P21/n
a,Å 7.9101(2) 16.9910(4) 9.6110(3) 11.229(2) 15.7469(4)
b,Å 8.4109(2) 11.1273(4) 18.9329(9) 12.259(3) 13.6976(4)
c,Å 14.6494(4) 16.6057(4) 9.6818(3) 7.0080(14) 16.5585(5)
R,deg 102.690(2)
(cid:6),deg 99.886(1) 97.557(2) 90.016(2) 104.11(3) 93.710(2)
γ,deg 110.743(2)
V,Å3 884.54(4) 3112.27(15) 1761.74(11) 935.6(3) 3564.10(17)
Z 2 4 4 2 4
λ,Å 0.71073 0.71073 0.71073 0.71073 0.71073
F calcd,gcm-3 2.136 1.748 2.145 2.019 1.631
crystsize,mm3 0.25×0.15×0.12 0.20×0.12×0.10 0.30×0.12×0.12 0.12×0.06×0.01 0.30×0.20×0.20
T,K 100 100 100 100 100
R1a 0.0129 0.0222 0.0115 0.0155 0.0262
wR2b 0.0296 0.0546 0.0306 0.0354 0.0593
GOFc 1.026 1.081 1.088 1.016 1.001
aR1)Σ||F
o
|-|F
c
||/Σ|F
o
|.bwR2){Σ[w(F
o
2-F
c
2)2]/Σ[w(F
o
2)2]}1/2.cGOF){Σ[w(F
o
2-F
c
2)2]/(n-p)}1/2,wherenisthenumberofreflections
andpisthetotalnumberofparametersrefined.
ν(C-N) and σ(C-H); 2586, 2622, 2871, 2975, 3083, and 3170 PerkinElmerLambda20UV-visspectrophotometer,usingsamples
ν(C-H) and ν(N-H). UV-vis (H
2
O), λ
max
, nm (ε, M-1 cm-1): dissolvedinmethanol(1-4)andwater(5a,7a,and9a)at298and
207 (59705), 216 (58039), 252 sh (19354), 296 (11495), 384 310K.Theaqueoussolutionbehaviorof5a,7a,and9awithrespect
(821).1HNMR(400MHz,d 6 -DMSO):δ9.85,5.78,-8.59,-14.92 to hydrolysis was studied at 298 and 310 K over 24-72 h by
ppm. UV-visspectroscopy(5a,7a,and9a)and1HNMRspectroscopy
Complex 8. ESI-MS in MeOH (positive), m/z: 398.2
(5a).Theinteractionof5awith9-methyladenine(1:1.5)wasstudied
[OsCl (Him) ]+,534.3[OsCl (Him) ]+,ESI-MSinMeOH(nega-
2 2 2 4 by 1H NMR spectroscopy in aqueous phosphate buffer (pH 6.0,
tive),m/z:396.3[OsCl(Him)-2H]-,465.8[OsCl(Him)-2H]-,
2 2 2 3 310K)over72h.InfraredspectrawereobtainedfromKBrpellets
530.0 [OsCl (Him) -2H]-. 1H NMR (400 MHz, d-DMSO): δ
2 4 6 with a PerkinElmer FTIR 2000 instrument (4000-400 cm-1).
15.88,11.45,8.97,4.14,-4.16,-13.0,-15.7,-17.97ppm.
Electrosprayionizationmassspectrometrywascarriedoutwitha
trans-[OsCl (Hbzim) ]Cl (9a). Benzimidazole (163 mg, 1.38
2 4
Bruker Esquire 3000 instrument (Bruker Daltonic, Bremen, Ger-
mmol)wasaddedtoasuspensionof4(214.6mg,0.34mmol)in
hexanol-1(5mL),andthemixturewasheatedat160°Cfor24h many) in methanol (5a-7a, 8, and 9a) and water (5a). Expected
togiveayellow-brownsolution.Then,thealcoholwasevaporated andexperimentalisotopedistributionswerecompared.The1HNMR
underreducedpressureat60°C.Additionofdiethylether(15-20 spectra were recorded at 400.13 MHz on a Bruker DPX400
mL)totheresidue,andtreatmentofthemixtureinanultrasound (UltrashieldTMMagnet)spectrometer.Cyclicvoltammogramswere
bath for 1.5 h resulted in a dirty-yellow solid. This was filtered measuredinatwo-compartmentthree-electrodecellusinga1.0
off,driedinvacuoandpurifiedbycolumnchromatographyonsilica mm diameter glassy-carbon disk working electrode, probed by
usingaseluentamixtureofCHCl 3 /CH 3 OH4:1andcollectingthe a Luggin capillary, and connected to a silver-wire pseudoref-
thirdfraction(R f )0.48)orCHCl 3 /CH 3 OH3:1(R f )0.67).The erence electrode, and a platinum auxiliary electrode. Measure-
product of the composition 9a·CH 3 OH·(C 2 H 5 ) 2 O, crystallized as ments were performed at room temperature, using an EG & G
dark-orangecrystalsbydiffusionofdiethyletherintothemethanol
PARC 273A potentiostat/galvanostat. Deaeration of solutions
solutionof9a,wasfilteredoff,washedwithdiethylether,anddried
was accomplished by passing a stream of argon through the
undernitrogenatmosphereat140°Cfor1h.Yield:180mg,70%.
