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New half sandwich-type Ru(II) coordination compounds characterized by thefac-Ru(dmso-S)3 fragment: influence of the face-capping group on thechemical behavior and in vitro anticancer activity†

Ioannis Bratsos,*a Camilla Simonin,a Ennio Zangrando,a Teresa Gianferrara,a Alberta Bergamob andEnzo Alessio*a

Received 3rd June 2011, Accepted 5th July 2011DOI: 10.1039/c1dt11043h

The Ru(II) complex fac-[RuCl(dmso-S)3(dmso-O)2][PF6] (P2) was found to be an excellent precursorfor the facile preparation in high yield of half sandwich-type compounds of the general formula fac-[RuCl(dmso-S)3(N)2][PF6] (e.g. (N)2 = 1,2-diaminoethane (en, 4), trans-1,2-diaminocyclohexane(dach, 5), or 2 NH3 (6)). Neutral half sandwich-type compounds of the general formula fac-[RuCl(dmso-S)3(N–O)] where N–O is an anionic chelating ligand (e.g. N–O = picolinate (pic, 7)) arebest prepared from the universal Ru(II)-dmso precursor cis-[RuCl2(dmso)4] (P1). These complexes, thatwere fully characterized in solution and in the solid state, are structurally similar to the anticancerorganometallic compounds [Ru(h6-arene)(chel)Cl][PF6]n but, in place of a face-capping arene, have thefac-Ru(dmso-S)3 fragment. In contrast to what observed for the corresponding arene compounds, thatrapidly hydrolyze the Cl ligand upon dissolution in water, compounds 4–6 are very stable and inert inaqueous solution. Probably their inertness is the reason why they showed no significant cytotoxicityagainst the MDA-MB-231 cancer cell line.

Introduction

To date, the most successful Ru-anticancer drugs are the twoRu(III) anionic coordination compounds developed in the late1980s NAMI-A and KP1019 (Fig. 1), they are however stillat the stage of early clinical trials. Their chemistry, anticanceractivity and presumed mechanisms of action have been extensivelyreviewed and will not be further addressed here.1–3 Suffice it to saythat both compounds showed in vivo activity remarkably differentfrom that of Pt drugs in preclinical animal models and that theyare currently undergoing phase 2 clinical evaluation.

In recent years, the focus in the search for novel rutheniumanticancer drugs has shifted from coordination to organometalliccompounds and from Ru(III) to Ru(II),4 and new classes of halfsandwich arene–Ru(II) compounds with promising anticanceractivity have emerged.5 In particular some compounds of thegeneral formula [Ru(h6-arene)(chel)Cl][PF6]n (chel = N–N, N–Oor O–O chelating ligand; n = 1 or 0, depending on the chargeof chel) showed excellent activity both in vitro against human

aDepartment of Chemical and Pharmaceutical Sciences, Via L. Giorgieri 1,34127, Trieste, Italy. E-mail: jbratsos@units.itbCallerio Foundation Onlus, Via A. Fleming 22-31, 34127, Trieste, Italy† Electronic supplementary information (ESI): Tables of X-ray crystal-lographic data in CIF format for compounds P2–BF4, 2–7; Tables ofcrystallographic data of compounds 2 and 3; Ortep drawing of complex5B. CCDC reference numbers 828778–828782, 828784 and 828786. ForESI and crystallographic data in CIF or other electronic format see DOI:10.1039/c1dt11043h

Fig. 1 Chemical structures of NAMI-A and KP1019.

cancer cells – including some platinum-resistant lines – and invivo against animal tumor models.6 Best results were obtained forchel = 1,2-diaminoethane (en). These compounds are believed tobe activated through hydrolysis of the chloride ligand and to targetDNA forming mono-functional adducts.7,8

A few years ago we started a structure-activity relation-ship investigation aimed to establish whether the h6-arenefragment of these organometallic half sandwich compoundsmight be effectively replaced by another neutral 6-electron

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donor face-capping ligand – or by three monodentate lig-ands (L) that form a stable fac-Ru(L)3 fragment – whilemaintaining the other ligands unchanged. Thus, we devel-oped series of new half sandwich Ru(II) coordination com-pounds of the general formula [Ru([9]aneS3)(chel)Cl][X],9,10

[Ru([9]aneS3)(chel)(dmso-S)][X]2, [Ru([9]aneS3)(chel)(dmso-S)],11

and [Ru([9]aneN3)(chel)(dmso-S)][X]212 (where chel = neutral N–

N chelating ligand such as en, 2,2¢-bipyridine (bipy) or substitutedbipy, or chel = O–O chelating dicarboxylate ligand such as oxalateor malonate, X = CF3SO3, PF6, Cl), in which the aromaticfragment was replaced either by the sulfur macrocycle 1,4,7-trithiacyclononane ([9]aneS3) or by the corresponding nitrogenmacrocycle 1,4,7-triazacyclononane ([9]aneN3, also known astacn). So far, only complexes that are capable of hydrolyzing themonodentate ligand at a reasonable rate and of acting as hydrogenbond donors through the chelating ligand (e.g. chel = en or dach,where dach = trans-1,2-diaminocyclohexane) were found to havemoderate antiproliferative activity in vitro.9,10,12

In this paper we describe the synthesis, characterization andsome in vitro cytotoxicity results for a series of new halfsandwich-type complexes of the general formula fac-[RuCl(dmso-S)3(N)2][PF6] (where (N)2 = en (4), dach (5), or 2 NH3 (6)) that, inplace of a face-capping arene or macrocycle, bear three S-bondeddmso ligands in facial geometry, an arrangement that is knownto be particularly stable on Ru(II) species (Fig. 2).13 In addition,for comparison reasons, the neutral complex, i.e. fac-[RuCl(dmso-S)3(N–O)] (7) where N–O is the anionic chelating ligand picolinate,was prepared and characterized.

