Osmium(III) analogues of KP1019: electrochemical and chemical synthesis, spectroscopic characterization, X-ray crystallography, hydrolytic stability, and antiproliferative activity.

Article pubs.acs.org/IC Osmium(III) Analogues of KP1019: Electrochemical and Chemical ‑ Synthesis, Spectroscopic Characterization, X ray Crystallography, Hydrolytic Stability, and Antiproliferative Activity Paul-Steffen Kuhn,† Gabriel E. Büchel,†,‡ Katarina K. Jovanovic,́§ Lana Filipovic,́§ Sinisǎ Radulovic,́§ Peter Rapta,⊥ and Vladimir B. Arion*,† † Faculty of Chemistry, Institute of Inorganic Chemistry, University of Vienna, Waḧ ringer Strasse 42, A-1090 Vienna, Austria ‡ Division for Physical Sciences & Engineering and KAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia § Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia ⊥ Department of Physical Chemistry, Slovak University of Technology, Radlinskeh́ o 9, SK-81237 Bratislava, Slovak Republic * S Supporting Information ABSTRACT: A one-electron reduction of osmium(IV) complexes trans-[OsIVCl (Hazole) ], where Hazole = 1H- 4 2 pyrazole ([1]0), 2H-indazole ([2]0), 1H-imidazole ([3]0),and 1H-benzimidazole ([4]0), afforded a series of eight new complexes as osmium analogues of KP1019, a lead anticancer drug in clinical trials, with the general formula (cation)[trans- OsIIICl (Hazole) ], where cation = H pz+ (H pz[1]), H ind+ 4 2 2 2 2 (H ind[2]), H im+ (H im[3]), Ph P+ (Ph P[3]), nBu N+ 2 2 2 4 4 4 (nBu N[3]), H bzim+ (H bzim[4]), Ph P+ (Ph P[4]), and 4 2 2 4 4 nBu N+ (nBu N[4]). All complexes were characterized by 4 4 elementalanalysis,1HNMRspectroscopy,electrosprayionizationmassspectrometry,UV−visspectroscopy,cyclicvoltammetry, whileH pz[1],H ind[2],andnBu [3],inaddition,byX-raydiffraction.Thereducedspecies[1] − and[4] − arestableinaqueous 2 2 4 mediaintheabsenceofairoxygenanddonotreactwithsmallbiomoleculessuchasaminoacidsandthenucleotide5′-dGMP. Cell culture experiments in five different human cancer cell lines (HeLa, A549, FemX, MDA-MB-453, and LS-174) and one noncancerous cell line (MRC-5) were performed, and the results were discussed and compared to those for KP1019 and cisplatin. Benzannulation in complexes with similar structure enhances antitumor activity by several orders of magnitude, implicatingdifferentmechanismsofactionofthetestedcompounds.Inparticular,complexesH ind[2]andH bzim[4]exhibited 2 2 significant antiproliferative activity in vitro when compared to H pz[1] and H im[3]. 2 2 ■ INTRODUCTION Chart 1. Ruthenium Complexes in Clinical Trials Over the past decades the field of ruthenium- and osmium- based antitumor agents has attracted continuous interest.1,2 The synthesis of new organometallic3−8 and coordination compounds9−12 as well as their spectroscopic properties and antiproliferative activityinvitroandinvivohavebeenreported. However,onlytwopotentialrutheniumanticanceragentsarein clinicaltrials,namely,[RuIIICl (Hind) ] − (NKP-1339/KP1019, 4 2 Hind=1H-indazole)13,14and(H im)[RuIIICl (DMSO)(Him)] 2 4 (NAMI-A, Him = 1H-imidazole, DMSO = dimethyl sulf- oxide)15 (Chart 1). The first one is active against a variety of advanced and metastatic solid malignancies, most promising in complexes was discovered in the 1970s,21 the synthesis, nonsmall cell lung cancer and gastrointestinal neuroendocrine spectroscopic,andfirstbiologicalinvestigationsoftheosmium- tumors,16 whereas the second one mainly acts as an (IV) analogue of KP101918 as well as the Os-NAMI-A-type antimetastatic drug with a current research focus on lung complexes19 have been reported quite recently. cancer.17 [OsIVCl 4 (Hazole) 2 ] showed comparable cytotoxicity to their Attempts to prepare related osmium complexes have also been undertaken, and these are well-documented in the Received: July17, 2014 literature.18−20 While potential antitumor activity of osmium Published: October 7, 2014 ©2014AmericanChemicalSociety 11130 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article ruthenium(III) congeners and a higher stability in aqueous drug’s bioavailability and corroborates the antiproliferative solution. However, the solubility of these neutral complexes in activityassaydatainvitro.28Inaddition,itwasintriguingtofind 1%DMSO/waterwasborderlinefortheinvitrotestingoftheir out whether the coordination mode of indazole remains intact antiproliferative activity.11,18 Furthermore, the first osmium- upon one-electron reduction. (VI)−nitrido complexes with azole heterocycles have been Hereinwereporttheelectrochemicalandchemicalsynthesis, found to possess potential antitumor properties.9 spectroscopic characterization, and X-ray diffraction studies of Although ruthenium and osmium belong to the same group complexes of the general formula (cation)[OsIIICl (Hazole) ], 4 2 of the periodic table, there are distinct differences between the where cation = azolium, tetrabutylammonium, or tetraphenyl- twometalsandtheircoordinationchemistry,suchastheability phosphonium, while Hazole = 1H-pyrazole, 2H-indazole, 1H- ofosmiumtostabilizehigheroxidationstates,themagnitudeof imidazole, and 1H-benzimidazole (Chart 3). The antiprolifer- spin−orbit coupling, the rate of metal−ligand exchange ative activity of complexes H pz[1], H ind[2], H im[3], and 2 2 2 reactions, and their behavior toward hydrolysis.20,22 H bzim[4] was tested in five different human cancer cell lines 2 Indazole is usually referred to as 1H-indazole, which is the (HeLa, A549, FemX, MDA-MB-453, and LS-174) and one predominant form in the gaseous phase and aqueous solution. nontumorigeniccellline(MRC-5),andtheobtaineddatawere Two other tautomers, 2H-indazole and 3H-indazole, are also c■ompared to those of KP1019 and cisplatin. well-documented(Chart2).23,24Therehasbeensomeevidence EXPERIMENTAL SECTION Chart 2. Tautomeric Forms of Indazole Materials. Solvents were obtained from commercial sources and usedasreceived.OsO (99.8%)waspurchasedfromJohnsonMatthey, 4 while1H-pyrazole,1H-indazole,1H-imidazole,and1H-benzimidazole as well as NaBH, nBuNBH, and nBuNCl from Sigma-Aldrich. All 4 4 4 4 chemicals were used without further purification. The starting compounds [OsIVCl(Hazole)] (Hazole = 1H-pyrazole ([1]0), 2H- 4 2 indazole ([2]0), 1H-imidazole ([3]0), and 1H-benzimidazole ([4]0)) were synthesized as previously reported in the literature.18 thattheindazoletautomeridentityhasanimpactonbiological (H 2 pz)[OsIIICl 4 (Hpz) 2 ] (H 2 pz[1]). NaBH 4 (10.5 mg, 0.27 mmol) properties.25−27 Recent investigations on the antiproliferative was added to a suspension of [OsIVCl 4 (Hpz) 2 ] ([1]0) (118 mg, 0.25 activity of [OsIVCl (Hind)] − , where Hind is 1H- or 2H- mmol)inmethanol(2mL).Theclearsolutionwasstirredfor30min indazole, showed diff 5 erent results in vitro and in vivo. The 2H- a H n C d l fi ( l 0 te .5 re m d. L T ) h w en as a ad so d l e u d t . io T n h o e f r p e y su ra lt z i o n l g e s ( o 1 l 8 id m w g a , s 0 fi .2 lt 6 er m ed m o o ff l) ,w in as 6 he M d tautomer was found to be superior in the three human cancer withwater anddiethyl ether,anddriedinvacuo.Singlecrystals ofX- celllinesCH1(ovariancarcinoma),SW480(coloncarcinoma), ray diffractionquality were collected directlyfromthemother liquor. andA549(nonsmall cell lungcancer), butinferior ina Hep3B Yield: 108 mg, 80%. Anal. Calcd for CH ClNOs (M = 537.28 g· SCID mouse xenotransplantation model.11 mol−1): C, 20.11; H, 2.44; N, 15.65. F 9 ou 1 n 3 d: 4 C, 6 20.29; H, 2.42; N, These results encouraged us to further investigate the 15.27%. Electrospray ionization mass spectrometry (ESI-MS) in influenceoftheindazoletautomeridentityonphysicochemical MeOH (negative): m/z 467.9 [OsIIICl 4 (Hpz) 2 ]−, 331.8 [OsIIICl 4 ]−; a [O nd sIV b C io l l ( o H gi a c z a o l le p ) ro ] p c e o r m tie p s ou o n f ds th w e e ir re o r s e m ga i r u d m ed c b o y m u p s le a x s e s s u . it T ab h l e e E in S fr I a -M red S ( i M n IR M ), eO ν, H cm ( − p 1: os 5 i 7 ti 1 v , e) 6 : 63 m ,7 /z 71, 4 9 3 0 2 3 .9 ,1 [ 0 O 46 sC ,1 l 3 1 ( 1 H 3 p , z 1 ) 1 2 6 ] 6 +. ,1 M 26 id 4 - , 4 2 1346,1399,1473,1511,3121,and3325.UV−vis(DMSO),λ ,nm precursors for the synthesis of osmium(III) analogues of (ε,M−1cm−1):256(7823),275(5775),299(5792),391sh(22 m 5 ax ).1H KP1019 by electrochemical and chemical reduction. These NMR (DO, 500.10 MHz): δ−22.33 (br.s, 2H), −7.82(br. s, 2H), reduced species were expected to be more soluble in aqueous −0.60(br 2 . s, 2H), 6.60(s, 1H, Hpz), 7.96(s, 2H, Hpz) ppm. media than the parent osmium(IV) compounds. The aqueous (H ind)[OsIIICl (Hind) ] (H ind[2]). NaBH (9.5 mg, 0.25 mmol) 2 4 2 2 4 solubility is an important property that has an impact on a wasaddedtoasuspensionof[OsIVCl(Hind)]([2]0)(112mg,0.20 4 2 a Chart 3. Starting Compounds and Final Products Studied in This Work aUnderlined complexes were investigated by X-ray crystallography. 11131 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article mmol)inmethanol(3mL).Theclearsolutionwasstirredfor30min 2H),4.39(br.s,2H),5.74(br.s,2H),7.55−7.60(m,2H),7.83−7.88 andfiltered.Thenasolutionof1H-indazole(24mg,0.21mmol)in6 (m, 2H), 9.40(s, 1H), 14.57 (br.s, 1H) ppm. MHCl(1mL)wasadded.Theresultingsolidwasfilteredoff,washed (Ph P)[OsIIICl (Hbzim) ] (Ph P[4]). NaBH (8 mg, 0.21 mmol) 4 4 2 4 4 withwateranddiethylether,anddriedinvacuo.SinglecrystalsofX- wasaddedtoasuspensionof[OsIVCl(Hbzim)]([4]0)(104mg,0.18 4 2 raydiffractionqualitywerecollectedfromthemotherliquorafterslow mmol)inwater(4mL).Theclearsolutionwasstirredfor30minand evaporation of the solvent. Yield: 73 mg, 54%. Anal. Calcd for filtered.Thentetraphenylphosphoniumchloride(72mg,0.19mmol) C H ClNOs·MeOH (M = 719.50 g/mol): C, 36.72; H, 3.22; N, wasaddedtothesolution.Theresultingsolidwasfilteredoff,washed 21 19 4 6 11.68. Found: C, 36.40; H, 3.52; N, 12.03%. ESI-MS in MeOH withwateranddiethylether,anddriedinvacuo.Yield:113mg,67%. (negative): m/z 567.9 [OsIIICl 4 (Hind) 2 ]−, 332.5 [OsIIICl 4 ]−. ESI-MS Anal.CalcdforC 38 H 32 Cl 4 N 4 OsP·H 2 O(M=925.72g/mol):C,49.30; in MeOH (positive): m/z 498.0 [OsIIICl(Hind)]+, 118.8 [Hind]+. H,3.70;N,6.05;O,1.73.Found:C,48.64;H,3.52;N,5.73;O,1.94%. 1 ( M ε 3 , I 8 R M 0 , , − ν 1 1 , 4 c c 4 m m 6, − − 1 1 1 : ) 4 : 6 7 2 0 8 5 4 , 9 , 1 6 5 ( 7 1 1 2 5 7 , , 8 7 2 1 3 9 6 8 ) 2 , , 9 3 8 , 6 0 a 8 9 n , d ( 8 1 3 9 2 3 7 5 3 , 1 0 1 6 . 0 ) 2 U 0 , 0 3 V , 9 − 1 6 0 v s 8 i h s 1 2 ( , ( D 9 1 3 1 M 7 3 9 8 S ) , O , 1 ) 6 1 , 1 7 λ 5 3 m , 2 ( a 1 x 9 , 2 6 n 3 1 m 5 ) , . E c [O m SI s − - I 1 M II : C 6 S l 1 4 i ] 4 n − , . 6 M E 8 S e 7 O I , - 7 M H 2 S 0 ( , n i 7 n e 4 g M 7 a , t e i 8 v O 2 e 8 H ) , : 9 ( m 9 p / 9 o z , si 1 t 5 1 i 6 v 0 e 7 6 ) .9 , : 1 m [ 2 O / 4 z s 7 I , I 3 I 1 C 3 4 9 l 4 3 . ( 1 6 H , [ 1 P b 4 z h i 8 4 m P 4, ) ] 2 + 1 ] . 5 − M 8 , 9 3 I , R 3 a , 1 n . ν d 8 , 1 2 H H) N ,1 M .3 R 9 ( ( D br 2 . O s, , 2 5 H 00 ) . , 1 7 0 .1 M 2 H (t z , ) 1 : H δ ,J − = 11 7 .0 H 2 z ( ) b , r 7 . .3 s, 5 2 ( H t, ) 1 , H − , 0 J .2 = 3 7 (b H r. z) s , , ( 3 6 1 3 9 3 7 6 . ) U , V 36 − 1 vi s s h (D (2 M 49 S 6 O ), ), 42 λ m 2 ax ( , 6 n 0 m 8), (ε 6 , 07 M ( − 5 1 2 c 5 m ). − 1 1 H ): 2 N 6 M 1 R (1 ( 3 D 7 M 94 S ) O , 3 -d 0 6 6 , 7.55(d,1H,J=8Hz),7.77(d,1H,J=8Hz),8.08(s,1H),13.00(br. 500.10 MHz): δ −17.77 (br. s, 2H), −5.18 (br. s, 2H), 2.04 (br. s, 2H),4.24(br.s,2H),4.40(br.s,2H),5.76(br.s,2H),7.68−8.02(m, s, 1H)ppm. add (H ed 2 im to )[O a s s II u IC sp l 4 e ( n H s i i m on ) 2 ] o ( f H [ 2 O im sI [ V 3 C ]) l . ( N H a i B m H ) 4 ] (1 ( 1 [3 m ]0 g ) ,0 ( . 1 3 2 0 8 m m m g o , l) 0 w .2 a 7 s 20H (n , B P u h 4 4 N P ) ) [O p s p I m IIC . l 4 (Hbzim) 2 ] (nBu 4 N[4]). nBu 4 NBH 4 (26 mg, 0.10 4 2 mmol) was added in two portions to a suspension of [Os- mmol)inmethanol(3mL).Theclearsolutionwasstirredfor30min andfiltered.Thenasolutionofimidazole(19mg,0.29mmol)in6M Cl 4 (Hbzim) 2 ] ([4]0) (30 mg, 0.05 mmol) in water (3 mL). After 5 HCl(1mL)wasadded.Theresultingsolidwascollectedbyfiltration, mintheresultingpalegreensolidwasfilteredoff,washedwithwater, methanol, and diethyl ether, and dried in vacuo. Yield: 32 mg, 77%. washed with water, methanol, and diethyl ether and dried in vacuo. Yield: 135 mg, 90%. Anal. Calcd for CH ClNOs·0.5MeOH (M = Anal.CalcdforC 30 H 48 Cl 4 N 5 Os·H 2 O(M=828.24g/mol):C,43.47; 9 13 4 6 H,6.08;N,8.45;O,1.91.Found:C,43.25;H,6.00;N,8.23;O,1.61%. 553.30g/mol):C,20.62;H,2.73;N,15.19.Found:C,20.40;H,2.45; ESI-MS in MeOH (negative): m/z 567.9 [OsIIICl(Hbzim)]−, 331.8 [ m N 1 O 0 / , 9 s z 2 II 1 , I 6 C 4 9 1 l . . 4 8 1 8 ( 3 H 6 [ 5 % H i , m . 2 ) 1 im 2 1 ] E 7 ] − S 3 + , . , I 3 - M 3 1 M 2 3 IR . 2 S 2 3 , , [ ν i O n , 1 s 4 c I m 3 II M C 7 − , l 1 e 4 : ] O 1 − 6 5 H . 