solution for 10 min prior to the measurements and then
Ayellowpowderof9a·n(C H)Owasobtainedafterevaporating
2 52 maintaining a blanket atmosphere of argon over the solution
volatilecomponentsofthethirdfraction,additionofdiethylether
during the measurements. The potentials were measured in 0.2
to the remaining residue, and treatment of this mixture in an
ultrasound bath. Anal. Calcd for C H Cl N Os (M ) 769.13 Mphosphatebuffersolutions(pH7.0)(7a,9a)andinacetonitrile
28 24 3 8 r
(0.15 M [nBu N][BF ]/CH CN) (5a, 6a, 9a), using as internal
g/mol):C,43.72;H,3.15;N,14.57.Found:C,43.56;H,3.08;N, 4 4 3
14.47.ESI-MSinMeOH(positive),m/z:498.2[OsCl (Hbzim) ]+, standardsmethylviologen(E 1/2 ox)-0.44VvsNHEinwater)29
2 2
734.3[OsCl 2 (Hbzim) 4 ]+;ESI-MSinMeOH(negative),m/z:496.3 and ferrocene (E 1/2 ox ) 0.69 V vs NHE in acetonitrile)30
[OsCl(Hbzim)-2H]-,732.1[OsCl(Hbzim)-2H]-.IRspectrum respectively, and are quoted relative to NHE.
2 2 2 4
in KBr, cm-1: 428, 456, 549, 615, 697, 739, 760, 775, 846, 857, CrystallographicStructureDetermination.X-raydiffraction
884,975,1012,1113,1144,1185,1250,1264,1306,1350,1421, measurements were performed on a Bruker X8 APEXII CCD
1464,1493,1508,1594,1619,1708,and1774ν(C-C),ν(C-N) diffractometer.Singlecrystalswerepositionedat40mmfromthe
and σ(C-H); 2837, 2911, 2982, 3102, 3133, and 3375 ν(C-H) detector, and 2798, 1490, 1920, 1282, and 1724 frames were
and ν(N-H). UV-vis (H 2 O), λ max , nm (ε, M-1 cm-1): 215 measured, each for 15, 60, 7, 80, and 2 s over 1° scan width for
(116635), 240 sh (55784), 294 (31907), 300 (31884), 312 sh
(15683),379(4089).1HNMR(400MHz,d-DMSO):δ9.2,8.0,
6
5.12,3.96ppm. (29) GuedesdaSilva,M.F.C.;Pombeiro,A.J.L.;Geremia,S.;Zangrando,
Physical Measurements. Elemental analyses were carried out E.; Calligaris, M.; Zinchenko, A. V.; Kukushkin, V.; Yu,J. Chem.
Soc.,DaltonTrans.2000,1363–1371.
attheMicroanalyticalServiceoftheInstituteofPhysicalChemistry
(30) Barrette,W.C.,Jr.;Johnson,H.W.,Jr.;Sawyer,D.T.Anal.Chem.
oftheUniversityofVienna.UV-visspectrawererecordedona 1984,56,1890–1898.
Inorganic Chemistry, Vol. 47, No. 16, 2008 7341
Stepanenko et al.
5a, 6a, 7a, 8, and 9a, correspondingly. The data were processed Scheme1.Transformationpathwaysof1-4into5a,6a,7a,8,and
usingSAINTsoftware.31Crystaldata,datacollectionparameters, 9a:a)mer-[OsCl3(Hazole)3],b)trans-[OsCl2(Hazole)4]Cl(5a,6a,7a,
andstructurerefinementdetailsaregiveninTable1.
9a),c)cis-[OsCl2(Hazole)4]Cl(8)
The structures were solved by direct methods and refined by
full-matrix least-squares techniques. Non-hydrogen atoms were
refinedwithanisotropicdisplacementparameters.Hydrogenatoms
wereplacedatcalculatedpositionsandrefinedasridingatomsin
the subsequent least-squares model refinements. The isotropic
thermal parameters were estimated to be 1.2 times the values of
the equivalent isotropic thermal parameters of the non-hydrogen
atomstowhichhydrogenatomsarebonded.Oneoftheimidazole
ligands in 8 was found to be disordered over 2 mirror-related
positions.AttemptstosolvethestructureinthespacegroupC2/c
ended up with more severe disorder and a higher R1 value. The
followingcomputerprogramswereused:structuresolution,SHELXS-
97;32a refinement, SHELXL-97;37b molecular diagrams, ORTEP;
computer: Pentium IV. Scattering factors were taken from the
literature.33
CellLinesandCultureConditions.HumanA549cells(non- comparisontountreatedcontrolmicrocultures,andIC 50 valueswere
small cell lung carcinoma) and SW480 cells (adenocarcinoma of calculatedfromconcentration-effectcurvesbyinterpolation.Evalu-
the colon) were kindly provided by Brigitte Marian, Institute of ationisbasedonmeansfromatleastthreeindependentexperiments,
CancerResearch,MedicalUniversityofVienna,Austria.CH1cells eachcomprisingsixreplicatesperconcentrationlevel.