Fig. 2 Schematic representation of half sandwich organometallic andcoordination compounds that differ for the nature of the face-cappingfragment. From left to right: h6-arene, [9]aneS3, [9]aneN3, and (dmso-S)3.

Experimental

Materials

The precursor cis,fac-[RuCl2(dmso-S)3(dmso-O)] (P1, from nowon simply cis-[RuCl2(dmso)4]) was synthesized according toa recently published upgraded procedure.14 The ligands 1,2-diaminoethane (en), (±)-trans-1,2-diaminocyclohexane (dach) andpyridine-2-carboxylic acid (picolinic acid, Hpic) were purchasedfrom Sigma-Aldrich and used as received. Gaseous ammoniawas obtained upon gentle heating of a mixture of NH4Cl(s) andKOH(s) connected to the flask containing the reaction mixture.

Instrumental methods

Mono- (1H, 13C) and bi-dimensional (1H-1H COSY, 1H-13C HSQC,1H-13C HMBC) NMR spectra were recorded on a Varian 500 MHzspectrometer. All spectra were run at room temperature (r.t.). 1Hchemical shifts in D2O were referenced to the internal standard 2,2-dimethyl-2,2-silapentane-5-sulfonate (DSS) at d = 0.00, while inCD3NO2 and CDCl3 were referenced to the peaks of residual non-

deuterated solvents (d = 4.33 and 7.26, respectively); 13C chemicalshifts in D2O were referenced to the peak of residual free dmso(d = 39.4), while in CD3NO2 to the peak of residual non-deuteratedsolvent (d = 62.8). UV-Vis spectra were obtained on a Jasco V-500UV-Vis spectrophotometer equipped with a Peltier temperaturecontroller, using 1.0 cm path-length quartz cuvettes (3.0 mL). Thesamples (1.0 mM) were prepared in distilled H2O and all spectrawere run at 25 ◦C. Infrared spectra were recorded on a Perkin-Elmer 983G spectrometer. Elemental analysis was performed atthe Dipartimento di Scienze e Tecnologie Chimiche, University ofUdine (Italy).

Synthesis of the complexes

fac-[RuCl(dmso-S)3(dmso-O)2][PF6] (P2). To a solution of500.0 mg of cis-[RuCl2(dmso)4] (P1, 1.03 mmol) in 30 mL ofmethanol, 400 ml of DMSO (5.63 mmol) and 260.0 mg of AgPF6

(1.03 mmol) were added, and the reaction mixture was refluxedfor 1.5 h in the dark. The AgCl precipitate was filtered off overcelite and the yellow filtrate was rotary concentrated to ca 5mL and saturated with diethyl ether added dropwise. A yellowsolid precipitated on standing at room temperature for 24 h,and was collected by filtration, washed with cold methanol anddiethyl ether and vacuum dried (231.0 mg, 67%). Found: C, 17.6;H, 4.57. C10H30ClF6O5PRuS5 (672.15) requires: C, 17.9; H, 4.50.Complex P2 is soluble in H2O, methanol, acetone, nitromethaneand dichloromethane, slightly soluble in chloroform, while it isinsoluble in ethanol. Selected IR (KBr) nmax/cm-1 1124 s and1094 s (S O(dmso-S)), 927 s (S O (dmso-O)), 483 w (Ru–O), 426 m(Ru–S). dH (500 MHz, D2O) P2: 3.42 and 3.41 (12 H, 2 partiallyoverlapping s, CH3 dmso-S), 3.26 (6 H, s, CH3 dmso-S), 2.88 (12H, s, CH3 dmso-O); P2a (fac-[RuCl(dmso-S)3(dmso-O)(H2O)]+,see text): 3.48 (3 H, s, CH3 dmso-S), 3.47 (3 H, s, CH3 dmso-S),3.39 (3 H, s, CH3 dmso-S), 3.37 (3 H, s, CH3 dmso-S), 3.35 (3 H, s,CH3 dmso-S), 3.33 (3 H, s, CH3 dmso-S), 2.92 (3 H, s, CH3 dmso-O), 2.89 (3 H, s, CH3 dmso-O); P2b (fac-[RuCl(dmso-S)3(H2O)2]+

see text): 3.50 (6 H, s, CH3 dmso-S), 3.42 (6 H, s, CH3 dmso-S),3.38 (6 H, s, CH3 dmso-S).