1 3 6 E 7 , , S ( I 6 1 - n 5 M 5 9 e 7 , S g 8 a 7 , i t 4 n i 8 a v n , M e d 7 ) e 9 : O 3 9 1 , H m 3 1 5 / 0 ( . z 4 p 5 o U , s 4 V i 1 t 6 i − 0 v 7 6 e v . 2 ) i 9 s : , 2 c [O m 96 s − 4 I 1 I , I : C a 6 l n 2 4 d ] 1 − , 3 . 7 1 E 4 4 S 8 6 I , - . M 8 U 1 S V 7 − , in 8 v 8 M is 4 e , ( O D 10 H M 07 S ( , O p 1 o ) 1 s , i 5 t λ i 1 m v , e ax 1 ) , : 2 n 5 m m 2 / , z ( 1 ε 2 3 , 4 0 M 2 9 . 4 , 3 − 1 1 [ 4 B c 3 m u 8 4 , − N 1 1 ) ] 4 : 2 + 8 . 2 6 M 6 , 0 1 IR 6 ( 2 1 , 3 ν 1 , , 2 ( ( 1 D H 2 M ) 2 , 5 S − ) O , 1 ) 3 4 , 7 .0 λ 8 7 m ( a ( x 3 b , 0 r n 4 . m ) s . , ( 2 1H ε H , ) M N ,2 M − . 1 8 R 5 cm ( ( D b − r 1 2 . ) O : s, , 2 2 5 6 H 0 3 0 ) ( . , 1 7 7 0 0 . 9 4 M 7 1 ) H ( , s z 2 , ) 9 2 : 3 H δ ( ) − 6 , 3 8 1 1 . 9 5 6 . 9 4 ), 3 ( 3 s ( 2 , b 6 1 r H . s s h ) , 1 0 ( − 4 . 3 5 5 5 6 5 . 5 2 ) − ) , 3 . 1 3 ( . 1 0 6 b H 6 5 r. ( s ( N m , 6 M 2 2 , H 9 8 R 9 H ) ) , , ( ) 0 D , 3 .9 2 M 3 5 . 2 0 S 2 ( O s t h , ( - b 1 d r ( 2 6 . 3 , H s 8 5 , , 2 0 2 J 7 0 H ) = . , 1 ) 0 7 , 3 3 6 H M . 0 1 z 4 H ) s − , h z 1 ) 3 : . ( . 3 2 2 4 δ 2 41 ( ( − 3 s m e ) 1 x , , 7 t 8 . , 4 8 H 8 3 5 H 0 ), ( , ( b 4 J 4 r .2 . 3 = 2 8 s 7 ) , ( , b 2 H 6 r H . z 0 ) ) s 8 , , , ppm. 2H), 4.39 (br.s, 2H), 5.74 (br.s, 2H) ppm. (Ph P)[OsIIICl (Him) ](Ph P[3]).NaBH (9.6mg,0.25mmol)was 4 4 2 4 4 Physical Measurements. Elemental analyses were performed by added to a suspension of [OsIVCl(Him)] ([3]0) (100 mg, 0.21 4 2 the Microanalytical Service of the Faculty of Chemistry of the mmol)inmethanol(4mL),andthereactionmixturewasstirreduntil University of Vienna and were carried out with a PerkinElmer 2400 thesolutionbecameclear.Ph 4 PCl(84mg,0.22mmol)wasadded,and CHN Elemental Analyzer. 1H NMR (500.10 MHz) spectra were the precipitated solid was collected by filtration, washed with recorded on a Bruker Avance III instrument at 25 °C. ESI-MS methanol, and dried in vacuo. Yield: 97.7 mg, 56%. Anal. Calcd for measurements were carried out on a Bruker AmaZon SL ion trap C H ClNOsP·HO (M = 825.60 g/mol): C, 43.64; H, 3.66; N, 30 28 4 4 2 spectrometer (Bruker Daltonics GmbH). The stability of the 6.79. Found: C, 43.52; H, 3.75; N, 6.61%. ESI-MS in MeOH complexes in aqueous media at 25 °C was determined by UV−vis (negative):m/z467.9[OsIIICl(Him)]−,331.8[OsIIICl]−.ESI-MSin 4 2 4 spectroscopyonaPerkinElmerLambda650spectrometerinanoptical MeOH (positive): m/z 339.1 [Ph 4 P]+. MIR, ν, cm−1: 613, 663, 690, cell of 1 cm path length in the wavelength range of 250−800 nm in 754, 816, 996, 1059, 1109, 1325, 1440, 1484, and 3192. UV−vis combination with a PerkinElmer PTP-6 Peltier System. (DMSO), λ max , nm (ε, M−1 cm−1): 270 (9234), 292 (5709), 331 sh Electrochemistry. Cyclic voltammograms (CVs) were measured (1011),379(299).1HNMR(DMSO-d,500.10MHz):δ−20.37(br. 6 in a three-electrode cell using a 2 mm diameter glassy carbon disk s,2H),−18.66(br.s,2H),0.48(br.s,2H),7.70−8.05(m,20H,Ph 4 P) working electrode, a platinum auxiliary electrode, and a Ag|Ag+ ppm. reference electrode containing 0.1 M AgNO. Measurements were (nBu 4 N)[OsIIICl 4 (Him) 2 ] (nBu 4 N[3]). nBu 4 NBH 4 (16 mg, 0.06 performedatroomtemperatureusinganEG& 3 GPARCpotentiostat/ mmol)wasaddedintwoportionstoasuspensionof[OsCl 4 (Him) 2 ] galvanostat 273A. Deaeration of solutions was accomplished by ([4]0) (15 mg, 0.03 mmol) in methanol (2 mL). After 5 min the purgingastreamofargonthroughthesolutionfor5minpriortoeach slightly turbid solution was filtered. The solution generated single experiment. The potentials were measured in a freshly prepared crystalsofX-raydiffractionqualityuponstandingatroomtemperature solutionof0.1M(nBuN)[BF]inDMSOusing[Fe(η5-CH)](E 4 4 5 5 2 1/2 for 48 h. =0.68VvsNHE)29asinternalstandardandarequotedrelativetothe (H 2 bzim)[OsIIICl 4 (Hbzim) 2 ] (H 2 bzim[4]). NaBH 4 (8 mg, 0.21 normal hydrogen electrode (NHE). mmol)wasaddedtoasuspensionof[OsIVCl 4 (Hbzim) 2 ]([4]0)(108 Theinsituspectroelectrochemicalelectronparamagneticresonance mg,0.19mmol)inmethanol(3mL).Theclearsolutionwasstirredfor (EPR)/UV−vis−NIR(NIR=near-infrared)experimentswerecarried 30 min and filtered. Then a solution of benzimidazole (24 mg, 0.20 out under argon atmosphere in a flat spectroelectrochemical cell, mmol)in6MHCl(1mL)wasadded.Theresultingsolidwasfiltered suitable for an optical transmission EPR resonator (ER 4104 OR-C off,washedwithwater,methanol,anddiethylether,anddriedinvacuo. 9609) of an EMX EPR spectrometer. The working electrode was a Yield: 67 mg, 50%. Anal. Calcd for C 21 H 19 Cl 4 N 6 Os·0.5MeOH (M = laminated Pt mesh with a small hole in the foil coincident with the 703.48g/mol):C,36.70;H,3.01;N,11.95.Found:C,36.74;H,2.73; lightbeam,whichlimitedtheactivesurfaceareaoftheelectrode.APt- N, 11.60%. ESI-MS in MeOH (negative): m/z 568 wirecounterelectrodeandaAgwirepseudoreferenceelectrodewere [OsIIICl(Hbzim)]−, 332 [OsIIICl]−. ESI-MS in MeOH (positive): used. The optical EPR resonator cavity was connected to the diode- 4 2 4 m/z119[Hbzim]+.MIR,ν,cm−1:591,730,1012,1108,1244,1305, array UV−vis−NIR spectrophotometer Avantes Avaspec (Avantes, 2 1371,1409,1444,1493,and3295.UV−vis(DMSO),λ ,nm(ε,M−1 Netherlands) byoptical fibers. A deuterium−halogen lamp DH 2000 max cm−1):259(14791),299(7130),306(7309),331sh(4582),361sh (Sentronic,Germany)wasusedasalightsource.UV−visspectrawere (2951),423(688),498(261).1HNMR(DMSO-d,500.10MHz):δ processed by the AvaSoft 7.7 software package. Both EPR and UV− 6 −17.85 (br. s, 2H), −5.23 (br. s, 2H), 2.02 (br. s, 2H), 4.22 (br. s, vis−NIR spectrometers were synchronized together by trigger pulses 11132 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article Table 1. Crystal Data and Details of Data Collection for H pz[1], H ind[2], and nBu N[3] 2 2 4 compound Hpz[1] Hind[2] nBuN[3] 2 2 4 empiricalformula CH ClNOs C H ClNOs C H ClNOs 9 13 4 6 21 19 4 6 22 44 4 5 Fw 537.25 687.42 710.62 spacegroup P2/c P1̅ Pbcn 1 α,Å 7.4126(4) 9.4631(3) 14.7786(5) b,Å 12.9016(6) 11.0678(4) 8.6629(3) c,Å 16.1850(8) 13.6308(5) 22.4948(8) α,deg 106.881(1) β,deg 94.636(2) 102.959(1) γ,deg 96.242(1) V,Å3 1542.78(13) 1307.98(8) 2879.91(17) Z 4 2 4 λ,Å 0.71073 0.71073 0.71073 ρ ,gcm−3 2.313 1.745 1.639 calcd crystalsize,mm 0.29×0.17×0.13 0.12×0.10×0.02 0.16×0.13×0.04 T,K 100(2) 100(2) 100(2) μ,mm−1 8.956 5.303 4.818 Ra 0.0132 0.0172 0.0219 1 wRb 0.0306 0.0410 0.0481 2 GOFc 1.041 1.010 1.066 aR =Σ∥F|−|F∥/Σ|F|.bwR ={Σ[w(F2−F2)2]/Σ[w(F2)2]}1/2.cGOF={Σ[w(F2−F2)2]/(n−p)}1/2,wherenisthenumberofreflections 1 0 c 0 2 0 c 0 0 c and pis the total numberof parameters refined. received from the potentiostat. A Heka PG310USB (Lambrecht, preparedinsteriledeionizedwater,supplementedwithpenicillin(192 Germany) potentiostat with a PotMaster 2.73 software package was U/mL),streptomycin(200mg/mL),4-(2-hydroxyethyl)piperazine-1- used for the coulometric studies, and a Heka PG285 potentiostat ethanesulfonic acid (HEPES) (25 mM), L-glutamine (3 mM), and (Lambrecht, Germany) with the same software equipment was used 10% of heat-inactivated fetal calf serum (FCS) (pH 7.2). The cells for the spectroelectrochemical studies. Sample solutions with were grownat 37 °C in5% CO in a humidified airatmosphere. 