originate from an ascites sample of a patient with a papillary
Results and Discussion
cystadenocarcinomaoftheovaryandwerekindlydonatedbyLloyd
R.Kelland,CRCCenterforCancerTherapeutics,InstituteofCancer Synthesis. (H azole) [OsIVCl ] (1-4) were obtained by
2 2 6
Research,Sutton,UK.Cellsweregrownin75cm2cultureflasks reaction of [(DMSO) H] [OsIVCl ] with azole heterocycles
2 2 6
(Iwaki/Asahi Technoglass) as adherent monolayer cultures in
in 1:3 molar ratio in dry ethanol at room temperature in
completeculturemedium,i.e.,MinimalEssentialMedium(MEM) 79-97% yields. Replacement of chlorido ligands in
supplementedwith10%heat-inactivatedfetalbovineserum,1mM
[OsCl]2-byazoleheterocycleswasrealizedunderprolonged
sodiumpyruvate,4mML-glutamineand1%non-essentialamino 6
acids(100×)(allpurchasedfromSigma-Aldrich).Cultureswere heating of (H 2 azole) 2 [OsCl 6 ] with the corresponding azole
maintained at 37 °C in a humidified atmosphere containing 5% in 1:4 molar ratio in isoamyl alcohol (130 °C for 7a, 8) or
CO . hexanol-1 (160-170 °C for 5a, 6a, 9a) to generate trans-
2
Cytotoxicity Tests in Cancer Cell Lines. Cytotoxicity was [OsCl 2 (Hazole) 4 ]Clin50-70%yields(5a,7a,9a)and<5%
determined by means of the colorimetric MTT assay (MTT ) yield(6a).Thesynthesisof6aisaccompaniedbyconcurrent
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide, reactions, which resulted in many fractions difficult to
purchasedfromFluka).Forthispurpose,cellswereharvestedfrom separate by column chromatography and finally in minor
culture flasks by trypsinization and seeded in 100 µL aliquots in yield of the desired product.
completeculturemediuminto96-wellmicrocultureplates(Iwaki/
The synthesis of this family of compounds proceeds in
AsahiTechnoglass).Celldensitiesof4.0×103cells/well(A549),
several steps including reduction of OsIV to OsIII and
1.5×103cells/well(CH1),and2.5×103cells/well(SW480)were
inevitable formation of mer-[OsCl (Hazole) ] (Scheme 1),
chosen in order to ensure exponential growth throughout drug 3 3
which can be detected in the reaction mixture along with
exposure. Cells were allowed to settle for 24 h, followed by the
the main product by TLC. The yield of mer-[OsIIICl -
additionofdilutionsofthetestcompoundsin100µL/wellcomplete 3
culture medium and incubation for 96 h. At the end of exposure, (Hazole) 3 ] can be significantly increased when the amount
drug solutions were replaced by 100 µL/well RPMI 1640 culture of azole heterocycle used relative to the amount of
medium (supplemented with 10% heat-inactivated fetal bovine (H azole) [OsIVCl ]isreducedandthereactioniscarriedout
2 2 6
serum)plus20µL/wellMTTsolutioninphosphate-bufferedsaline undermilderconditions.Thereactionpathwaystomeridional
(5 mg/ml PBS). After incubation for 4 h, medium was removed, isomers were proposed and will be discussed in another
andthereducedformazanproductformedbythemetabolicactivity work.34 We note here the key role of the mer-isomer of
of vital cells was dissolved in 150 µL DMSO per well. Optical [OsIIICl (Hazole) ]intheformationof[OsIIICl (Hazole) ]Cl
3 3 2 4
densities at a wavelength of 550 nm were measured with a
compounds.
microplate reader (Tecan Spectra Classic), using a reference
Thefourthazoleheterocyclecoordinatestoosmiumupon
wavelength of 690 nm to correct for unspecific absorption. The
substitution of the chlorido ligand, which is in the trans
quantity of vital cells was expressed in terms of T/C values by
position to the azole ligand in mer-[OsCl (Hazole) ]. The
3 3
(31) SAINT-Plus(Version7.06a)andAPEX2;Bruker-NoniusAXSInc.: substitution of one of the two mutually trans-arranged
Madison,Wisconsin,USA,2004.
(32) (a) Sheldrick, G. M. SHELXS-97, Program for Crystal Structure chlorido ligands is less favored (<4% yield for 8) and was
Solution; University of Go¨ttingen: Go¨ttingen, Germany, 1997. (b) observed only for imidazole as a less bulky and strongest
Sheldrick,G.M.SHELXS-97,ProgramforCrystalStructureRefine-
ment;UniversityofGo¨ttingen:Go¨ttingen,Germany,1997. net electron-donor ligand (Scheme 1).
(33) International Tables for X-ray Crystallography; Kluwer Academic Itshouldbenotedthatthesynthesisofrutheniumspecies
Press:Dordrecht,TheNetherlands,1992;Vol.C,Tables4.2.6.8and
5b,7b,and9bwasperformedundermildconditions,starting
6.1.1.4.