The preparation of P2-BF4 is very similar to that of P2 usingAgBF4 instead of AgPF6. Crystals suitable for X-ray analysis wereobtained from a DMSO solution of P2–BF4 saturated with acetoneand n-hexane.

cis,cis-[RuCl2(dmso-S)2(en)] (1) and trans-[RuCl2(dmso-S)2(en)](2). To a suspension of P1 (503.0 mg, 1.04 mmol) in ethanol(60 mL), a moderate excess of 1,2-diaminoethane (100 mL,1.50 mmol) was added and the mixture was refluxed for 4 h; thesolution, obtained upon warming, gradually turned from yellowto orange. Concentration under vacuum to ca 20 mL led to thespontaneous precipitation of a small amount of the cis,cis-isomer1 as a yellow solid. The solid was collected by filtration, washedwith cold ethanol and diethyl ether and vacuum dried (55.0 mg,13.4%). Addition of diethyl ether to the orange filtrate inducedthe formation of the trans,cis-isomer 2 as an orange solid, whichwas collected by filtration, washed with cold ethanol and diethylether and vacuum dried (75.0 mg, 18.6%). Crystals of 2 suitablefor X-ray analysis were obtained by slow diffusion of diethyl etherin the solution.

1: Found: C, 18.8; H, 5.26; N, 7.24. C6H20Cl2N2O2RuS2 (388.34)requires: C, 18.6; H, 5.19; N, 7.21. Complex 1 is soluble in

9534 | Dalton Trans., 2011, 40, 9533–9543 This journal is © The Royal Society of Chemistry 2011

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water, chloroform, nitromethane, slightly soluble in methanol andethanol. dH (500 MHz, D2O) 4.50 (1 H, br, NH en), 4.41 (1 H, br,NH en), 3.93 (1 H, br, NH en), 3.39 (3 H, s, CH3 dmso-S), 3.35 (3H, s, CH3 dmso-S), 3.32 (3 H, s, CH3 dmso-S), 3.27 (3 H, s, CH3

dmso-S), 2.98 (2 H, br, CH2 en), 2.55 (2 H, br, CH2 en).2: Found: C, 18.7; H, 5.20; N, 7.19. C6H20Cl2N2O2RuS2 (388.34)

requires: C, 18.6; H, 5.19; N, 7.21. Complex 2 is soluble in water,methanol, chloroform, nitromethane, slightly soluble in ethanol,while it is insoluble in dichloromethane. dH (500 MHz, D2O) 4.37(4 H, br, NH2 en), 3.29 (12 H, s, CH3 dmso-S), 2.90 (4 H, br, CH2

en).

[{cis,fac-RuCl2(dmso-S)3}2(l-en)] (3). 1,2-diaminoethane(6.9 mL, 0103 mmol) was added to a solution of 100.0 mg of P1(0,206 mmol) in methanol (5 mL) and the solution was allowedto stand at room temperature for a prolonged time (ca. 5 days).During this time, crystals suitable for X-ray analysis formed inthe solution, and were then removed by filtration, washed with amixture of methanol/diethyl ether (1 : 1) and finally with diethylether and vacuum dried (53.6 mg, 59.5%). Found: C, 19.3; H,5.10; N, 3.17. C14H44Cl4N4O6Ru2S6 (Mr 872.85) requires: C, 19.3;H, 5.08; N, 3.21. Complex 3 is soluble in water, chloroform,nitromethane, slightly soluble in methanol, dichloromethane,while it is insoluble in ethanol and acetone. UV-Vis lmax (H2O)/nm302 and 352 (e/dm3 mol-1 cm-1 442 and 713). Selected IR (KBr)nmax/cm-1 3262 m, 3223 m and 3147 m (NH2), 3032 m, 3005 mand 2920 m (CH2), 1083 s (S O(dmso-S)), 424 m (Ru–S). dH (500MHz, D2O) 3.68 (4 H, br, NH2 en), 3.47 (12 H, s, CH3 dmso-S),3.44 (12 H, s, CH3 dmso-S), 3.41 (12 H, s, CH3 dmso-S), 3.04 (4H, br, CH2 en). dH (500 MHz, CDCl3) 3.50 (4 H, br, NH2 en), 3.43(12 H, s, CH3 dmso-S), 3.42 (12 H, s, CH3 dmso-S), 3.40 (12 H,s, CH3 dmso-S), 3.15 (4 H, br, CH2 en). dH (500 MHz, CD3NO2)3.46 (4 H, br, NH2 en), 3.38 (24 H, two partially overlapped s,CH3 dmso-S), 3.33 (12 H, s, CH3 dmso-S), 3.08 (4 H, br, CH2 en).dC (101 MHz, CD3NO2) 48.0 (CH3 dmso-S), 46.8 (CH3 dmso-S),46.5 (CH3 dmso-S), 46.0 (CH2 en).

