Osmium‐Nitrosyl Complexes with Glycine, Picolinic Acid, ­L‐Proline and D‐Proline: Synthesis, Structures and Antiproliferative Activity

Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 ARTICLE DOI:10.1002/zaac.201300054 Osmium-Nitrosyl Complexes with Glycine, Picolinic Acid, -Proline and L -Proline: Synthesis, Structures and Antiproliferative Activity D Anatolie Gavriluta,[a] Maria Novak,[b] Jean Bernard Tommasino,[a] Samuel M. Meier,[b] Michael A. Jakupec,[b] Dominique Luneau,*[a] and Vladimir B. Arion*[b] Keywords: Osmium; Amino acids; Bioinorganic chemistry; Cytotoxicity Abstract.Thereactionsof[Os(NO)Cl ]2–withglycine(GlyH),pico- theoretically possible, has been isolated, namely mer(Cl), 5 linicacid(PicoH),l-proline(l-ProH)andd-proline(d-ProH)afforded trans(NO,O)-[Os(NO)Cl (AA)]–. Spectroscopic and electrochemical 3 four novel complexes of the general formula [Os(NO)Cl (AA)]–, properties, behavior in aqueous solution and antiproliferative activity 3 whereAA=Gly,Pico,l-Proandd-Pro,respectively.X-raydiffraction inthreehumancancercelllinesarealsoreported. studieshaverevealedthatinallcasesthesameisomertypefromthree Introduction ingtofindoutthereasonsfortheobserveddifferenceincyto- toxicity.Nitricoxideplaysimportantrolesinbiochemicalpro- The antitumor activity of metal based drugs depends upon cesses.[5] Itis a typicalexample of a non-innocentligand[6] in manyparameters,[1]includingthereactivitytowardsaminoac- coordination chemistry. It binds to metals linearly (NO+) or ids, the most important low-molecular-weight biological li- adopting a bent mode (NO–). The linearly coordinated NO+ gands. Investigation of these reactions can help in establish- ligand is a poor σ-donor, but a very strong π-acceptor. NO+ mentofthenatureofthespeciesdeliveredintothecells,since shows a range of structural trans effects (STEs) depending the compounds can be modified inside the human body, in upon the identity of the trans ligand.[7] Generally trans M–L getting insight into the mechanisms of detoxification and me- bonds to π-acceptor ligands (NO, CO) are lengthened due to tabolism of applied drugs, and in identification of potential competition for π-electron density, while π-donation from cellular targets responsible for cytotoxicity.[2] For instance, a chlorido or hydroxido ligands, stimulated by the strong π-ac- platinum(II) compound with l-methionine, Pt(L-met) , was 2 ceptornatureofNO+,resultsininverseSTEs,namely,shorten- isolated from human urine, while studying the metabolism of ingofboth transbondsM–NO+andM–L . Inaddition,the cisplatininthehumanbody.[3]Theeffectofcellculturemedia, trans metal-nitrosyl unit acts as a reaction mediator or regulator of consistingmainlyofaminoacids,salts(Na+,K+,Mg2+,Ca2+), geometry around the metal atom.[8] Linkage isomerization of vitamins and glucose, on the metal-based drugs is a standard the N- and O-bonded nitrosyl ligands is also well-documen- test now to probe their resistance to a chemical environment ted.[9]Despitegreatinterestofresearchersinthistypeofcom- on application in vitro in cell culture experiments. pounds, very little is known about the reactivity of rutheni- Quite recently, we prepared a series of ruthenium(II)- and um(II)-nitrosyl compounds with amino acids,[9b,10] while data osmium(II)-nitrosyl complexes with azole ligands and found on the related osmium counterparts to the best of our knowl- outthattheyshowastrikingdifferenceinantiproliferativeac- edge have not been reported so far. tivity, in strong contrast to previous comparative studies of homologous ruthenium(II) and osmium(II) complexes.[4] Theonlywell-characterizedosmiumcompoundswithamino Withinamoreextendedprograminitiatedrecently,wearetry- acidsareosmyl(VI)complexeswithglycine,DL-alanine,DL- valine, DL-leucine, DL-isoleucine, DL-phenylalanine ligands * Prof.Dr.D.Luneau reported more than 30 years ago,[11] and osmium(II)-arene E-Mail:dominique.luneau@univ-lyon1.fr compounds with l-prolinate, namely, [(η6-p-cymene)Os(L- * Prof.Dr.V.Arion E-Mail:vladimir.arion@univie.ac.at prolinate)] 3 [BF 4 ] 3 ,[12] and [OsCl(CO)(L-phenylalaninate)- [a] UniversitéClaudeBernardLyon1 (PPh ) ].