2 approximate concentration of 0.5 mM, prepared with 0.2 M MTT Assay. Antiproliferative activity of tested osmium(III) (nBuN)[PF] supporting electrolyte in DMSO, were purged with complexes, KP1019, and cisplatin was determined using 3-(4,5- 4 6 argon for 5 min prior to each experiment. A silver wire dymethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma- pseudoreference electrode was calibrated against an Fc/Fc+ redox Aldrich) assay.34 Cells were seeded into 96-well cell culture plates couple. (ThermoScientificNunc)atacelldensityof4000c/w(HeLa),6000 Crystallographic Structure Determination. X-ray diffraction c/w(A549),4000c/w(MDA-MD-453),5000c/w(FemX,MRC-5), measurements were performed on a Bruker D8 VENTURE CCD and 7000c/w (LS-174),in100μLof culturemedium.After24 hof diffractometer. Single crystals were positioned at 35, 40, and 50 mm growth, cells were exposed to the serial dilutions of the tested fromthedetector,and731,960,and1735framesweremeasured,each complexes, KP1019, and cisplatin. The osmium(III) complexes and for 2.5, 30, and 10 s over 0.5, 0.25 and 0.5° scan width for KP1019 were dissolved in DMSO at a concentration of 30 mM as (H pz)[OsIIICl (Hpz) ] (H pz[1]), (H ind)[OsIIICl (Hind) ] stock solution, and prior to use they were diluted with nutrient 2 4 2 2 2 4 2 (Hind[2]), and nBuN[OsIIICl(Him)] (nBuN[3]), respectively. mediumtothedesiredfinalconcentrations(inrangeupto300μM). 2 4 4 2 4 The data were processed using SAINT software.30 Crystal data, data Hind[2]wasdissolvedinDMSOunderanargonatmospheretoavoid 2 collection parameters, and structure refinement details are given in directcontactofthesubstancewithoxygen.Cisplatin(CDDP)stock Table1.Thestructuresweresolvedbydirectmethodsandrefinedby solution was madein 0.9%NaCl ata concentration of1.66mM and full-matrixleast-squarestechniques.Non-hydrogenatomswererefined afterward was diluted with nutrient medium to the desired final with anisotropic displacement parameters. H atoms were inserted in concentrations (in range up to 100 μM). Each concentration was calculated positions and refined with a riding model. The following tested in triplicate. Serial dilutions were made in culture medium so computer programs and hardware were used: structure solution, thatthefinalconcentrationofDMSOperwellwaslessthan1%(v/v) SHELXS-97, and refinement, SHELXL-97;31 molecular diagrams, and in all experiments. After incubation periods of 48 h, 20 μL of MTT ORTEP.32ThecrystalstructureofHind[2]containedsolvent,which solution (5 mg/mL in phosphate buffer, pH 7.2) was added to each 2 couldnotbelocalized.Therefore, SQUEEZEoption implementedin well. Samples were incubated for 4 h at 37 °C, with 5% CO in a 2 PLATON33 was applied in the finalrefinementstep. humidifiedatmosphere.Formazancrystalsweredissolvedin100μLof Interaction with Small Biomolecules. The reactivity of 10%sodiumdodecyl sulfate (SDS). Absorbances wererecorded after complexes toward different amino acids (L-histidine, L-methionine, L- 24 h, on an enzyme-linked immunosorbent assay (ELISA) reader cysteine) was investigated. In particular, Hpz[1] (2−3 mg/mL) and (ThermoLabsystemsMultiskanEX200−240V),atthewavelengthof 2 Hbzim[4] (1 mg/mL) were dissolved in DO and treated with an 570 nm. IC values (μM) were determined from the cell survival 2 2 50 equimolar amount of the respective amino acid, and the 1H NMR diagrams. The percentages of surviving cells relative to untreated spectrawererecordedover14−16hatroomtemperature.TheirDNA controls were determined. The IC value, defined as the 50 bindingabilitywasexamined by1HNMRspectroscopy bytreatment concentration of the compound causing 50% cell growth inhibition, with 2equiv of 5′-dGMP solution in DO. w■as estimated fromthe dose−response curves. 2 Cell Lines and Culture Conditions. Human cervical carcinoma (HeLa),humanmelanoma(FemX),humanalveolarbasaladenocarci- RESULTS AND DISCUSSION noma (A549), human colorectal adenocarcinoma (LS-174), human Synthesis and Characterization of the Complexes. breastcancer(MDA-MB-453)celllines,andnormalhumanfetallung fibroblastcellline(MRC-5)weremaintainedasmonolayerculturein WhileosmiumanaloguesofNAMI-Ahavebeendocumentedin the Roswell Park Memorial Institute (RPMI) 1640 nutrient medium theliterature,19thecorrespondingosmium(III)counterpartsof (Sigma Chemicals Co, USA). RPMI 1640 nutrient medium was KP1019 have not yet been reported. This is mainly because of 11133 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article distinctdifferencesincoordinationchemistryofrutheniumand osmiumwithchloridoandazoleligandsandacleartendencyto lower oxidation states for ruthenium compared to osmium upon sequential substitution of chlorido ligands by azole heterocycles in [MIVCl ]2− (M = Ru, Os). 6 Starting from (H azole) [OsIVCl ] and by exploring the 2 2 6 Anderson-type transformations, two families of osmium(IV) complexes, namely, (H azole)[OsIVCl (Hazole)]20 and trans- 2 5 [OsIVCl (Hazole) ],18 were synthesized. The availability of 4 2 compounds [1]0−[4]0 (Chart 3) has prompted us toward the final step to synthesize osmium(III) analogues of KP1019, namely, one-electron electrochemical or chemical reduction. Dimethyl sulfoxide solutions of [1]0−[4]0 show intense charge-transfer absorptions in the visible region of optical spectra reaching extinction coefficients (ε) ofover 10000 M −1 cm −1(Figure1).18Ofnoteisastrongblueshiftofbandsinthe Figure2.UV−visspectroelectrochemistryof[1]0.