fac-[RuCl(dmso-S)3(en)][PF6] (4). To a solution of P2(150.0 mg, 0.223 mmol) in 15 mL dichloromethane, 15 mL of 1,2-diaminoethane (0.224 mmol) was added. Colorless plate crystalssuitable for X-ray analysis formed spontaneously from the undis-turbed solution after overnight standing at room temperature. Thecrystals were collected by filtration, washed with dichloromethaneand vacuum dried (122.2 mg, 95.1%). Found: C, 16.8; H, 4.61;N, 4.92. C8H26ClF6N2O3PRuS3 (575.98) requires: C, 16.7; H, 4.55;N, 4.86. Complex 4 is soluble in water, acetone, nitromethane,while it is insoluble in methanol, ethanol, dichloromethane andchloroform. UV-Vis lmax (H2O)/nm 279 and 328 (e/dm3 mol-1

cm-1 245 and 273). Selected IR (KBr) nmax/cm-1 3335 m, 3274 m,3233 m and 3152 w (NH2), 3022 m, 2974 w and 2930 w (CH2),1093 s and 1082 s (S O(dmso-S)), 424 m (Ru–S). dH (500 MHz,D2O) 3.45 (6 H, s, CH3 dmso-S), 3.44 (6 H, s, CH3 dmso-S), 3.34(6 H, s, CH3 dmso-S), 3.05–2.95 (2 H,m, CH2 en), 2.92–2.82 (2H, m, CH2 en). dH (500 MHz, CD3NO2) 4.22 (2 H, vb, NH2 en),3.85 (2 H, vb, NH2 en), 3.40 (6 H, s, CH3 dmso-S), 3.39 (6 H, s,CH3 dmso-S), 3.27 (6 H, s, CH3 dmso-S), 3.18–3.10 (2 H, m, CH2

en), 3.05–2.97 (2 H, m, CH2 en). dC (101 MHz, D2O) 47.5 (CH3

dmso-S), 46.7 (CH3 dmso-S), 46.4 (CH3 dmso-S), 44.7 (CH2 en),44.6 (CH2 en). dC (101 MHz, CD3NO2) 47.7 (CH3 dmso-S), 47.6(CH3 dmso-S), 46.9 (CH3 dmso-S), 45.7 (CH2 en).

fac-[RuCl(dmso-S)3(dach)][PF6] (5). The same procedure asfor 4 was followed, using 28.2 mL of trans-1,2-diaminocyclohexane(0.24 mmol) (122.0 mg, 86.8%). Found: C, 22.8; H, 5.17; N, 4.41.C12H32ClF6N2O3PRuS3 (630.08) requires: C, 22.9; H, 5.12; N, 4.45.Complex 5 is soluble in water, acetone, nitromethane, DMSO,slightly soluble in methanol and ethanol while it is insoluble inchloroform and dichloromethane. UV-Vis lmax (H2O)/nm 280 and328 (e/dm3 mol-1 cm-1 240 and 265). Selected IR (KBr) nmax/cm-1

3322 m, 3246 s, 3229 m and 3148 w (NH2), 1100 s br, 1061 s and1073(s) (S O(dmso-S)), 431 m (Ru–S). dH (500 MHz, D2O) 5.47(1 H, d br, N1Heq), 5.11 (1 H, d br, N2Heq), 4.19 (1 H, t, N1Hax),3.64 (1 H, t, N2Hax), 3.47 (3 H, s, CH3 dmso-S), 3.45 (6 H, 2overlapped s, 2 ¥ CH3 dmso-S), 3.41 (3 H, s, CH3 dmso-S), 3.38 (3H, s, CH3 dmso-S), 3.30 (3 H, s, CH3 dmso-S), 2.63–2.43 (2 H, m,C2H/C1H), 2.27 (1 H, d, C6Hax), 2.14 (1 H, d, C3Hax), 1.72 (2 H,d, C4Hax/C5Hax), 1.50–1.16 (4 H, m, C6Heq/C3Heq/C4Heq/C5Heq).dC (101 MHz, D2O) 60.7 (C1H), 58.5 (C2H), 48.3 (CH3 dmso-S),47.2 (CH3 dmso-S), 47.1 (CH3 dmso-S), 46.5 (CH3 dmso-S), 46.0(CH3 dmso-S), 45.4 (CH3 dmso-S), 34.4 (C3H2/C6H2), 24.4 and24.3 (C4H2 + C5H2).

fac-[RuCl(dmso-S)3(NH3)2][PF6] (6). Gaseous ammonia wasbubbled through a stirred solution of P2 (150.0 mg, 0.223 mmol) in15 mL of dichloromethane for 20 min, during which time a whitishprecipitate started to form. The mixture was stirred overnight atr.t. and then the precipitate was collected by filtration, washed withdichloromethane and vacuum dried (112.2 mg, 91.5%). Crystalssuitable for X-ray analysis formed spontaneously, within ca 24 h,from the unstirred reaction mixture. Found: C, 13.2; H, 4.37; N,5.13. C6H24ClF6N2O3PRuS3 (549.94) requires: C, 13.1; H, 4.40; N,5.09. Complex 6 is soluble in water, acetone, nitromethane, slightlysoluble in methanol, while it is insoluble in ethanol, chloroformand dichloromethane. UV-Vis lmax (H2O)/nm 279 and 330 (e/dm3

mol-1 cm-1 271 and 276). Selected IR (KBr) nmax/cm-1 3369 m,3342 m, 3329 m, 3316 m and 3187 m (NH3), 1105 s and 1090 s(S O(dmso-S)), 426 m (Ru–S). dH (500 MHz, D2O) 3.43 (6 H,s, CH3 dmso-S), 3.40 (6 H, s, CH3 dmso-S), 3.36 (6 H, s, CH3

dmso-S). dH (500 MHz, CD3NO2) 3.36 (12 H, 2 overlapped s, 2 ¥CH3 dmso-S), 3.30 (6 H, s, CH3 dmso-S), 2.97 (6 H, br, 2 ¥ NH3).