[13] 3 2 LaboratoiredesMultimatériauxetInterfaces(UMR5615) Allthis promptedustoinitiate thestudyofthe reactionsof CampusdeLaDoua 69622VilleurbanneCedex,France [Os(NO)Cl ]2– withsome potentiallybidentate aminoacids in 5 [b]UniversityofVienna order to elucidate the effect of coordinated NO+ on the nature InstituteofInorganicChemistry WähringerStrasse42 of the favored co-ligand in trans-position and on the favored 1090Vienna,Austria isomer type formed, to isolate and fully characterize the reac- Supporting information for this article is available on the WWW tionproductsandevaluatetheirantiproliferativeactivityinhu- under http://dx.doi.org/10.1002/zaac.201300054 or from the au- thor. mancancercelllines.Hereinwereportonthesynthesisoffour Z.Anorg.Allg.Chem.0000,(cid:2),((cid:2)),0–0 ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim 1 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 ARTICLE D.Luneau,V.B.Arionetal. novelosmium(II)-nitrosylcomplexeswithglycinate,picolinate X-ray Structure Analysis (taken for comparison), l-prolinate and d-prolinate ligands Three isomeric structures are theoretically possible for (Scheme1), their X-ray diffraction structures, spectroscopic [Os(NO)Cl (AA)]– (AA = Gly, l-Pro, d-Pro) and and electrochemical properties, behavior in aqueous solution 3 [Os(NO)Cl (Pico)]–: one fac-isomer with three chlorido li- and antiproliferative activity in A549 (non-small cell lung), 3 gands coordinated to osmium in facial configuration and two SW480 (colon) and CH1 (ovarian) cancer cells. withthreechloridoligandsboundtometalinmeridionalfash- ion. In the first hypothetical meridional isomer NO is located intranspositiontotheNatomoftheaminoacidorPico,while in thesecond NOis positioned transto thecarboxylic oxygen atomoftheAAorPicoligands.[8]TheresultsofX-raydiffrac- tion studies of 1–4 are shown in Figure1 and Figure2. The crystallographic data are quoted in Table1, while selected bond lengths and bond angles are summarized in Table2. Crystals of 1–4 consist of complex anion [Os(NO)Cl (AA)]– 3 (AA=Gly,l-Pro,d-Pro)or[Os(NO)Cl (Pico)]–(Pico=picol- 3 inate) and tetrabutylammonium cation. The complexes do not contain solvent in the unit cell. The isolated complexes from the reactions of [Os(NO)Cl ]– with corresponding amino 5 acid and PicoH have been characterized as mer(Cl), trans(NO,O)-(nBu N)[Os(NO)Cl (AA)] and mer(Cl), 4 3 trans(NO,O)-(nBu N)[Os(NO)Cl (Pico)].[8] Three chlorido li- 4 3 gandsoccupymeridionalpositions,whilethenitrosylligandis arranged trans to the carboxylic oxygen atom of the corre- sponding AA or Pico ligand. Scheme1. Results and Discussion Synthesis of the Complexes Although we are interested in preparation of osmium-ni- Figure1.ORTEPdrawingsofthecomplexanion[Os(NO)Cl (Gly)]– trosyl complexes with the whole series of amino acids, we in 1 (left) and of the complex anion [Os(NO)Cl (Pico)]– in 2 3 (right) 3 initiated our study by selecting a non-chiral amino acid (gly- withatomlabeling.Thethermalellipsoidsareshownat30%and50% cine),andachiralaminoacidwithacyclicstructure(proline), probabilitylevel,respectively. which is able to generate a second chiral center when coordi- natedtometal.Inaddition,picolinicacidwasusedasaligand with the same potential denticity, but containing a sp2-hy- bridized nitrogen atom. Complexes 1 and 2 were prepared by thereactionsof(nBu N)[Os(NO)Cl ]withGlyHandPicoHin 4 5 boiling n-butanol in 80 and 72% yield, respectively. Com- pounds 3 and 4 were synthesized analogously and purified by column chromatography on silica in satisfactory yields (55% for 3 and 44% for 4). ESI mass spectra measured in negative ion mode showed strong peaks with m/z 401, 449, 441 and 441 attributed to [Os(NO)Cl (AA)]–, where AA = Gly, Pico, Figure2. ORTEP drawings of the complex anions [Os(NO)Cl (L- 3 3 l-Pro and d-Pro, respectively. Another salient feature is the Pro)]–(left)and[Os(NO)Cl 3 (D-Pro)]–(right)in3and4,respectively, with atom labeling. The thermal ellipsoids are shown at 50% prob- presence of peaks with high m/z values at 1044 (1), 1140 (2), abilitylevel. 