(34) Chiorescu,I.;Stepanenko,I.N.Manuscriptinpreparation. from[RuCl 3 (EtSPh) 3 ](forimidazolederivative)or(H 2 azole)-
7342 InorganicChemistry,Vol.47,No.16,2008
Osmium(III)ComplexeswithAzoleHeterocycles
Figure1.ORTEPplotsofthefirstcrystallographicallyindependentcation
in trans-[OsCl2(Hpz)4]Cl (5a) [left] and of the complex cation in trans- Figure2.ORTEPplotsofthecomplexcationsintrans-[OsCl2(Him)4]Cl
[OsCl2(Hind)4]Cl(6a)[right]withtheatom-numberingschemes.Thermal (7a) [left] and trans-[OsCl2(Hbzim)4]Cl (9a) [right] with the atom-
displacement ellipsoids are drawn at the 50% probability level. Selected numberingschemes.Thermaldisplacementellipsoidsaredrawnatthe50%
bondlengths(Å)andangles(deg)for5aand6a,correspondingly:Os1-Cl1 probabilitylevel.Selectedbondlengths(Å)andangles(deg)in7aand9a,
2.3722(4),Os1-Cl22.3591(4),Os1-N12.0657(14),Os1-N62.0649(13), respectively:Os-Cl12.3558(5),Os-Cl22.3576(5),Os-N12.0712(14),
N6-Os-N1 89.05(5), N6-Os1-Cl1 90.94(4), N1-Os1-Cl1 89.30(4), Os-N3 2.0693(14), N1-Os-N3 89.87(6), N1-Os-N1′ 179.14(7),
ΘCl1-Os1-N1-N225.42(12),ΘCl1-Os1-N6-N7161.11(12);Os-Cl12.3477(9), N1-Os-Cl1 90.43(3), N1-Os-Cl2 89.57(3), ΘCl1-Os-N1-C1 45.68(13),
Os-N12.073(3),Os-N32.075(3),N1-Os-N386.48(11),N1-Os-Cl1 ΘCl1-Os-N3-C450.13(13);Os-Cl12.3683(8),Os-Cl22.3479(8),Os-N1
89.86(9),N3-Os-Cl190.04(9),ΘCl1-Os1-N1-N2 -30.7(3),ΘCl1-Os1-N3-N4 2.081(3),Os-N32.071(3),Os-N52.076(3),Os-N72.089(3),N1-Os-N7
155.7(3). 92.04(11),N1-Os-N3177.97(11),N1-Os-Cl188.72(8),N1-Os-Cl2
90.26(8), ΘCl1-Os-N1-C1 51.2(3), ΘCl1-Os-N5-C15 49.5(3), ΘCl1-Os-N3-C8
44.3(3),ΘCl1-Os-N7-C2240.9(3).
[RuCl (Hazole) ](whereHazole)Hpz,Hbzim).19Likewise,
4 2
the mer-[RuCl (Hazole) ] was an intermediate in the syn- cations. Selected bond lengths (Å) and angles (deg) are
3 3
thesis of trans-[RuCl (Hazole) ]Cl. Starting from (H ind)- quoted in the legends to Figures 1 and 2. 7a crystallized in
2 4 2
[RuCl (Hind) ] and indazole in refluxing aqueous ethanol, themonoclinicspacegroupC2/candisisostructural(Figure
4 2
trans-[RuCl 2 (Hind) 4 ] was obtained. Oxidation of the latter S1intheSupportingInformation)withtherelatedruthenium
with hydrogen peroxide in methanol in the presence of compound 7b.19
hydrochloric acid and indazole resulted in 6b.17 Formation Theosmium(III)ionsin[OsCl (Hpz) ]+,[OsCl (Hind) ]+,
2 4 2 4
of cis-[RuCl 2 (Hazole) 4 ]Cl or isomerization of trans- [OsCl 2 (Him) 4 ]+, and [OsCl 2 (Hbzim) 4 ]+ of 5a-7a and 9a
[RuCl 2 (Hazole) 4 ]intothecisisomerhasnotbeenobserved have the expected distorted octahedral coordination geom-
yet. etry, with four azole heterocycles bound through their
The ruthenium compounds are kinetically more labile nitrogen atoms in the equatorial plane and two chloride
towardligandexchangeorisomerizationthantheanalogous ligands in axial positions. A propeller-like arrangement of
osmiumspecies.Theminoryieldofcis-[OsCl 2 (Him) 4 ]Cl(8) thetetrazolecore,formedonthetiltoftheazoleringswith
produced under harsh conditions along with predominant respect to the OsN plane to reduce the mutual repulsions,
4
formation of the trans isomer 7a suggests that the transient ischaracteristicforthecationsin5a-7aand9a.Thepitch
cis isomers in the case of ruthenium were not discovered of the four azole planes varies between 18.8 and 20.9° in
because of their quick isomerization into trans species.
thefirstcrystallographicallyindependentcomplexcationof
Crystal Structures. The structures of the complexes 5a,andbetween24.6and34.8°inthesecond,24.4and31.2
reported herein are of interest because of the paucity of in 6a, 42.0 and 44.9 in 7a, and between 43.8 and 48.8° in
documentedX-raydiffractiondataonosmium(III)complexes
9a. The trans-coordinated pyrazole and indazole ligands in
and especially on osmium(III)-azole derivatives. Reported
5a and 6a, correspondingly, are parallel to one another,
data on the latter are confined to TpOsIII(Him)Cl 35 and
2 whereas imidazole and benzimidazole planes are almost
[OsIIICl (Hazole) ], where Hazole ) pyrazole, indazole,
3 3 perpendicular to one another in 7a and 9a, respectively.