fac-[RuCl(dmso-S)3(pic)] (7). To a solution of P1 (100.0 mg,0.206 mmol) in 5 mL methanol, 25.4 mg of H(pic) (0.206 mmol)and 28.7 mL of triethylamine (20.8 mg, 0.206 mmol) were addedand the mixture was vigorously stirred for 24 h. During thistime, the product precipitated spontaneously from the yellow-orange solution as a white solid, which was collected by filtra-tion, washed with acetone and diethyl ether and vacuum dried.(79.9 mg, 78.7%). Crystals of 7 suitable for X-ray analysis formedspontaneously from the unstirred reaction mixture. Found: C,29.0; H, 4.55; N, 2.79. C12H22ClNO5RuS3 (493.02) requires: C,29.2; H, 4.50; N, 2.84. Complex 7 is soluble in water, chloroform,dichloromethane and nitromethane, slightly soluble in methanoland ethanol, while it is insoluble in acetone. UV-Vis lmax (H2O)/nm270 and 328 (e/dm3 mol-1 cm-1 3 902 and 2 959). Selected IR (KBr)nmax/cm-1 1668 s (COOasym), 1334 s (COOsym), 1102 s and 1082 s(S O(dmso-S)), 430 m (Ru–S). dH (500 MHz, D2O) 9.57 (1 H,dd, J = 5.6, 0.7 Hz, C6H), 8.24 (1 H, td, J = 7.7, 1.4 Hz, C4H),8.16 (1 H, dd, J = 7.8, 1.1 Hz, C3H), 7.88 (1 H, ddd, J = 7.5, 5.6,1.7 Hz, C5H), 3.58 (3 H, s, CH3 dmso-S), 3.52 (3 H, s, CH3 dmso-S), 3.49 (3 H, s, CH3 dmso-S), 3.45 (3 H, s, CH3 dmso-S), 3.13

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Table 1 Crystallographic data for compounds P2–BF4, 2–7

P2–BF4 2 3 4 5·0.5(CH2Cl2) 6 7

Empirical formula C10H30BClF4O5-RuS5

C6H20Cl2N2O2-RuS2

C14H44Cl4N2O6-Ru2S6

C8H26ClF6N2O3-PRuS3

C12.50H33Cl2F6N2-O3PRuS3

C6H24ClF6N2O3-PRuS3

C12H22ClNO5-RuS3

fw 613.97 388.33 872.81 575.98 672.53 549.94 493.01Crystal system Monoclinic Monoclinic Orthorhombic Monoclinic Monoclinic Orthorhombic TetragonalSpace group P21/n P2/n Pbca P21/n P21/c Pbca P42/na/A 10.9250(11) 12.000(4) 14.3210(11) 9.2800(8) 21.399(4) 10.8150(11) 21.307(4)b/A 18.0620(17) 9.201(3) 26.898(3) 16.9870(11) 16.056(3) 17.1720(15)c/A 12.0490(12) 25.854(5) 16.1320(15) 12.5940(15) 15.126(2) 20.8050(17) 7.959(2)b (◦) 91.175(11) 91.04(2) 92.718(10) 92.37(2)V/A3 2377.1(4) 2854.1(14) 6214.1(9) 1983.1(3) 5192.6(15) 3863.8(6) 3613.3(13)Z 4 8 8 4 8 8 8Dcalcd/g cm-3 1.716 1.807 1.866 1.929 1.721 1.891 1.813m(Mo-Ka)/mm-1 1.260 1.751 4.315 1.387 1.173 1.419 1.383F(000) 1248 1568 3536 1160 2728 2208 2000q range, deg 2.03–29.64 1.58–27.10 2.13–26.44 2.50–28.00 1.85–24.71 1.96–25.10 1.35–29.63no. of reflns collcd 30671 30772 22588 22094 69933 40326 36298no. of indep reflns 6594 6083 2294 4605 8475 3136 4966Rint 0.0440 0.0514 0.0487 0.0540 0.0700 0.1040 0.0590no. of reflns (I > 2s(I)) 4612 4294 2063 2779 4060 1299 3100no. of refined params 254 279 319 232 562 216 214goodness-of-fit (F2) 0.927 0.906 1.137 0.861 0.829 0.781 0.867R1, wR2 (I > 2s(I))a 0.0356, 0.0869 0.0333, 0.0745 0.0261, 0.0663 0.0414, 0.0924 0.0500, 0.1169 0.0485, 0.1023 0.0287, 0.0631residuals, e/A3 0.438, -0.312 0.437, -0.379 0.338, -0.534 0.505, -0.462 0.806, -0.639 0.649, -0.502 0.326, -0.296

a R1 = R‖F o| - |F c‖/R |F o|, wR2 = [R w(F o2 - F c

2)2/R w(F o2)2]

12

(3 H, s, CH3 dmso-S), 2.91 (3 H, s, CH3 dmso-S). dC (101 MHz,D2O) 174.8 (COO), 153.9 (C6H), 151.3 (C2), 141.6 (C4H), 130.5(C5H), 128.5 (C3H), 48.2 (CH3 dmso-S), 47.3 (CH3 dmso-S), 46.9(CH3 dmso-S), 45.5 (CH3 dmso-S), 45.4 (CH3 dmso-S), 44.4 (CH3

dmso-S).