1124 (3) and 1124 (4) due to the formation of ion clusters 2[Os(NO)Cl (AA)]–+nBu N+.Complexes1–4showastrong Uponcoordinationofglycinate,l-ord-prolinatetoosmium 3 4 NO stretching vibration at 1822, 1804, 1793 and 1794cm–1, analmostplanarfive-memberedringisformed.Thedeviation respectively. The observed wavenumbers of the ν(NO) band ofatomsfromthemeanplanethroughOs,N1,C1,C2andO1 arewellcomparablewiththoseofotherosmium-nitrosylcom- in 1 and through Os, N1, C4, C5, O1 in 3 and 4 does not pounds reported previously,[14] and implies a nitrosonium or exceed0.053Å,0.061and0.063Å,respectively.Nevertheless nitrosyl character of the NO ligand.[15] aweaktendencytowardsadoptionofahalf-chairconformation 2 www.zaac.wiley-vch.de ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim Z.Anorg.Allg.Chem.0000,0–0 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 Osmium-NitrosylComplexeswithGlycine,PicolinicAcid,l-Prolineandd-Proline Table1.CrystalDataandDetailsofDataCollectionfor1–4. 1 2 3 4 empiricalformula C H Cl N O Os C H Cl N O Os C H Cl N O Ru C H Cl N O Ru 18 40 3 3 3 33 36 2 4 3 32 36 2 4 3 31 35 3 4 4 formulaweight 643.08 797.76 696.62 735.05 temperature/K 293(2) 100(2) 110(2) 100(2) crystalsize/mm 0.28(cid:2)0.21(cid:2)0.08 0.15(cid:2)0.15(cid:2)0.10 0.61(cid:2)0.47(cid:2)0.38 0.15(cid:2)0.15(cid:2)0.08 crystalsystem orthorhombic monoclinic orthorhombic orthorhombic spacegroup Pna2 P2 /n P2 2 2 P2 2 2 1 1 1 1 1 1 1 1 λ/Å 0.71073 0.71073 0.71073 0.71073 a/Å 10.259(2) 10.9354(7) 10.3601(9) 10.3972(3) b/Å 16.797(3) 19.6189(13) 14.873(2) 14.8726(5) c/Å 15.765(3) 13.4422(9) 17.987(1) 17.9473(60) β/° 108.147(1) V/Å3 2716.6(9) 2740.5(3) 2771.5(5) 2775.25(15) Z 4 4 4 4 ρ /g·cm–3 1.572 1.675 1.637 1.635 calcd. μ/mm–1 5.009 4.973 4.916 4.909 F(000) 1280 1376 1368 1368 Θrange/° 3.13–25.00 2.11–25.50 3.55–26.00 2.26–30.23 reflectionscollected 7548 29601 18368 49303 reflectionsunique 4097 5105 5388 8192 R a) 0.0230 0.0399 0.0217 0.0210 1 wR b) 0.0563 0.1105 0.0492 0.0378 2 GOFc) 1.000 1.089 1.019 0.907 a) R = Σ||F |–|F||/Σ|F |. b) wR = {Σ[w(F 2–F2)2]/Σ[w(F 2)2]}1/2. c) GOF = {Σ[w(F 2–F2)2]/(n–p)}1/2, where n is the number of reflections 1 o c o 2 o c o o c andpisthetotalnumberofparametersrefined. Table2.Selectedbondlengths/Åandangles/°in1–4. (DMSO) H[OsCl (DMSO)(NO)][17] are well-comparable with 2 4 those in 1–4 (Table2). Atom1–Atom2 1 2a) 3 4 Upon coordination of l- or d-prolinate to osmium via the Os–O1 2.016(4) 2.010(4) 2.014(2) 2.0138(18) nitrogen atom the latter, in addition to C4, becomes a chiral Os–N1 2.085(4) 2.107(6) 2.107(3) 2.104(2) center. The literature data[18] indicate that in the majority of Os–N2 1.707(5) 1.729(6) 1.735(3) 1.719(2) N2–O3 1.176(7) 1.164(8) 1.164(4) 1.180(3) cases the nitrogen atom adopts the same configuration as the Os–Cl1 2.3703(15) 2.3522(18) 2.3740(10) 2.3794(7) asymmetric prolinate carbon (in our case C4). In rare cases, Os–Cl2 2.3876(13) 2.3709(17) 2.3685(9) 2.3766(6) however,thenitrogenandtheasymmetriccarbonoftheprolin- Os–Cl3 2.3675(15) 2.3567(15) 2.3620(10) 2.3657(7) ate adopt opposite configurations.[12] In 3 and 4 the atoms C4 Atom1–Atom2– andN1adoptthesameconfiguration,namelyS S andR R , C N C N Atom3 respectively. O1–Os–N1 79.65(17) 75.0(3) 80.83(10) 81.05(7) The complex anions in 1 form a chain which runs almost Os–N2–O3 178.4(6) 172.5(7) 176.4(3) 176.5(2) parallelwitha-axis(Figure3).Twohydrogenbondinginterac- a) The geometric parameters are strongly affected by the observed tions are evident, namely N1–H···Cl2i with N1···Cl2i 3.223Å, disorderinthecoordinatedPicoligand. N1–H···Cl2i 123.0° and N1–H···Cl1i with N1···Cl1i 3.292Å, N1–H···Cl2i175.2°(atomsmarkedwithihavebeengenerated of the five-membered chelate ring is obvious. The angle be- with symmetry transformation x + 0.5, –y + 1.5, z). Similarly, tweenplanesthroughO1,Os,N1andN1,C4,C5,O1at170.6, in3and4thecomplexanionsassembleinachainviaintermo- 169.5 and 169.2° in 1, 3 and 4 is well comparable to that lecular hydrogen bonding N1–H···O2i with the following pa- in (η6-benzene)chlorido(L-prolinato)ruthenium(II) at 166°.