imidazole, or benzimidazole.34,36 In addition, these are to TheaverageOs-Clbondlengths[2.365(7)(5a),2.3477(9)
our knowledge the first structures of osmium compounds
(6a), 2.3567(9) (7a), and 2.358(10) Å (9a)] are well
containingfourazoleandtwochloridoligandsatthemetal
comparable to those found in (H im)[trans-OsCl (Him)-
center.37 The crystal structures of 5a-7a and 9a consist of 2 4
(DMSO)][2.3627(11)Å]26andinmer-[OsCl (NH ) (Me S)]
[OsCl 2 (Hpz) 4 ]+, [OsCl 2 (Hind) 4 ]+, [OsCl 2 (Him) 4 ]+, and [2.368(8) Å].29 The average Os-N bond in 3 5a- 3 7a 2 and 2 9a
[OsCl (Hbzim) ]+ cations, respectively, and Cl- anions.
2 4 [2.0653(4),2.074(3),2.0703(9),and2.079(5)Å,correspond-
Cocrystallized solvent molecules were found in the crystal
ingly] is well comparable with the average Ru-N bond in
structures of 6a (disordered CH OH and H O) and 9a
3 2 therelatedrutheniumcomplexestrans-[RuCl (Hpz) ]Cl(5b),
(CH OHand(C H ) O).Figure1displaysperspectiveviews 2 4
3 2 5 2 trans-[RuCl (Hind) ]Cl (6b), trans-[RuCl (Him) ]Cl (7b),
of the [OsCl (Hpz) ]+ and [OsCl (Hind) ]+ cations and 2 4 2 4
2 4 2 4 and trans-[RuCl (Hbzim) ]Cl (9b) [2.0603(24), 2.0705(3),
Figure 2 those of [OsCl (Him) ]+ and [OsCl (Hbzim) ]+ 2 4
2 4 2 4 2.0685(3), and 2.0771(36) Å, correspondingly]17,19 and is
significantly shorter than similar bonds trans to sulfur-
(35) Bennett,B.K.;Pitteri,S.J.;Pilobello,L.;Lovell,S.;Kaminsky,W.;
Mayer,J.M.J.Chem.Soc.,DaltonTrans.2001,3489–3497. bound DMSO in the osmium(II) complexes trans,cis,cis-
(36) Chiorescu,I.;Stepanenko,I.N.;Arion,V.B.;Krokhin,A.A.;Keppler,
[OsCl (Hind) (DMSO) ] and trans,cis,cis-[OsCl (Hpz) -
B.K.J.Biol.Inorg.Chem.2007,12(Suppl1),S226. 2 2 2 2 2
(37) CSDVersion5.29;November2007. (DMSO) 2 ]at2.130(4),2.137(4),and2.1202(17),2.1365(17)
Inorganic Chemistry, Vol. 47, No. 16, 2008 7343
Stepanenko et al.
of 2.371(2) Å is significantly longer than the Os-Cl bond
in trans-[OsCl (Him) ]+ [2.3567(9) Å], (H im)[trans-
2 4 2
OsCl (Him)(DMSO)] [2.3627(11) Å],26 or in mer-
4
[OsCl (NH ) (Me S)] [2.368(8) Å].29 The average Os-N
3 3 2 2
bondin8[2.071(21)Å]iswellcomparablewiththeaverage
Ru-N bond in the related ruthenium complexes trans-
[RuCl (Him) ]Cl (7b) and trans-[RuCl (Hbzim) ]Cl (9b)
2 4 2 4
[2.0685(3) Å and 2.0771(36) Å, correspondingly],19 but
slightly longer than the average Ru-N bond in trans-
[RuCl (Hind) ] [2.0639(1) Å].17
2 4
UV-visSpectra.Theelectronicabsorptionspectraofthe
aqueoussolutionsof5a,7a,and9aarecharacterizedbyfour
absorption bands with maxima within 200-215, 240-252,
288-300,and373-380nm.Thefirstthreeabsorptions(the
Figure3.Thestructureofthecationincis-[OsCl2(Him)4]Cl(8),showing second band of 7a and 9a is a shoulder) are overlapping
theatom-numberingscheme.Selectedbondlengths(Å)andangles(deg): bands due to intraligand transitions of coordinated azole
Os-Cl12.371(2),Os-N12.092(10),Os-N62.050(6),Os-N112.069(9),
N6-Os-N11 88.1(2), N11-Os1-N1 172.2(3), N6-Os-Cl1 88.65(13), heterocycles and charge-transfer (CT) bands. The UV-vis
N1-Os-Cl1 84.7(3), N1i-Os-Cl1 95.5(3),ΘCl1-Os-N1-C2 54.7(8), spectrum of 9a has the third benzenoid band.42 The fourth
ΘCl1-Os-N6-C7 -126.8(5),andΘCl1-Os-N11-C1245.58(6);symmetrycode: band is also attributed to CT transitions. Their extinction
i)x,-y,z.
coefficients are in the range from 820 to 4089 M-1 cm-1,
Å,correspondingly.38Likeforrutheniumcongeners,intrans- which are too intense for pure d-d transitions. The d-d
[OsCl (Hazole) ]Cl the average M-N bond lengths are in bands of osmium(III) are often overlapped with the low-
2 4
the following rank order (5a < 6a) and (7a < 9a), which energy CT absorptions since the low-spin osmium(III) has
large spin-orbital contribution from intraconfigurational
agreeswellwiththerelativeelectron-donorcharacterofthe
corresponding N-ligand pairs [E (Hind) > E (Hpz) and transitions.BecauseofthedecreaseofCTtransitionenergy
E (Hbzim)>E (Him)]andtheirb L asicities[pK L (H ind+)< in the series 5a-9a-7a bathochromic shifts from 289 to
L L a 2
pK(H pz+) and pK(H bzim+) < pK(H im+)]18,19,39–41 296andfrom373to384nmforthethirdandfourthbands
a 2 a 2 a 2
were observed.