(a)Electrochemical potential dependence of optical spectra with corresponding cyclic voltammogram(0.2M(nBuN)[PF]inDMSO,scanrate2mVs−1). Figure 1. UV−vis spectra of the osmium(IV)-complexes [1]0 (red), 4 6 (b) Optical spectrum of [1]0 in 0.2 M (nBuN)[PF]/DMSO before [2]0 (blue), [3]0 (green), and [4]0 (black) adapted fromref 18. (blackline)andafterbulkelectrolysisatthefi 4 rstredu 6 ctionpeak(0.3V vs NHE). The colored traces represent the optical spectra of the electrochemically generated species [1]−, measured at different time visibleregionofthespectrumof[2]0relativetoHpz,Him,and points after electrolysis. Hbzim complexes [1]0, [3]0, and [4]0, the origin of which is presumably due to a large variation of the dipole moment (of the order of several Debye) for an electron transfer from 2H- sample[1]0waselectrolyzedin0.2M(nBu N)[PF ]inDMSO 4 6 indazole to osmium.11 Electrochemical reduction of complexes in a special coulometric cell using a large platinum mesh [1]0−[4]0at0.3Vledtoastrongdecreaseofabsorptioninthe working electrode at 0.3 V versus NHE. After a complete − − − visible region for [1] , [3] , [4] and that with maximum at reduction the solution was inserted into the 1 cm UV cuvette, − about628nmfor [2] andtoan increase ofbandsinthe UV- which was then immediately tightly closed to avoid contact rangeofthespectrumtoextinctioncoefficientsexceeding5000 with air oxygen, and UV−vis spectra were recorded at M −1 cm −1 due to intraligand transitions of the azole increasing time points. Figure 2b shows the absorption spectra heterocycles. oftheinitial[1]0(blackline)andone-electronreducedspecies − Strongabsorption bandsduetoosmium(IV) regenerated by [1] (colored lines) measured over time. Marginal changes in − reoxidation of osmium(III) species by air oxygen provided the spectrum of electrochemically generated species [1] further evidence for the reversible one-electron OsIV/III (colored lines) confirmed the high stability of the product in reduction. DMSO. The stability data indicated that isolation of the More detailed investigation was performed with complex osmium(III) species inthe solidstateafter reduction ofparent [1]0.Areversible cathodicreduction(theratioofcathodicand osmium(IV) complexes would be possible. anodic currents in the corresponding cyclic voltammogram is Therefore, we directed our efforts to the chemical reduction closetoone)wasobservedevenatalowscanrate(2mVs −1) of complexes [1]0−[4]0 and isolation of the reduced species inDMSOasshowninFigure2a.Upontheinsitureductionat [1] −−[4] − byadditionofacertainsaltasreductantandcation the first cathodic peak in DMSO the dominating absorption supplier. By reacting suspensions of [3]0 or [4]0 in water or band at 386 nm decreased via an isosbestic point at 329 nm methanolwithasmallexcesstetrabutylammoniumborohydride (Supporting Information, Figure S1). Simultaneously, a new pale green solids of the composition nBu N[3] and nBu N[4] 4 4 band at 301 nm appeared. In addition, upon the voltammetric were isolated. Attempts to replace the tetrabutylammonium scanreversalacompleterecoveryofthe[1]0opticalbandswas cationbythecorrespondingazoliumionviathesodiumsaltsas observedindicatingreversibleelectrochemicalbehaviorandthe reportedforrelatedosmium(IV)complexes20failed.Reduction high stability of cathodically generated [1] − (Figure 2a). of complexes [3]0 and [4]0 with NaBH in methanol or water 4 − To investigate the stability of [1] , produced electrochemi- followed by addition of tetraphenylphosphonium chloride callyatroomtemperatureunderargon,weperformedanexsitu afforded complexes PPh [3] and PPh [4] in 56 and 67% 4 4 spectroelectrochemical experiment, where the corresponding yields, respectively. In addition, complexes [1] −−[4] − were 11134 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article prepared and isolated as azolium salts in 50 to 90% yields by the monoclinic space group P2 /c, while H ind[2] and nBu N- 1 2 4 reduction of [1]0−[4]0 in methanol with NaBH and [3]crystallizedinthetricliniccentrosymmetricspacegroupP1̅ 4 subsequent addition of the azole heterocycle in 6 M andorthorhombicspacegroupPbcn,respectively.Thereisone hydrochloric acid. complex anion and one azolium counterion in the asymmetric All complexes were characterized by 1H NMR spectroscopy, unitofH pz[1]andH ind[2],whilehalfofcomplexanionand 2 2 andtheirspectrashowsignalsoftheazoliumcounterionaswell half of counterion are in nBu N[3]. The osmium ion in 4 as of the coordinated azole ligands. The signals of the nBu N[3] lies on a center of inversion, and the centrosym- 4 coordinated azole heterocycles are broad and markedly metriccomplexanionisofpointgroupsymmetryC,whilethe i upfield-shifted due to the paramagnetism of the osmium(III) nitrogen atom of the nBu N+ resides on a 2-fold rotation axis 4 center (d5, S = 1/2). (C ). The osmium(III) ion in all three complex anions has an 2 UV−vis spectra of H pz[1], H ind[2], H im[3], and octahedralcoordinationgeometrywithfourchloridoligandsin 2 2 2 H bzim[4] in DMSO shown in Figure 3 are almost identical the equatorial plane and two azole heterocycles in axial 2 to those for [1] −−[4] − generated upon the electrochemical positions. reduction of [1]0−[4]0. The lengthening of the Os−N and Os−Cl bonds in [1] − , − − [2] , and [3] compared to those in one-electron oxidized species [1]0, [2]0, and [3]0 is worth noting (Table 2). At the same time the Os−N and Os−Cl bonds in [3] − are well- comparable with Ru−N and Ru−Cl at 2.078(5) and 2.360(2), 2.379(2) Å in [RuIIICl (1H-imidazole) ] − ,35 and similar to 4 2 those in [RuIIICl (5-nitro-1H-imidazole) ] − .36 4 2 As in [1]0, [2]0, and [3]0,18 both trans-azole ligands in [1] − , − − [2] ,and[3] arearrangedparalleltoeachotherincontrastto otherrelatedstructures,forexample,(Ph P)[trans-RuIIICl (1H- 4 4 indazole) ], in which the azole ligands are significantly 2 twisted.37 As in [2]0, the indazole ligand in [2] − adopts a quinoid tautomeric form and binds to osmium(III) via nitrogen atom Figure3.UV−visspectraoftheosmium(III)species[1]−(red),[2]− N1. Thismode ofcoordination has been documented onlyfor (blue),[3]− (green), and [4]− (black). osmium(IV)sofar,11,18andthereasonsremaintobeelucidated by theoretical studies of their electronic structure. The usual ESImassspectrameasuredinbothpositiveandnegativeion coordination mode of neutral indazole to a transition metal is modesareinagreementwiththeproposedcompositionforthe via N2. Deprotonated indazole species act as a bridging ligand eight isolated