[16] rameters:N1···O2i3.001Å,N1–H···O2i146.3°(atomsmarked The Os–N1 and Os–O1 bonds at 2.085(4) and 2.016(4) in 1 with i have been generated with symmetry transformation x – are markedly shorter than in OsO (Gly) at 2.114(3) and 0.5,–y+1.5,–z+1)andN1···O2i3.010Å,N1–H···O2i145.7° 2 2 2.038(3)Å, respectively.[11] The nitrosyl ligand is coordinated (atoms marked with i have been generated with symmetry linearlyto osmiumin allcompounds studied(Table1).So the transformation x + 0.5, –y + 1.5, –z + 1), respectively (Fig- NO ligand acts as a poor σ-donor, but as a very strong ure3). πacceptor.[7]TheOs–N2andN2–O3bondsin1–4arerespec- Thediamagnetismof1–4in solution,asconfirmedbytheir tively shorter and longer than in (PPh ) [OsCl (NO)] charac- “normal” 1H NMR spectra without detectable paramagnetic 4 2 5 terized as {Os(NO)}6 species with linear OsNO angle shiftsorlinebroadeningoftheprotonresonancesevenatroom (178.5(8)°).[14c] The average equatorial Os–Cl bond in temperature, the wavenumbers of NO stretching vibrations, (PPh ) [OsCl (NO)]at2.387(4)ÅisclosertoOs–Clbondsin along with linearity of Os–N–O moiety provide compelling 4 2 5 1–4 (Table2) than the Os–Cl bond in trans position to NO at evidence for the formulation {Os(NO)}6 in terms of the Ene- 2.270(1)Å, although the structure was found disordered over mark–Feltham notation,[19] where 6 is the sum of the number two positions. Likewise, the equatorial Os–Cl bonds in ofelectronsinthedorbitalsofthemetalandintheπ*orbital Z.Anorg.Allg.Chem.0000,0–0 ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.zaac.wiley-vch.de 3 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 ARTICLE D.Luneau,V.B.Arionetal. Figure3.Portionsofcrystalstructuresof1(left),3(middle)and4(right),showingthehydrogenbondinginteractions. of NO. This formulation is in accord with OsII (d6, S = 0) bondedtoNO+(S=0)orNO0(S=½)coupledantiferromag- netically or via a closed shell interaction to OsIII (S = ½). Aqueous Solution Behavior Theaqueoussolutionbehaviorwithrespecttohydrolysisof 1–4 was studied over 24h by UV/Vis spectroscopy and elec- trospray ionization ion trap (ESI-IT) mass spectrometry at 293and310K,respectively.Theelectronicabsorptionspectra suggestthatthecomplexesareinertwithrespecttohydrolysis (Figures S1–S2).Furthermore, the lack ofhydrolysis was evi- denced by ESI-IT MS, which featured solely peaks due to Figure4. ESI-IT massspectra of 1–4 inaqueous solution containing [Os(NO)Cl (AA)]–ionsovertheentireincubationperiod(Fig- 3 1%DMSO,measuredafter24h.Thespectrafeaturesolelyions,cor- ure4). The complexes were also stable in the presence of responding to [Os(NO)Cl (AA)]–, indicating that the compounds do 3 4equiv. ascorbic acid, a biological reducing agent. Incubation not hydrolyze during this time period. The inset shows the isotopic of 1–4 with cytochrome c (cyt) resulted in the formation of distributionofcomplexanionin4andthesimulatedspectrum(grey). Experimental ESI-IT values include a standard deviation of non-selective adducts probably via electrostatic interactions m/z(cid:3)0.05. between the negatively charged [Os(NO)Cl (AA)]– ions and 3 the multiply positively charged cyt (Figure S3). Aqueous solutions of complexes 3 and 4 at neutral pH are indeed optically active and display Cotton effects for both enantiomers (Figure5). As expected they are roughly mirror images over the 240–450 nm region of the circular dichroism (CD)spectra,whiletheirUV/Visspectraareverysimilar(Fig- ure S2). Electrochemical Measurements The cyclic voltammograms of 1–4 in CH CN are shown in 3 Figure6 and Figure7. The redox processes occur exclusively on the complex anions [Os(NO)Cl (AA)]–, where AA = gly- 3 Figure5.CDspectraofcomplexes3(blackline)and4(greyline)in cinate,picolinate,l-prolinateandd-prolinate.Forallthecom- water at pH 7.0 {concentration 4(cid:2) 10–4 M; T = 298 K, I = 0.10m pounds, the cyclic voltammograms display a one-electron (KCl)inH O}. 