Of note are also the intramolecular hydrogen bonding
interactions N2-H···Cl1 (Figure 1) and N17-H···Cl2 in The analogous ruthenium compounds have the same
two crystallographically independent cations of 5a [N2-H character of UV-vis spectra,19 but because of the stronger
0.880,H···Cl12.681,N2···Cl13.159Å,∠N2HCl1115.34°; ligandfieldsplittingforathird-rowtransitionmetalCTbands
N17-H0.880,H···Cl22.741,N17···Cl23.176Å,∠N17HCl2 of [OsCl 2 (Hazole) 4 ]Cl are blue-shifted. CT bands of
111.88°]. In addition, the crystal structures of 5a, 6a, and [RuCl 2 (Hazole) 4 ]ClweredescribedasLMCTtransitionsand,
in contrast to the investigated complexes (5a, 7a, and 9a),
9a are stabilized by interionic hydrogen bonds of the type
N-H···Cl (Tables S1-S3 in the Supporting Information). they are blue-shifted with increasing basicity and electron
donorpropertiesoftheN-ligands([RuCl (Hpz) ]Cl,401nm;
Each chloride ion in 7a forms four hydrogen bonds with 2 4
[RuCl (Hbzim) ]Cl, 363 nm; [RuCl (Him) ]Cl, 346 nm).19
four imidazole rings of four neighboring complex cations 2 4 2 4
For comparison, the compounds (H azole)[trans-OsCl -
(Figure S2 in the Supporting Information). 2 4
(Hazole)(DMSO)]didnotshowadependenceofCTenergy
8 crystallized in the monoclinic space group Cm. The
crystalstructureof8consistsof[OsCl (Him) ]+cationsand on the electron-donor character of the N-ligands.26
2 4
Cl- anions. Figure 3 displays a perspective view of the TheLMCTbandsfor[MCl 2 (Hazole) 4 ]Clareprobablydue
[OsCl (Him) ]+cation.Selectedbondlengths(Å)andangles
toπClfdtransitions.Besides,theelectrontransitionfrom
2 4
π orbitals of azole to metal d orbitals can also take place.
(deg) are quoted in the legend to Figure 3.
The osmium(III) ion in cis-[OsCl (Him) ]+ has the ex- The hole in t 2g 5 configuration of the metal is presumably
2 4 responsiblefortheselow-energytransitions.MLCTdfπ*
pecteddistortedoctahedralcoordinationgeometry,withtwo
(azole) transitions might also be expected.
cis-arranged chlorido ligands and two imidazole ligands in
TheabsenceofclearcorrelationsbetweentheCTenergy
equatorial positions and other two imidazoles in axial
and the observed M-Cl and M-N length variation in
positions, one of which was found disordered over two
positionswith50:50%occupancy.TheOs-Clbondlength [MCl 2 (Hazole) 4 ]Cl can presumably be explained by the
dominationofsomeCTbands,whichmakestheinterpreta-
(38) Stepanenko,I.N.;Cebrian-Losantos,B.;Arion,V.B.;Krokhin,A.A.; tion of electronic absorption spectra difficult.
Nazarov,A.A.;Keppler,B.K.Eur.J.Inorg.Chem.2007,3,400– ElectrochemicalStudy.Thecyclicvoltammogramsof7a,
411.
(39) Reedijk,J.InComprehensiVeCoordinationChemistry;Wilkinson,G.; 9a in 0.2 M phosphate buffer (pH 7) and 5a, 6a, 9a in
Gillard,R.D.;McCleverty,J.A.,Eds.;PergamonPress:Elmsford, acetonitrile(0.15M[nBu N][BF ]/CH CN)atacarbondisk
NY,1987;2,73-98. 4 4 3
working electrode, recorded with a scan rate of 0.2 V/s,
(40) Reisner,E.;Arion,V.B.;Keppler,B.K.;Pombeiro,A.J.L.Inorg.
Chim.Acta2008,361,1569–1583.