complexes. In particular, positive ion spectra for in polynuclear complexes38,39 and in very rare cases as a H 2 ind[2]showedpeakswithm/z498.0and118.8attributedto monodentate indazolate ligand coordinated via N1 or N2.40,41 [OsIIICl 2 (Hind) 2 ]+ and [H 2 ind]+, while those measured in the Avariednumberofhydrogen-bondinginteractionsisevident negative ion mode displayed signals at m/z 567.9 and 332.5, in the crystal structures of H pz[1], H ind[2], and nBu N[3], which are due to [OsIIICl 4 (Hind) 2 ] − and [OsIIICl 4 ] − , which are shown in Supporti 2 ng Inform 2 ation, Figures S 4 2−S5. respectively. The presence of tetraphenylphosphonium and Thehydrogen-bondingparametersaresummarizedinSupport- tetrabutylammonium cations in Ph 4 P[3], Ph 4 P[4] and nBu 4 N- ing Information, Tables S1−S3. [3], nBu 4 N[4] was confirmed by strong peaks with m/z 339.1 Stabilityin Aqueous MediaandDMSO. The stability of and 242.3, respectively, in their mass spectra. metalcomplexesunderphysiologicalconditionsisanimportant X-ray Crystallography. The results of X-ray diffraction parameter in the development of potential metal-based drugs. studies of complexes H 2 pz[1], H 2 ind[2], and nBu 4 N[3] are ThehydrolyticbehaviorofH 2 pz[1]andH 2 bzim[4]inaqueous showninFigure4.Selectedbondlengths(Å)andbondangles mediawasmonitoredby1HNMRspectroscopyover14h,and (deg) are quoted in Table 2. Complex H 2 pz[1] crystallized in no change in chemical shifts of the proton resonances was observed (Figures S6 and S7, see Supporting Information). However,asolidprecipitatedslowlyfromtheaqueoussolution of H bzim[4], which could be attributed to [4]0 by ESI-MS 2 measurements. Note that aquation of KP1019 studied previously42 resulted in formation of mer,trans- [RuIIICl (Hind) (H O)], which was isolated and characterized 3 2 2 by X-ray crystallography. The coordinated water molecule can be replaced by other nucleophilic ligands,42 and, in particular, by nitric oxide.43 It was suggested that the monoaqua species might play a role in the mechanism of action of KP1019.42,43 Some ruthenium-based potential anticancer drugs were shown recently44 to undergo a fast replacement of mono- dentate ligands by DMSO solvent molecules upon dissolution. Therefore, the results of the in vitro MTT assays when using Figure 4. ORTEP view of complex anions [OsIIICl(Hpz)]− (left), stock solutions of metal complexes in DMSO should be 4 2 [OsIIICl (Hind) ]− (middle), and [OsIIICl (Him) ]− (right) in interpreted with care. The stability of complexes in DMSO is Hpz[1] 4 , Hind[ 2 2], and nBuN[3]. Thermal e 4 llipsoid 2 s are drawn at important for validation of the in vitro biological assays.44 2 2 4 50%probability level. Complexes H pz[1], H ind[2], H im[3], and H bzim[4] 2 2 2 2 11135 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article − Table 2. Selected Bond Lengths (Å) and Angles (deg) in the Coordination Polyhedron of Osmium(III) in Complexes [1] , [2] − , and [3] − and in One-Electron Oxidized Species [1]0, [2]0, and [3]0 with Osmium in Oxidation State 4+ Reported Previously18 complex [1]− [2]− [3]− [1]0 [2]0 [3]0 Os−N 2.069(2)2.059(2) 2.071(2)2.061(2) 2.0752(17) 2.059(5) 2.050(5) 2.069(2) Os−Cl 2.3872(6)2.3868(6)2.3834(6) 2.3760(6)2.3666(7)2.3992(6) 2.3780(5) 2.3301(14) 2.3439(16) 2.3381(6) 2.3411(6) 2.3883(6) 2.3735(5) 2.3326(14) 2.3395(15) 2.3139(6) remainintactwhendissolvedinDMSO.NegativeionESImass intact in solution over the investigated time frame in the spectra of these four complexes in DMSO solutions diluted presence of L-histidine and L-cysteine as well as L-methionine with methanol 1:100 showed intense peaks attributed to (Figure 6). The hydrolytic stability of H pz[1] and lack of 2 [OsIIICl (Hazole) ] − andlack ofions thatcould hypothetically 4 2 resultfromsubstitutionofchloridoorazoleligandsbyDMSO. 1HNMRspectraofH pz[1]inDMSO-d remainedunchanged 2 6 over16hprovidingfurtherevidenceforthecomplexstabilityin solution (Figure 5). 1H NMR measurements of H bzim[4] 2 Figure 6. 1H NMR spectra of reaction mixtures of H 2 pz[1] with L- histidine,L-methionine,L-cysteine,andtheDNAnucleotide5′-dGMP in DO measuredover 15−16 h. 2 reactivity toward biomolecules are presumably responsible for Figure5.1HNMRspectraofH 2 pz[1]inDMSOmeasuredover16h. its low antiproliferative activity in the investigated human cancer cell lines (vide infra). Since benzannulated complexes over 14 h gave the same results (Figure S8, see Supporting have shown appreciable antiproliferative activities in several Information), although the color of the solution turned red humancancercelllines(videinfra),similarmeasurementswere during the investigated time frame, indicating minor conductedforH bzim[4].However,therewasnodifferencein 2 reoxidation of [4] − to [4]0 in the presence of air oxygen. reactivityofH bzim[4]comparedtothatofH pz[1]observed. 2 2 Further UV−vis measurements over 168 h confirmed slow As found during the investigations of the hydrolytic stability, a reoxidation of the benzannulated osmium(III) complex to solid precipitated slowly from the H bzim[4] solutions with 2 osmium(IV) species (Supporting Information, Figure S9, amino acids, which could be attributed to [4]0 by ESI-MS lower) in contrast to H pz[1], which remained intact over measurements, and no amino acid adduct formation was seen. 2 the same time frame (Supporting Information, Figure S9, Cyclic Voltammetry. The cyclic voltammograms of the upper). complexes H pz[1],H ind[2],H im[3],and H bzim[4] in0.1 2 2 2 2 Reactions with Small Biomolecules. Many drugs are M(nBu N)[BF ]/DMSOataglassycarbonworkingelectrode 4 4 administered intravenously, and once they have entered the with a scan rate of 0.2 V s −1 display a reversible one-electron bloodstream, amino acids and proteins are their first potential oxidationwaveattributedtotheOsIII→OsIVprocesswithE 1/2 reaction partners. The reactions of potential anticancer agents values ranging from 0.31 to 0.50 V versus NHE and an withsmallbiomolecules,suchasaminoacids,havebeenstudied irreversible reduction wave attributed to the OsIII → OsII in solution by 1H NMR spectroscopy45−47 and occasionally by process with E potential values between −1.16 and −1.53 V p isolation and characterization of the resulting products in the versus NHE (Figure 7, Table 3, and Supporting Information, solid state.48 L-Histidine, L-methionine, and