2 4 www.zaac.wiley-vch.de ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim Z.Anorg.Allg.Chem.0000,0–0 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 Osmium-NitrosylComplexeswithGlycine,PicolinicAcid,l-Prolineandd-Proline Figure6.Cyclicvoltammetryof1(left)and2(right)at100mVs–1onGCelectrode(3mm)in0.1mTBAPF inCH CN. 6 3 Figure7.Cyclicvoltammetryof3(left)and4(right)at100mVs–1onGCelectrode(3mm)in0.1mTBAPF inCH CN. 6 3 irreversible reduction wave (determined by comparing the peak current height (i ) with that of standard Fc/Fc+ couple p under the same experimental conditions) attributed to the {Os(NO)}6(cid:2){Os(NO)}7processwithredoxpotentialvalues quoted in Table3. Table3.Electrochemicaldatafor1–4a). Complex cathodicpeakpotentials anodicpeakpotentials (E ) (E ) (E ) pc pa 1/2 1 –1.97b) 1.54b)(sh) 1.67d) 2 –1.37b) 1.58c) 3 –1.95b) 1.45b) 1.60d) 4 –1.88b) 1.44b) 1.59d) a)Peakpotential(V)andhalf-wavepotentialE recordedinCH CN 1/2 3 at293Kwithaglassycarbonelectrode,0.1mTBAPF assupporting 6 electrolyte;allpotentialsarevs.SCE,scanrate0.1V·s–1;b)Irrevers- ible system (E ); c) Reversible system; d) Quasireversible system. pa Ferrocene/ferricinium (E ox = +0.445 V) couple was used as an in- 1/2 Figure8.UV/Visspectraof2inCH CN,before(solidtrace)andafter ternalstandard. 3 (dottedtrace)exhaustiveelectrolysis(1h). Uponoxidationcomplex2showedaone-electronreversible wave (Figure6, right) with E = 1.58 V vs. SCE, which can 505 nm (see Figure8). Thecyclic voltammetry followed after 1/2 beassignedtothe{Os(NO)}6(cid:2){Os(NO)}5transfer.Theone- electrolysis showed the same reversible wave indicating the electrontransferprocesswasconfirmedbytheexhaustiveelec- stability of the oxidized species of 2 under inert atmosphere. trolysis performed at 1.9 V. This oxidation was accompanied Similar electrochemical behavior was reported for bydevelopmentofanewvisibleabsorptionbandwithλ at [OsCl (NO)]2–.[14c,20] max 5 Z.Anorg.Allg.Chem.0000,0–0 ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.zaac.wiley-vch.de 5 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 ARTICLE D.Luneau,V.B.Arionetal. For complexes 1, 3 and 4 the cyclic voltammetry data is oxygenisamorepreferredligandintranspositiontothecoor- more complicated. Indeed, we observe two oxidation waves dinatedNO+ligandthanthechlorideligand,pyridinenitrogen, (see Figure6 and Figure7). Moreover, the coulometry per- secondary or primary amine. The only isomer isolated from formedat1.9V/SCEgaveavalueofn =4(n =apparent reactions of [Os(NO)Cl ]2– with amino acids is mer(Cl), app app 5 electron number). Electrochemical oxidation behavior of trans(NO,O)-(nBu N)[Os(NO)Cl (AA)]. No evidence for for- 4 3 aminoacidsisknownand describedasverycomplicated.The mationofothertheoreticallypossibleisomershasbeenfound. oxidationoftheaminogroupfollowedbytheoxidationofthe The work demonstrated that amino acids can have an impact carboxylic group are possible.[21] At this stage, we can assign on the fate of potential antitumor drugs in vitro and in vivo. thefirstpeaktotheoxidationoftheaminogroupoftheamino Investigation of reactivity of ruthenium and osmium com- acidate ligand and the second oxidation peak to {Os(NO)}6 plexes towards other amino acids including those with other (cid:2) {Os(NO)}5 electrochemical oxidation. In the case of 2 the donor atoms and with potentially higher denticity is worth- picolinate ispresumably stabilized bythe heterocycle,and we while and may shed light on the difference on cytotoxicity of donotobservethisoxidationinthewindowofredoxpotentials ruthenium-andosmium-nitrosylcomplexeswithazolehetero- studied here. cycles.Toenableadefinitiveassessmentofthewayandextent how the amino acids alter the cytotoxic properties, missing links between these two related series of complexes will have Antiproliferative Activity to be prepared and comparatively studied. Osmium(II)-nitrosyl complexes (1–4) with amino acids and PicoH were studied for their capacity to inhibit cancer cell Experimental Section growth in vitro in the human cancer cell lines CH1 (ovarian carcinoma), SW480 (colon carcinoma) and A549 (non-small Starting Materials cell lung cancer), yielding the IC values listed in Table4. 50 All compounds show the highest effect in the generally rather (nBu 4 N) 2 [Os(NO)Cl 5 ] was synthesized as previously reported in the chemosensitiveCH1cells,whileSW480cellsandthebroadly literature.