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7344 InorganicChemistry,Vol.47,No.16,2008
Osmium(III)ComplexeswithAzoleHeterocycles
Table2.CyclicVoltammetricDatafor5a-7aand9a Table3.CytotoxicityofOsmiumComplexes5a,7a,and9aand
RutheniumComplexes5b,7b,and9binThreeHumanCancerCell
E 1/2(OsII/III)a, E 1/2(OsIII/IV)a, E 1/2(OsII/III)c, E 1/2(OsIII/IV)c,
Lines
complex (∆E p)b (∆E p)b (∆E p)b (∆E p)b
5ad -0.2(70) 1.35(60)
IC50(µM)a
6ad 0.1(60) 1.55(70) compound A549 CH1 SW480
7ae -1.17(60) 0.36(70) 5a 3.2(0.8 1.0(0.2 2.3(0.2
9a -1.21(60) 0.31(70) -0.4(60) 1.1(70) 5b 18(2 2.0(0.2 2.9(0.2
aPotentialsE 1/2(E 1/2 )(E pa +E pc)/2,whereE paandE pcaretheanodic 7a 60(11 39(2 38(2
andcathodicpeakpotential,respectively)measuredatascanrate0.2Vs1- 7b 2.1(0.4 7.1(1.0 1.7(0.3
in0.2Mphosphatebuffersolutions(pH7.0),usingmethylviologen(E 1/ 9a 12(2 2.3(0.2 3.0(0.6
2ox)-0.44VvsNHEinwater)asinternalstandard,aregiveninvolts 9b 4.6(0.2 9.1(1.9 2.0(0.1
m an i d lli q v u o o lt t s e . d c r P el o a t t e iv n e tia to ls N a H re E m .b ea ∆ s E ur p e v d al a u t es a ( s ∆ ca E n p ) rat E e pa 0. - 2 E V p s c 1 ) - ar i e n g 0 i . v 1 e 5 n M in a50%inhibitoryconcentrationsintheMTTassay(96hexposure).Values
[nBu4N][BF4]/CH3CN, using ferrocene (E 1/2 ox ) 0.69 V vs NHE in aremeans(standarddeviationsofatleastthreeindependentexperiments.
acetonitrile)asinternalstandard,andquotedrelativetoNHE.dComplex
ispoursolubleinphosphatebuffersolution.eComplexispoursolublein [OsCl (Hpz) ]+ in the positive ion ESI mass spectrum of
2 4
CH3CN.
an aqueous solution of 5a.
1H NMR spectra of 5a in D O (310 K) were monitored
2
over72h.Thespectraobtainedimmediatelyafterdissolution
of 5a and after 72 h were identical, providing further
evidence against hydrolysis of the complex in aqueous
medium.1HNMRspectraofamixtureof5aand9-methyl-
adenine (as a DNA model base) in 1:1.5 molar ratio in
phosphate buffer (pH 6.0, 310 K) were measured. The
absence of a new set of signals expected for coordinated
9-methyladenine even after 72 h indicated inertness of 5a
towardthepurinenucleobasesundertheconditionsemployed
(Figure S3 in the Supporting Information).
Figure4.MultipleUV-visspectraofaqueoussolutionof5ameasured Antiproliferative Activity. The osmium(III) complexes
over72hwith1htimeinterval. 5a,7a,and9aaswellastheirruthenium(III)analogues5b,
7b, and 9b were studied for their antiproliferative activity
display a reversible one-electron reduction wave with
inthreehumancancercelllinesbymeansoftheMTTassay.
potential values ranging from -1.21 to -1.17 V (in
IC values are listed in Table 3, and concentration-effect
phosphate buffer) or -0.4 to 0.1 V (in CH CN), which is 50
3 curves are depicted in Figure 5. All compounds have
assigned to the OsIII f OsII process, and a one-electron
noteworthy antiproliferative effects, with IC values in the
oxidation wave with potential values ranging from 0.31 to 50
10-6to10-5Mconcentrationrange.Thecisplatin-sensitive
0.36V(inphosphatebuffer)or1.10to1.55V(inCH CN),
3 ovariancarcinomacelllineCH1tendstobesomewhatmore
which is attributed to the OsIII f OsIV process (Table 2).
sensitivetothesecomplexesthantheintrinsicallycisplatin-
The redox potentials of the complexes are in the following
resistant cell lines A549 (non-small cell lung cancer) and
rank order: E (7a) g E (9a) (in phosphate buffer) and
1/2 1/2 SW480 (colon cancer), with the notable exception of the
E (6a) > E (5a) > E (9a) (in CH CN), which agrees
1/2 1/2 1/2 3 rutheniumcomplexes7band9b.Theactivitypatternofthese
with the relative electron-donor character of the N-ligands
two compounds is all the more remarkable, as CH1 cells
(E (Hind)>E (Hpz)>E (Hbzim)>E (Him)).40Theredox
L L L L aregenerallychemosensitivetoabroadvarietyofanticancer
responses are characterized by a peak-to-peak separation
agents.
(∆E )of60-70mVandananodicpeakcurrent(i )thatis
p pa Variationofthecentralmetalyieldsnouniformpicture:
almost equal to the cathodic peak current (i ), as expected
pc osmium is at least slightly more favorable in the case of
for reversible electron-transfer processes. The one-electron
pyrazole complexes, whereas ruthenium results in a
natureofelectron-transferprocesseswasverifiedbycompar-
markedly higher antiproliferative activity in the case of
ingthepeakcurrentheight(i p )withthatofstandardmethyl imidazolecomplexes.Structure-activityrelationshipswith
viologen or ferrocene/ferrocenium couples under identical
regard to variation of the azole ligand are not uniform
experimental conditions.