Crystallographic measurements

All crystal structures (P2–BF4, 2 and 4–7) were determined atroom temperature on an Enraf Nonius CAD4 single crystaldiffractometer (Mo-Ka radiation, l = 0.71073 A), except thecollection of intensity data for 3 that was performed at the X-ray diffraction beamline of synchrotron Elettra (Trieste) (at 100K, l = 1.00000 A). Cell refinement, indexing and scaling of all thedata sets were performed using programs Denzo and Scalepack.15

All the structures were solved by direct methods and subsequentFourier analyses16 and refined by the full-matrix least-squaresmethod based on F 2 with all observed reflections.16 Hydrogenatoms were placed at calculated positions. The DF map of 5revealed the presence of a lattice molecule of dichloromethanebeside the two independent complexes. In compound 2 onediaminoethane carbon atom (C12) was found disordered over twopositions and refined with occupancies of 0.674(14)/0.326(14).All the calculations were performed using the WinGX System,Ver 1.80.05.17 Crystal data and details of refinements are given inTable 1.

In vitro studies

Cell cultures. The human tumor cell line used for the presentin vitro studies is the breast carcinoma MDA-MB-231. Cells weremaintained in a humidified atmosphere with 5% CO2 at 37.0 ◦C,in Dulbecco’s modified Eagle’s medium (DMEM), (EuroClone,Whetherby, UK) supplemented with 10% fetal bovine serum (FBS)(Invitrogen Italia, Milano, Italy), 2 mM L-glutamine (EuroClone,

Whetherby, UK), 100 UI mL-1 penicillin and 100 mg mL-1

streptomycin (EuroClone, Whetherby, UK), 1% non-essentialaminoacids (EuroClone, Wetherby, UK). Cells from confluentmonolayers were removed from flasks by a trypsin-EDTA solution(SIGMA, St. Louis, MO, USA); cell viability was determined bythe trypan blue exclusion test. For experimental purposes cellswere sown in multiwell culture clusters.

Antiproliferative activity. Cell growth was determined by MTTassay, a colorimetric assay based on the ability of the viable cellsto reduce a soluble yellow tetrazolium salt to blue formazancrystals.18,19 Cells were seeded (2000 cells/100 mL per well) onto96-well plates (Corning Costar, Milano, Italy) in complete mediumwith 5% of FBS. 24 h after the sowing, cells were treated by adding100 mL of complete medium containing the test compounds at theappropriate concentrations.

Final tested concentrations, ranging between 10 mM and 300mM, were obtained by several dilutions from a stock solutionprepared at 10-1 M in DMSO. Maximum DMSO concentration inthe cell incubation medium was £ 0.3% v/v, a value that does notaffect cell growth in controls.

After 72 h of treatment, the MTT dye, 10 mL per 100 mL ofmedium of a 5 mg mL-1 solution prepared in PBS, was addedand plates were incubated for 4 h at 37.0 ◦C. Absorbance wasmeasured, after dissolving the blue formazan crystals with DMSO(Sigma Chemical Co.), at 570 nm using a spectrophotometerSpectra Count Packard R© Bell (Meriden, CT, USA).

Results and discussion

Synthesis and characterization of the complexes

The primary aim of this work was that of finding suitablepreparations for half sandwich-type complexes of the generalformula fac-[RuCl(dmso-S)3(N)2][PF6], where either N is a neutral

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monodentate N-ligand or (N)2 is a neutral N–N bidentate chelator.In the previously investigated half sandwich compounds – bothorganometallic and coordination – best anticancer activities wereobtained when N–N is capable of acting as hydrogen bond donor(e.g. N–N = en or dach), presumably by reinforcing the interactionof the aquated species with target DNA.7 Accordingly, we firstfocused on en. The widely known Ru(II)-dmso precursor cis-[RuCl2(dmso)4] (P1) contains already the desired fac-Ru(dmso-S)3

fragment. However, consistent with its known reactivity towardsN-ligands,13 treatment of P1 with a slight excess of en in refluxingsolvents (e.g. ethanol) typically led to mixtures of the two neutralisomers cis,cis-[RuCl2(dmso-S)2(en)] (1) and trans-[RuCl2(dmso-S)2(en)] (2), with 2 largely prevailing. Compound 1 could beisolated, even though in low yield, by exploiting its lower solubilityin ethanol: when the reaction was performed in this solvent, a smallamount of pure 1 precipitated spontaneously upon careful con-centration of the mother liquor (Scheme 1). Pure 2 was obtained inhigh yield when the reaction was performed in refluxing toluene, asit precipitated spontaneously.20 The unexceptional X-ray structureof one of the two closely comparable independent complexes of 2is shown in Fig. 3 (Selected coordination distances and angles forboth – labeled A and B – are reported in Table S1†).