[14c,22]OsO 4 (99.8%)waspurchasedfromJohnsonMatthey; NH OH·HCl, K C O ·H O, dimethylsulfoxide, picolinic acid and d- chemoresistantA549 cellsare lessaffected bythe compounds 2 2 2 4 2 prolinewerefromAcros;nBu N[PF] ,glycineandl-prolinefromAl- investigated here. However, the complex anions hardly seem 4 6 drichandnicotineadeninedinucleotide(reducedform),ascorbicacid to make a contribution to the cytotoxicity in CH1 cells which andhorseheartcytochromecfromSigma.Thesynthesisofcomplexes are sensitive to the cytotoxic effects of the TBA counterion. wasperformedunderanargonatmospherebyusingstandardSchlenk Only in the SW480 cells, which are insensitive to the latter, a techniques. The solvents for ESI-IT MS studies were MilliQ water certaincytotoxiccomponentattributabletothecomplexanions (18.2MΩ; Millipore Advantage A10, 185UV Ultrapure Water Sys- is discernible but on a very modest level, with all IC values tem,Molsheim,France)andmethanol(HPLCgrade,Fisher). 50 being higher than 100μM. In A549 cells, cytotoxicity is gen- erally negligible, with IC values (cid:4)300μM. A rough 50 Syntheses comparison of 1–4 with osmium nitrosyl complexes containing an indazole ligand, namely [cation]+ cis- or trans- nBu N[Os(NO)Cl (Gly)] (1): A mixture of (nBu N) [Os(NO)Cl ] 4 3 4 2 5 [OsCl 4 (NO)(Hind)]–, where [cation]+ = Na+ or H 2 ind+, which (0.88g, 1.00mmol) and glycine (0.11g, 1.50mmol) in n-butanol hadshownsurprisinglylowpotenciesincontrasttoruthenium (10mL) was heated at reflux for 24 h. The solution was allowed to analoguesinthesamecelllines,4revealssimilarlymodestde- cooltoroomtemperatureandtransferredintoanopenbeaker.After3 grees of cytotoxicity. days thered crystals formedwere filteredoff and washedwith H 2 O/ ethanol1:2(3(cid:2)10mL),diethylether(3(cid:2)5mL)anddriedinvacuo. Table4. Inhibition of cancer cell growth by compounds 1–4 and Yield 0.52g (80%). C H N O Cl Os (643.11): C 33.70 (calcd. 18 40 3 3 3 Bu NCl in three human cancer cell lines; 50% inhibitory concentra- 33.62)H6.12(6.27)N6.50(6.53)%.ESI-MSinCH CN(negative): 4 3 tions(means(cid:3)standarddeviations),obtainedbytheMTTassay(ex- m/z=400.8[Os(NO)Cl (Gly)]–,364.9[Os(NO)Cl (Gly)-HCl]–,329.0 3 3 posuretime:96h). [Os(NO)Cl (Gly)–2HCl]–.IR:ν˜ =504,596,614,738,771,884,919, 3 Compound IC /μM 1022, 1192, 1286, 1344, 1379, 1478, 1577, 1680, 1822, 2874, 2958, 50 3117,3190cm–1.UV/VisinCH CN:λ (ε)=270(478),311(222), A549 CH1 SW480 381 (116), 506 (56) nm (m 3 –1·cm– m 1 a ) x . 1H NMR ([D ]DMSO, 1 629(cid:3)13 89(cid:3)11 140(cid:3)36 6 2 314(cid:3)76 64(cid:3)11 174(cid:3)38 500.13MHz): δ = 0.94 (t, 12H D , J = 7.3Hz), 1.32 (sxt, 8H C , J = 3 (cid:4)320 114(cid:3)37 237(cid:3)47 7.3Hz),1.58(qui,8H B ,J=7.8Hz,),3.18(t,8H A ,J=8.4Hz,),3.35 4 (cid:4)640 148(cid:3)38 274(cid:3)40 (2 H, H 2 ), 7.56 (s, 2 H, H 3 ), ppm. 13C{1H} NMR ([D 6 ]DMSO, Bu 4 NCl (cid:4)640 91(cid:3)16 (cid:4)320 125.77MHz): δ = 13.47 (C D ), 19.18 (C C ), 23.06 (C B ), 46.64 (C 2 ), 57.53(C ),179.32(C ),ppm.15NNMR([D ]DMSO,50.69MHz):δ A 1 6 = 375.43 (s, N ), ppm. X-ray diffraction quality single crystals were Conclusions 3 selectedmanuallyfromtheisolatedproductunderamicroscope. Anentrytoanew familyofosmium(II)-nitrosylcomplexes nBu N[Os(NO)Cl (Pico)] (2): A mixture of (nBu N) [Os(NO)Cl ] 4 3 4 2 5 with amino acids has been realized by the synthesis of com- (0.88g,1.00mmol)andpicolinicacid(0.19g,1.50mmol)inn-buta- plexes of the general formula (nBu 4 N)[Os(NO)Cl 3 (AA)], nol(10mL)washeated atrefluxfor24h.The solutionwasallowed where AA = glycinate, l- and d-prolinate. The carboxylate tocooltoroomtemperatureandtransferredintoanopenbeaker.After 6 www.zaac.wiley-vch.de ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim Z.Anorg.Allg.Chem.0000,0–0 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 Osmium-NitrosylComplexeswithGlycine,PicolinicAcid,l-Prolineandd-Proline 2daystheredcrystalsformedwerefilteredoffandwashedwithH O/ Crystallographic Structure Determination 2 ethanol1:2(3(cid:2)10mL),diethylether(3(cid:2)5mL)anddriedinvacuo. Yield 0.50g (72%). C H N O Cl Os (691.16): C 38.33 (calcd. X-raydiffractionmeasurementsofcompounds1and3wereperformed 22 40 3 3 3 38.23)H5.60(5.83)N5.98(6.08)%.ESI-MSinCH CN(negative): withanOxford-DiffractionXCALIBURdiffractometer,whilethoseof 3 m/z = 449.0 [Os(NO)Cl (Pico)]–. IR: ν˜ = 470, 533, 618, 683, 717, 2and4withaBrukerX8APEXIICCD-diffractometer.Singlecrystals 3 740, 765, 865, 885, 920, 1022, 1058, 1107, 1152, 1256, 1291, 1318, werepositionedat40mmfromthedetector and738and869frames 1380, 1470, 1611, 1687, 1804, 2873, 2962cm–1. UV/Vis in CH CN: weremeasured,eachfor20and10sover1°scanwidthfor2and4, 3 λ (ε)=255(7606),349(1844),499(207)nm(m–1·cm–1).1HNMR respectively.The datawere processedusing theCrysAlisRED pack- max ([D ]DMSO,500.13MHz):δ=0.94(t,12H ,J=7.3Hz),1.32(sxt, age[23] and SAINT software.