either.Inthecisplatin-resistantcelllines(A549,SW480),
Resistance to Hydrolysis and Reactivity Toward 9- activity of the osmium complexes decreases in the
Methyladenine in Aqueous Media. The aqueous solution followingrankorder:5a(Hpz)g9a(Hbzim)>7a(Him),
behavior of 5a, 7a, and 9awith respect to hydrolysis was whereas their ruthenium congeners rank in the reverse
studied at 298 and 310 K over 24-72 h by UV-vis order. In CH1 cells, the rank order also differs between
spectroscopy and in addition for 5a by 1H NMR spec- osmiumandrutheniumcomplexes,butimidazoleligands
troscopy. The complexes are quite stable in aqueous are unfavorable in any case. All three ruthenium com-
solution as can be seen from their electronic absorption plexes (5b, 7b, 9b) exert lower antiproliferative effects
spectra (Figure 4, for 5a). Immediate hydrolysis can be thantheindazoleanalogue6b(IC :0.67and0.69µMin
50
excluded because of the presence of the parent peak CH1 and SW480 cells, respectively).17
Inorganic Chemistry, Vol. 47, No. 16, 2008 7345
Stepanenko et al.
Figure5.Concentration-effectcurvesofosmiumcomplexes5a,7a,and9a(leftpanels)andrutheniumcomplexes5b,7b,and9b(rightpanels)inA549
(A,B),CH1(C,D)andSW480(E,F)cells,obtainedbytheMTTassay(96hexposure).Valuesaremeans(standarddeviationsofatleastthreeindependent
experiments.
A fairly well correlation of reduction potentials and permittedtheelucidationofessentialdifferencesbetween
cytotoxicpotenciesobservedwithinahomologousseriesof related osmium and ruthenium complexes. Whereas their
rutheniumcomplexescontainingvaryingnumbersofindazole trans isomers were observed for all azole heterocycles,
andchloridoligands17hadsubstantiatedtheconceptoffine- the formation of cis isomers was discovered only for the
tuning biological activity of ruthenium complexes by osmium complex with the strongest net electron-donor
adjusting their ligand composition to achieve the desired imidazole ligand. We found that osmium complexes,
redox properties and thereby stimulated the preparation which are markedly more inert than related ruthenium
of new analogues with different azole ligands in varying compounds toward substitution reactions (hydrolysis,
numbers.19 Because a number of four indazole ligands interaction with DNA bases), in accordance with their
hadresultedinastrongantiproliferativeactivityincancer positionintheperiodictable,partiallyshowacomparable
cells in vitro, we have focused here on complexes with antiproliferative activity.
four azole ligands. In conformity with expectations, the [OsIIICl (Hpz) ]Clcomplexshowedahigheractivitythan
2 4
lesseasilyreduciblecomplexeswithpyrazole,imidazole, the ruthenium(III) counterpart in three cell lines, whereas
and benzimidazole show lower antiproliferative activity the osmium(III) imidazole complex is less cytotoxic
than that reported previously for their indazole analogue. thantherutheniumanalogueinanycase.Thelackofaclear-
Although activities in the cisplatin-sensitive ovarian cutcorrelationbetweenantiproliferativeactivityandreduc-
carcinoma cell line CH1 approximate a correlation with tionpotentialswithinthetwoseriesof[OsIIICl (Hazole) ]Cl
2 4
reductionpotentials,anyattempttofindfurtherconclusive and[RuIIICl(Hazole)]Clcomplexeswithvariedazoleligands
2 4
relationships is complicated by the fact that activities indicates that the reduction potential cannot be the main
strongly depend on the cell line, however. The latter also determinantfactorforbiologicalactivityandthattheinfluence
appliestotheosmiumcompounds,indicatingthatvariation ofadditionalparameters(e.g.,cellularuptake,interactionswith
of the azole ligand has less predictable consequences for molecular targets) is of great importance. Although synthetic
biological activity and that the reduction potential is not limitationsimpededtheassayofantiproliferativeactivityinvitro
the only important parameter. of5aandcis-isomer8,itisworthreportingtheavailabledata,
because this may inspire novel synthetic approaches to these
Conclusions complexes,allowingforlargerscalesynthesisandthenevalu-
ationoftheircytotoxicity.
A[OsIIICl (Hazole) ]Clfamilyofcomplexeswasprepared
2 4
by reactions of (H azole) [OsCl ] with the corresponding
2 2 6
azole heterocycle. The full characterization of [OsCl - Acknowledgment. We thank Dr. S. Shova for helpful
2
(Hazole) ]Cl, both in the solid state and in solution, discussion.
4
7346 InorganicChemistry,Vol.47,No.16,2008
Osmium(III)ComplexeswithAzoleHeterocycles
SupportingInformationAvailable:Hydrogenbondsandtheir X-ray crystallographic files in CIF format for 5a-7a, 8, and 9a.
geometrical parameters in 5a, 6a, 9a, superposition of crystallo- This material is available free of charge via the Internet at
graphicallyindependenthalvesof7aand7b,N-H···Clhydrogen
http://pubs.acs.org.
bondinginteractionsin7a,1HNMRspectraof5awith9-methy-
ladenine,assignmentofUV-visspectraof1-4,descriptionofIR
spectraof1-4,5a,7a,9a,andcomparisonofthosewithfreeazoles, IC8006958
Inorganic Chemistry, Vol. 47, No. 16, 2008 7347