Fig. 3 X-ray structure of one of the two independent neutral complexestrans-[RuCl2(dmso-S)2(en)] (2) (thermal ellipsoids at 35% probabilitylevel).

It is also known that, under milder conditions, monodentateN-ligands can selectively replace the dmso-O of precursor P1,affording complexes of the general formula cis,fac-[RuCl2(dmso-S)3(N)].21 We found that treatment of P1 with en at ambienttemperature led to the isolation in moderate yield of the dinuclearspecies [{cis,fac-RuCl2(dmso-S)3}2(m-en)] (3, Scheme 1), that wascharacterized also through an X-ray structure (Fig. 4 and TableS2†).22 Even though not unprecedented, the bridging bindingmode of en is not very common either. A search in the CCDCdatabase (Cambridge Database, Version 5.32, November 2010)23

retrieved 25 (non polymeric) X-ray structures of metal complexeswith bridging en moieties, and only one of these includes Ru. Sincein this complex the two Ru(II) atoms are also linked through abridging N atom (intermetallic distance of 3.480 A),24 compound 3is, to our knowledge, the first structurally characterized example ofa dinuclear Ru complex in which the two fragments are connectedby a single bridging en linker that spans the metals at 7.0383(7) A.

Scheme 1 Synthetic strategy for the preparation of compounds 1–3 and7.

The bond lengths of the two units are similar to those foundin the other complexes here described. In particular the Ru–Nbond lengths (2.132(4) and 2.126(4) A) are comparable within theire.s.d.’s to those measured for the chelating en in the two complexesof 2 (range 2.120(3)–2.146(3) A) and only slightly shorter thanthose in the cationic complex 4 (see below). It is worth noting inthe bridging ligand the wide Ru–N–C bond angles (mean 119◦)and the N–C–C–N torsion angle in its skeleton (77.8(5)◦); thislatter is possibly induced by packing requirement.

This preliminary screening clearly confirmed that complex P1is not a suitable precursor for obtaining the target complexes,because neutral ligands are typically unable to replace the chlorideligands of P1. Thus, we reasoned that a possible alternativestrategy might involve the scarcely exploited cationic precursorfac-[RuCl(dmso-S)3(dmso-O)2][X] (P2, X = CF3SO3, PF6, BF4),

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Fig. 4 X-ray structure of the dinuclear complex [{cis,fac-RuCl2-(dmso-S)3}2(m-en)] (3) (thermal ellipsoids at 50% probability level).

easily obtained by treatment of P1 with an equivalent of AgX inthe presence of DMSO (Scheme 2). In practice, one Cl of P1 isreplaced by a dmso-O; the two adjacent dmso-O ligands of P2are relatively labile and smoothly replaced by stronger s-donorligands.

Compound P2 was originally described as BF4 salt in 1979 byBarnes and Goodfellow25 but almost neglected afterwards. We setup procedures for the preparations of different salts of P2 with X =CF3SO3, PF6, BF4; for solubility reasons, afterwards we focusedon the PF6 salt for preparing the substituted derivatives.

A perspective view of the complex molecule is shown in Fig. 5and a selection of coordination distances and angles is reportedin Table 2. The ruthenium atom is coordinated with irregularoctahedral geometry to five dmso ligands: three of them arebonded through S in a facial geometry and two through O. TheRu–S1 and Ru–S2 bond distances (2.2524(7), 2.2474(7) A) andthe Ru–O ones (mean 2.1395(17) A) are within the range found inthe fac-[Ru(dmso-S)3(dmso-O)3]2+ cation.26 The trans-located Ru–S3 (2.2818(8) A) and Ru–Cl1 (2.4159(8) A) bonds have lengthsthat compare well with those measured in cis-[RuCl2(dmso)4].27

Correspondingly, the S1–Ru–S2 angle is 97.13(3)◦, considerablywider than the opposite O5–Ru–O4 angle of 82.03(7)◦. The othertwo S–Ru–S angles (89.84(3) and 93.07(3)◦) are closer to the idealoctahedral values.

The chemical behavior of compound P2 in aqueous solutionconfirms the relative lability of the dmso-O ligands, and the kinetic

Table 2 Selected coordination bond distances (A) and angles (◦) forcompound P2–BF4

Ru–S(1) 2.2524(7) Ru–Cl(1) 2.4159(8)Ru–S(2) 2.2474(7) Ru–O(4) 2.1398(17)Ru–S(3) 2.2818(8) Ru–O(5) 2.1393(17)S(1)–Ru–S(2) 97.13(3) S(3)–Ru–Cl(1) 173.49(3)S(1)–Ru–S(3) 89.84(3) S(3)–Ru–O(4) 86.72(6)S(1)–Ru–Cl(1) 95.70(3) S(3)–Ru–O(5) 88.06(5)S(1)–Ru–O(4) 172.11(5) Cl(1)–Ru–O(4) 87.35(6)S(1)–Ru–O(5) 90.77(5) Cl(1)–Ru–O(5) 88.45(5)S(2)–Ru–S(3) 93.06(3) O(4)–Ru–O(5) 82.03(7)S(2)–Ru–Cl(1) 89.64(3) S(4)–O(4)–Ru 118.24(11)S(2)–Ru–O(4) 90.14(5) S(5)–O(5)–Ru 123.75(11)S(2)–Ru–O(5) 172.02(5)