[24] Crystal data, data collection param- 6 D 8H , J = 7.3Hz), 1.58 (qui, 8H , J = 7.8Hz,), 3.17 (t, 8H , J = eters,andstructurerefinementdetailsaregiveninTable1.Thestruc- C B A 8.4Hz,), 7.91 (t.d, 1 H, J = 6.5Hz, H ), 8.09 (t.d, 1 H, J = 7.6Hz, tureswere solvedby directmethods andrefined byfull-matrix least- 5 H ), 8.12 (t.d, 1 H, J = 7.7Hz, H ), 8.36 (d, 1 H, J = 5.5Hz, H ), squares techniques. The Pico ligand and tetrabutylammonium cation 3 4 6 ppm.13C{1H}NMR([D ]DMSO,125.77MHz):δ=13.48(C ),19.19 in 2 were found to be disordered over two positions with S.O.F. 6 D (C ), 23.06 (C ), 57.55 (C ), 126.63 (C ), 129.85 (C ), 141.46 (C ), 0.5:0.5. The disorder was resolved by using restraints SADI im- C B A 4 5 3 147.87 (C ), 148.77 (C ), 170.01 (C ), ppm. Suitable crystals for X- plementedinSHELXL.Non-hydrogenatomswererefinedwithaniso- 2 6 7 raydiffractionstudywereobtainedbyslowevaporationofthemother tropic displacement parameters. H atoms were localized from differ- liquoratroomtemperature. encemap.Thefollowingcomputerprogramswereused:structuresolu- tion, SHELXS-97;[25] refinement, SHELXL-97;[26] molecular dia- nBu N[Os(NO)Cl (L-Pro)] (3): A mixture of (nBu N) [Os(NO)Cl ] grams,ORTEP;[27]computer:IntelCoreDuo. 4 3 4 2 5 (0.88g, 1.00mmol) and l-proline (0.17g, 1.50mmol) in n-butanol (10mL) was heated at reflux for 24 h. The solvent was evaporated Crystallographic data for the structures in this paper have been de- under reduced pressure and the residue was purified by column positedwiththeCambridgeCrystallographicDataCentre,CCDC,12 chromatography on silica, using as eluent a mixture of CHCl /THF UnionRoad,CambridgeCB21EZ,UK.Copiesofthedatacanbeob- 3 3:2 and collecting the first fraction (R = 0.74). Yield 0.38g (55%). tained free of charge on quoting the depository numbers CCDC- f C H N O Cl Os (682.17): C 37.00 (calcd. 36.97) H 6.14 (6.35) N 922215(1),CCDC-922218(2),CCDC-922216(3)andCCDC-922217 21 43 3 3 3 6.00 (6.16)%. ESI-MS in CH CN (negative): m/z = 440.8 [Os(NO) (4). 3 Cl (L-Pro)]–,404.9[Os(NO)Cl (L-Pro)-HCl]–,369.0[Os(NO)Cl (L- 3 3 3 Electrochemistry Pro) - 2HCl]–. IR: ν˜ = 410,467, 500, 531, 581, 617, 702, 738, 798, 867,937,987,1038,1057,1136,1266,1303,1317,1346,1382,1471, Cyclic voltammetry measurements were performed at room tempera- 1662, 1793, 2873, 2959, 3173cm–1. UV/Vis in CH CN: λ , (ε) = 3 max ture using an AMEL 7050 all-in one potentiostat, using a standard 276(sh,579),306(225),386(96),507(52)nm(m–1·cm–1).1HNMR three-electrodesetupwitha3mmdiameterglassycarbonelectrode,a ([D ]DMSO,500.13MHz):δ=0.94(t,12H ,J=7.3Hz),1.32(sxt, 6 D platinum auxiliary electrode and a SCE (saturated calomel electrode) 8H , J = 7.3Hz), 1.58 (qui, 8H , J = 7.8Hz,), 1.85 (m, 3 H, H ’’, C B 5 as the reference electrode. Deareation of solutions was accomplished H ’, H ’), 2.09 (m, 1 H, H ’’), 2.87 (m, 1 H, H ’), 3.17 (t, 8H , J = 6 5 6 4 A bypassingastreamofN throughthesolutionfor30minpriortothe 8.4Hz,),3.54(m,1H,H ’’),3.86(qua,1H,J=7.2Hz,H ),8.74(d, 2 4 2 measurement and then maintaining a blanket atmosphere of N over 1H,J=7.1Hz,H ),ppm.13C{1H}NMR([D ]DMSO,125.77MHz), 2 3 6 the solution during the measurement. 1 or 2mM solutions of 1–4 in δ, ppm: 13.48 (C D ), 19.19 (C C ), 23.06 (C B ), 26.09 (C 5 ), 29.27 (C 6 ), 0.1mTBAPF assupportingelectrolytehavebeenusedformeasure- 53.58(C ),57.55(C ),64.17(C ),180.74(C ),ppm.X-raydiffraction 6 4 A 2 1 ments.Undertheseexperimentalconditionsthe[Fe(η5-C H ) ]0/+re- qualitysinglecrystalswereselectedmanuallyfromtheisolatedprod- 5 52 doxcouple(E =+0.445Vvs.SCE)wasusedasaninternalreference uctunderamicroscope. 