L-cysteine are Figures S6−S8). The obtained values are in good agreement knowntoformcomplexeswithrutheniumandothertransition with previously reported data for complexes [1]0−[4]0.18 The metals49,50 and play an important role in the mechanism of OsIII → OsIV waves are characterized by a peak-to-peak action of anticancer agents.51−54 In addition, the reactivity of separation (ΔE ) of 79−87 mV and an aniodic peak current P KP1019towardDNAbasesandthioethershasbeenreported.55 (i ) that is almost equal to the cathodic peak current (i ), as pa pc Therefore, the interactions of complex H pz[1] with the three expected for electrochemically reversible processes. The 2 amino acids L-histidine, L-methionine, and L-cysteine and the reversiblecharacteroftheredoxprocesswasfurtherconfirmed DNA nucleotide 5′-dGMP were investigated by 1H NMR byalinearplotobtainedforthepeakcurrent(i )versussquare- p spectroscopy in addition to the previously described aquation root of scan rate (ν1/2). experiments. Theone-electron natureofthe electron-transfer process was Solutions of H pz[1] were treated with equimolar amounts verified by comparing the peak current height with that of 2 oftherespectiveaminoacid,andthereactionsweremonitored standard ferrocene/ferrocenium couple under identical exper- by 1H NMR spectroscopy for at least 14 h. H pz[1] remained imental conditions. In our detailed spectroelectrochemical 2 11136 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article compounds. Complexes H pz[1] and H im[3] showed no 2 2 cytotoxic activity in the tested range of concentrations (up to 300 μM). IC values for H ind[2] and H bzim[4] were 50 2 2 obtainedinthetestedrangeofconcentrations(upto100μM), both having the highest cytotoxic effect in human melanoma cells, FemX (20.4 ± 6.0 μM and 25.8 ± 5.5 μM, respectively). Only H ind[2] exhibited cytotoxic activity against human 2 colorectal adenocarcinoma cells (LS-174), which could be a promisingresult,concerningdecreasedsensitivityofthistumor type to treatment. H ind[2] and H bzim[4] showed similar 2 2 cytotoxicactivitytocisplatininnormalfetallungfibroblastcell line (MRC-5), expressing lower toxicity than KP1019, which mightbeanadvantagefortheinvestigatedcomplexes,sincethe Figure7.CyclicvoltammogramofHpz[1]inDMSOcontaining0.10 2 M (nBuN)[BF] at a scan rate of 0.20 V s−1 using a glassy carbon MRC-5 cell line was used as a noncancerous model for the in 4 4 working electrode; scanone is shown in black, scantwo in gray. vitro toxicity evaluation. Considering the cytotoxicity results, it is obvious that study (vide supra), the controlled potential electrolysis benzannulation (regarded here as a fusion of an aromatic ring performed at the first reduction peak for [1]0 showed the to 1H-pyrazole or 1H-imidazole ring) causes a great difference consumption of one electron per molecule (n = 0.95). inantitumoractivityinvitrobetweenthecomplexeswithsimilar All investigated osmium(III) complexes appear to undergo structures. In particular, H 2 ind[2] is more cytotoxic than solvolysis upon reduction due to cathodically induced metal complexH 2 pz[1],andH 2 bzim[4]ismoreactivethanH 2 im[3]. dechlorination yielding the mono-DMSO species, as reported It implies that the investigated complexes might differently in the literature for related ruthenium complexes.42 The interactwithDNA,intermsofformingdifferentDNAadducts, appearanceofthenewspeciescanbeassignedtotheoxidation or produce reactive oxygen species with different efficiency. peak a at −0.23 to 0.03 V (Figure 7, gray line, Table 3, and Therefore, our next goal is investigating and revealing the Supporting Information, Figures S10−S12). mechanistic basis underlying their cytotoxicity profile. More- The OsIII → OsII redox potentials as well as those of the over, since there are studies confirming that NAMI-A has reductively induced solvolysis products have been calculated significant antimetastatic potential besides its low cytotoxicity, usingtheLever’sparametrizationapproach(eq1);56[S M (OsIII/ complexes H 2 pz[1] and H 2 im[3], having related structures, OsII) = 1.29;57 I (OsIII/OsII) = −0.39;57 E (Cl − ) = −0.24;56 should also be considered for further antimetastatic activity E (Hpz) = 0.20; M 56 E (2H-ind) = 0.18;11 E L (Him) = 0.12;56 investigations.59,60 v E a L L l ( u H es bz a i r m e ) qu = ote 0 d .10 in ;56 T L a E b L l ( e S 3 -D a M nd SO ar ) e i = n g 0 o .5 o 7 L d . a T g h re e em ca e l n cu t l w at i e t d h ind P a r z e o v l i e o ) u 2 s ] ly exh re ib p i o t r s t i e g d nifi d c a a t n a t s a h n o ti w p e ro d lif t e h r a a t tiv t e ra a n c s t - i [ v O ity sIV in C v l 4 it ( r 2 o H ,18 - the experimental data.58] with the IC 50 values in a concentration range comparable with that of the clinically studied ruthenium complex (H ind)- E = S M ·∑E L + I M (eq 1) [RuIIICl 4 (Hind) 2 ] (KP1019). Those results are also simi 2 lar to the cytotoxicity profile of osmium(III) counterpart H ind[2] MTT Assays. The antiproliferative activity of osmium(III)- 2 investigatedherein,althoughthecelllinesandincubationtimes complexes H 2 pz[1], H 2 ind[2], H 2 im[3], H 2 bzim[4], KP1019 in the present study are different. andcisplatinwasevaluated for48hofcontinuousdrugaction, ■ usingcolorimetricMTTassay.Thestudywasperformedinfive CONCLUSIONS human neoplastic cell lines (HeLa, FemX, A549, MDA-MB- 453, LS-174), and one human fetal lung fibroblast cell line The synthesis of osmium(III) analogues of the ruthenium(III) (MRC-5), which was used as a noncancerous model for the in investigational drug KP1019 has been realized via electro- vitro toxicity evaluation. Results are summarized in Table 4 in chemical and chemical reduction of previously reported termsofIC valuesforthe48hincubationperiod.IC values osmium(IV) complexes, trans-[OsIVCl (Hazole) ]. Note, how- 50 50 4 2 are calculated as mean values obtained from two to three ever, that the direct analogue of KP1019, namely, (H ind)- 2 independent experiments and presented with their standard [OsIIICl (1H-indazole)] has not been prepared yet. The 4 deviations (SDs). reasons behind stabilization of 2H-indazole tautomeric form Overall, the results indicate that osmium(III) complexes upon coordination to osmium(IV) and osmium(III) can possesslowercytotoxicactivityinalltestedcelllines,compared presumably be established