1/2 forpotentialmeasurements. nBu 4 N[Os(NO)Cl 3 (D-Pro)] (4): A mixture of (nBu 4 N) 2 [Os(NO)Cl 5 ] Electrospray Ionization Ion-Trap Mass Spectrometry (0.88g, 1.00mmol) and d-proline (0.17g, 1.50mmol) in n-butanol (10mL)washeatedatrefluxfor24h.Thesolventwasevaporatedat MassspectrawererecordedwithanAmaZonSLelectrosprayioniza- reducedpressureandtheresiduewaspurifiedbycolumnchromatog- tion ion-trap (ESI-IT) mass spectrometer (Bruker Daltonics GmbH, raphy on silica by using as eluent a mixture of CHCl /THF 3:2 and Bremen, Germany) in positive and negative ion modes. The samples 3 collecting the first fraction (R = 0.74). Yield 0.30g (44%). were injected into the mass spectrometer by direct infusion at f C H N O Cl Os (682.17): C 37.47 (calcd. 36.97) H 6.34 (6.35) N 6μL·min–1.Typicalinstrumentparameterswereasfollows:HVcapil- 21 43 3 3 3 6.03 (6.16)%. ESI-MS in CH CN (negative): m/z = 440.9 lary (cid:3)4.5kV, nebulizer 8psi, dry gas 8L·min–1, dry temperature 3 [Os(NO)Cl (D-Pro)]–, 404.9 [Os(NO)Cl (D-Pro) - HCl]–, 369.0 200°C, trap drive 57.6, RF Level 77% and average accumulation 3 3 [Os(NO)Cl (D-Pro) - 2HCl]–. IR: ν˜ = 406, 467, 500, 539, 581, 617, times 1.5–3.7ms. Protein samples were measured using slightly dif- 3 702, 738, 798, 868, 937, 987, 1037, 1057, 1087, 1137, 1266, 1303, ferent parameters: HV capillary (cid:3)3.5kV, nebulizer 6psi, dry gas 1317,1346,1382,1471,1662,1794,2873,2959,3173cm–1.UV/Vis 6L·min–1, dry temperature 180°C, trap drive 79.1, RF Level 100% inCH CN:λ (ε)=272(sh,493),312(209),387(96),507(51)nm and an average accumulation time 10μs. Raw data was processed 3 max (m–1·cm–1).1HNMR([D ]DMSO,500.13MHz):δ=0.94(t,12H ,J usingDataAnalysis4.0(BrukerDaltonicsGmbH,Bremen,Germany). 6 D = 7.3Hz),1.32 (sxt, 8H ,J = 7.3Hz), 1.58(qui, 8H , J= 7.8Hz,), Protein mass spectra were recorded over 0.5min and deconvoluted C B 1.85(m,3H,H ’’,H ’,H ’),2.09(m,1H,H ’’),2.87(m,1H,H ’), using the maximum entropy deconvolution algorithm with automatic 5 6 5 6 4 3.17 (t, 8H , J = 8.4Hz,), 3.54 (m, 1 H, H ’’), 3.86 (qua, 1 H, J = datapointspacingandaninstrumentpeakwidthof0.2.Stocksolutions A 4 7.2Hz, H ), 8.74 (d, 1 H, J = 7.2Hz, H ), ppm. 13C{1H} NMR of1–4(1%dmso,200μM), ascorbicacid(800μM)andcytochrome 2 3 ([D ]DMSO, 125.77MHz): δ = ppm: 13.48 (C ), 19.19 (C ), 23.06 c (200μM) were prepared in water. The compounds were incubated 6 D C (C ), 26.09 (C ), 29.27 (C ), 53.58 (C ), 57.55 (C ), 64.17 (C ), with ascorbic acid at a 1:4 and with cytochrome c at a 2:1 metal-to- B 5 6 4 A 2 180.74 (C ), ppm. Suitable crystals for X-ray diffraction study were biomoleculeratioandESI-ITmassspectrawererecordedafter1,3,6 1 growninTHF/hexane. and24h.Thefinalmetalconcentrationwas50μMineachincubation Z.Anorg.Allg.Chem.0000,0–0 ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.zaac.wiley-vch.de 7 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 ARTICLE D.Luneau,V.B.Arionetal. solution.Thesamplesweredilutedwithwater,andwithawater:meth- [2] L.E.H. Paul,J. Furrer, B.Therrien, J. Organomet.Chem. DOI: anol:formicacid(50:50:0.2)mixtureinthecaseofproteinsamples,to 10.1016/j.jorganchem.2012.12.011. ametalconcentrationof5μMpriortoinjectionintotheESI-ITMS. [3] a)W.W.Alden,A.J.Repta,Chem.-Biol.Interact.1984,48,121– 124;b)J.E.Melvic,O.E.Petersen,Inorg.Chim.Acta1987,137, 115–118. Cell Lines and Culture Conditions [4] G.E. 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Received:January30,2013 Commun.2012,48,5219–5246. PublishedOnline:(cid:2) 8 www.zaac.wiley-vch.de ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim Z.Anorg.Allg.Chem.0000,0–0 Job/Unit:Z13054 /KAP1 Date:08-04-1317:42:05 Pages:9 Osmium-NitrosylComplexeswithGlycine,PicolinicAcid,l-Prolineandd-Proline A. Gavriluta, M. Novak, J.B. Tommasino, S.M. Meier, M.A. Jakupec, D. Luneau,* V.B. Arion* ........................ 1–9 Osmium-Nitrosyl Complexes with Glycine, Picolinic Acid, l- Prolineandd-Proline:Synthesis,StructuresandAntiprolifera- tive Activity Z.Anorg.Allg.Chem.0000,0–0 ©0000WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.zaac.wiley-vch.de 9