by ongoing ab initio studies on the to the lead compound KP1019. While the latter exhibits electronic structure of trans-[OsIVCl (2H-indazole) ], which 4 2 antiproliferative activity in all tumor cell lines, strong tumor- will be reported in due course. While osmium(IV) complexes type selectivity was noted among active osmium(III) showstrongabsorptionsinthevisibleregionofopticalspectra, a Table 3. Cyclic Voltammetric Data for H pz[1], H ind[2], H im[3], and H bzim[4] 2 2 2 2 complex E Os(III/II) E Os(III/II) E a E a E Os(IV/III),(ΔE) p calc p calc 1/2 p Hpz[1] −1.23 −1.11 0.03 −0.07 0.50(87) 2 Hind[2] −1.16 −1.16 −0.01 −0.12 0.42(85) 2 Him[3] −1.53 −1.32 −0.23 −0.27 0.31(79) 2 Hbzim[4] −1.44 −1.37 −0.16 −0.33 0.37(82) 2 aThepotentials are quoted inV vs NHE. 11137 dx.doi.org/10.1021/ic501710k|Inorg.Chem.2014,53,11130−11139 Inorganic Chemistry Article Table 4. Results of MTT Assay Presented as IC (μM) Values Obtained after 48 h of Treatment 50 IC a[μM](mean±SD) 50 complex HeLa A549 FemX MDA-MB-453 LS-174 MRC-5 Hpz[1] >300 >300 >300 >300 >300 >300 2 Hind[2] 83.04±0.9 >100 20.4±6.0 >100 70.3±5.5 33.8±0.2 2 Him[3] >300 >300 >300 >300 >300 >300 2 Hbzim[4] 56.6±4.0 >100 25.8±5.5 >100 >100 31.5±3.0 2 KP1019 23.9±3.0 24.3±9.7 6.4±2.8 40.8±15.0 48.0±10.8 13.1±4.7 CDDP 7.8±2.3 17.2±0.7 10.8±0.9 21.0±5.7 22.4±7.2 30.3±3.0 aThesign>(infrontofthemaximumvalueoftheconcentration)indicatesthatIC valueisnotreachedintheexaminedrangeofconcentrations. 50 withextinctioncoefficientsexceeding10000M −1cm −1insome (3)Scolaro, C.;Bergamo, A.;Brescacin, L.;Delfino,R.;Cocchietto, cases, the osmium(III) complexes are characterized by much M.;Laurenczy,G.;Geldbach,T.J.;Sava,G.;Dyson,P.J.J.Med.Chem. lower intensity absorptions in the visible region of the optical 2005, 48, 4161−4171. spectra. Single-crystal X-ray diffraction studies of H pz[1], (4) Peacock, A. F.A.; Melchart, M.; Deeth, R. J.; Habtemariam, A.; H ind[2], and nBu N[3] have confirmed their formu 2 lation. Parsons, S.; Sadler,P. J. Chem.Eur. J. 2007,13,2601−2613. 2 4 (5) (a) Therrien, B.; Süss-Fink, G.; Govindaswamy, P.; Renfrew, A. One-electronreductionresultedinappreciableexpansionofthe K.; Dyson, P. J. Angew. Chem., Int. Ed. 2008, 47, 3773−3776. coordination sphere about osmium manifested in the length- ening of the Os−N and Os−Cl bonds by 0.005−0.016 and ( T b h ) er G rie o n v , en B d .; er S , m P i . t ; h, Ed G a . fe S , . F I . n ; or M g. ak C h h u im be . la A , ct B a . 2 C 0 . 14 E , .; 40 D 9 y , so 1 n 1 , 2− P 1 . 2 J 0 .; . 0.04−0.05Å,respectively.Therewasnochangeincoordination (c)Govender,P.;Sudding,L.C.;Clavel,C.M.;Dyson,P.J.;Therrien, modeofindazoleobserveduponreductionof[2]0to[2] − .The B.;Smith,G.S.DaltonTrans.2013,42,1267−1277.(d)Tamasi,G.; cytotoxicity profiles of the investigated complexes suggest that Carpini,A.;Valensin,D.;Messori,L.;Pratesi,A.;Scaletti,F.;Jakupec, osmium(III) analogues of KP1019 appear particularly interest- M. A.;Keppler, B.;Cini, R.Polyhedron 2014,81, 227−237. ing for future investigations of their biological activity in vitro (6)VanRijt,S.H.;Peacock,A.F.A.;Johnstone,R.D.L.;Parsons,S.; a■nd for further clinical development. Sadler, P.J. Inorg. Chem. 2009,48,1753−1762. (7)Filak,L.K.;Mühlgassner,G.;Bacher,F.;Roller,A.;Galanski,M.; ASSOCIATED CONTENT Jakupec,M.A.;Keppler,B.K.;Arion,V.B.Organometallics2011,30, * 273−283. S Supporting Information (8) Ibao, A.-F.; Gras, M.; Therrien, B.; Süss-Fink, G.; Zava, O.; UV−visspectroelectrochemistryof[1]0(FigureS1),hydrogen- Dyson, P.J. Eur. J. Inorg. Chem. 2012,2012,1531−1535. bonding interactions in H pz[1], H ind[2], and nBu N[3] 2 2 4 (9) (a) Ni, W.-X.; Man, W.-L.; Yiu, S.-M.; Ho, M.; Cheung, M. T.- (Figures S2−S5), hydrogen-bonding parameters (Tables S1− W.;Ko,C.-C.;Che,C.-M.;Lam,Y.-W.;Lau,T.-C.Chem.Sci.2012,3, S3), cyclic voltammograms of H 2 ind[2] (Figure S6), H 2 im[3] 1582−1588.(b)Cardoso,C.R.;Lima,M.U.S.;Cheleski,J.;Peterson, (Figure S7), and H 2 bzim[4] (Figure S8), 1H NMR spectra of E.J.;Venan̂ cio,T.;Farell,N.R.;Carlos,R.M.J.Med.Chem.2014,57, H pz[1] in D O measured over 16 h (Figure S9), 1H NMR 4906−4915. 2 2 spectraofH bzim[4]inDMSO-d measuredover14h(Figure (10) Suntharalingam, K.; Johnstone, T. C.; Bruno, P. M.; Lin, W.; 2 6 S10) and in D O measured over 16 h (Figure S11), UV−vis Hemann, M. T.; Lippard, S. J. J. Am. Chem. Soc. 2013, 135, 14060− 2 measurements of H pz[1] and H bzim[4] in DMSO over 168 14063. h (Figure S12), and 2 crystallograp 2 hic data in CIF format. This (11) Büchel, G. E.; Stepanenko, I. N.; Hejl, M.; Jakupec, M. A.; material is available free of charge via the Internet at http:// Keppler,B.K.;Heffeter,P.;Berger,W.;Arion,V.B.J.Inorg.Biochem. 2012, 113,47−54. p■ubs.acs.org. (12)Stepanenko,I.N.;Krokhin,A.A.;John,R.O.;Roller,A.;Arion, V. B.; Jakupec, M. A.; Keppler, B. K. Inorg. Chem. 2008, 47, 7338− AUTHOR INFORMATION 7347. Corresponding Author (13) Galanski, M.; Arion, V.; Jakupec, M.; Keppler, B. Curr. Pharm. *E-mail: vladimir.arion@univie.ac.at. Des. 2003,9, 2078−2089. Notes (14) Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.; T■he authors declare no competing financial interest. Keppler, B. K. Dalton Trans. 2008, 183−194. (15)Alessio,E.;Mestroni,G.;Bergamo,A.;Sava,G.Curr.Top.Med. Chem. 2004,4, 1525−1535. ACKNOWLEDGMENTS (16) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, We thank A. Dobrov for ESI mass spectra measurements, A. W.; Keppler, B. K. Chem. Sci. 2014, 5,2925−2932. Roller for collection of the X-ray data, and M. Malarek for (17) Bergamo, A.; Gaiddon, C.; Schellens, J. H. M.; Beijnen, J. H.; readingthemanuscriptandhelpfuldiscussion.Wearethankful Sava, G. J. Inorg. Biochem. 2012, 106,90−99. to the Ministry of Science and Technology of Serbia for (18) Büchel, G. E.; Stepanenko, I. N.; Hejl, M.; Jakupec, M. A.; financial support from Grant No. III41026. P.R. thanks the Keppler, B. K.; Arion, V. B. Inorg. Chem. 2011,50, 7690−7697. (19) Cebriań -Losantos, B.; Krokhin, A. A.; Stepanenko, I. N.; ScienceandTechnologyAssistance Agency(ContractNo.SK- Eichinger,R.;Jakupec,M.A.;Arion,V.B.;Keppler,B.K.Inorg.Chem. AT-0027-12) and Slovak Grant Agency VEGA (Grant No. 1/ 0■307/14) for financial support. 2 ( 0 2 0 0 7 ) , 4 B 6 u , ̈c 5 h 0 e 2 l, 3− G 5 . 0 E 3 . 3 ; . Stepanenko, I. N.; Hejl, M.; Jakupec, M. A.; Arion, V. 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