Fig. 5 X-ray structure of the cationic fragment offac-[RuCl(dmso-S)3(dmso-O)2][BF4] (P2–BF4) (thermal ellipsoids at35% probability level).

and thermodynamic stability of the fac-Ru(dmso-S)3 fragment(Fig. 6). Consistent with the geometry found in the solid stateand with a previous report,28 the 1H NMR spectrum of P2 in D2Opresents three singlets (6H each, two partially overlapped) for themethyls of the three facial dmso-S ligands (d = 3.42, 3.41 and3.26) and one singlet (12H, d = 2.88) for the two adjacent dmso-O ligands. As the equatorial plane is not a plane of symmetryfor the complex, the two pairs of equivalent dmso ligands in thisplane (two S- and two O-bonded) have diastereotopic methyls: thetwo singlets expected for the dmso-S ligands are partially resolved,whereas those of the dmso-O ligands are completely overlapped. Intime, the original signals are progressively replaced by those of the

Scheme 2 Synthetic strategy for the preparation of compounds 4–6 from precursor P2.

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Fig. 6 1H NMR (D2O, 500 MHz) spectral changes in the dmso regionduring the aquation of fac-[RuCl(dmso-S)3(dmso-O)2]+. The symbols (*),( ) and ( ) indicate the dmso resonances of the P2, P2a and P2b species,respectively.

less symmetrical mono-aqua species fac-[RuCl(dmso-S)3(dmso-O)(H2O)]+ (P2a, six singlets for the three inequivalent dmso-Sligands and two singlets for the dmso-O), and eventually by thoseof fac-[RuCl(dmso-S)3(H2O)2]+ (P2b) that has the same symmetryas P2 and shows three equally intense singlets (d = 3.50, 3.42 and3.38) for the fac-Ru(dmso-S)3 fragment. These spectral changesare accompanied by the growth of the singlet for free dmso atd = 2.71, whose final integration is 2/3 of that of the dmso-Ssignals. The aquation process goes to completion in ca 3 h at25 ◦C, and the NMR spectrum remains unchanged afterwardsfor hours: no significant hydrolysis of the chloride (that wouldlead to the symmetrical fac-[Ru(dmso-S)3(H2O)3]2+ species whichis characterized by a singlet at d = 3.44)29 or of the dmso-S ligands(that would increase the free dmso peak) were observed.

Encouraged by these results, we investigated the reactivity ofP2 towards en and dach and found that treatment of P2 withone equivalent of either ligand in CH2Cl2 solution at ambienttemperature led in a few hours to the selective replacement ofthe two adjacent dmso-O ligands and to the spontaneous pre-cipitation of fac-[RuCl(dmso-S)3(en)][PF6] (4) or fac-[RuCl(dmso-S)3(dach)][PF6] (5), respectively, in excellent yields.

We also reasoned that replacement of the two dmso-O ligandsof P2 with two NH3 molecules would provide a complex withimproved H-bonding capability compared to 4 and 5. Actually,during our present studies, the corresponding organometallic Ru–arene compound [RuCl(h6-p-cymene)(NH3)2][PF6] described byKeppler and co-workers was prepared with the same purpose.30

We found that compound fac-[RuCl(dmso-S)3(NH3)2][PF6] (6)precipitates spontaneously in a few minutes upon treatment ofa CH2Cl2 solution of P2 with gaseous ammonia at ambienttemperature.

The crystal structures of complexes 4–6 are depicted in Fig. 7–9and selected bond lengths and angles are shown in Table 3. The

Table 3 Selected coordination bond distances (A) and angles (◦) forcompounds 4–6

4 5, A 5, B 6

Ru–S(1) 2.2780(12) 2.291(2) 2.288(2) 2.286(2)Ru–S(2) 2.2917(11) 2.2887(19) 2.284(2) 2.294(2)Ru–S(3) 2.2740(12) 2.2829(19) 2.2853(19) 2.277(3)Ru–Cl(1) 2.4169(11) 2.4068(18) 2.417(2) 2.421(2)Ru–N(1) 2.158(4) 2.150(5) 2.145(5) 2.157(6)Ru–N(2) 2.153(3) 2.125(5) 2.167(6) 2.174(6)S(1)–Ru–S(2) 93.13(4) 93.91(7) 93.02(8) 93.96(9)S(1)–Ru–S(3) 92.91(4) 91.89(7) 95.28(7) 95.37(10)S(1)–Ru–Cl(1) 92.47(4) 91.53(7) 88.84(7) 90.53(9)S(1)–Ru–N(1) 171.50(10) 171.88(13) 171.93(17) 173.68(19)S(1)–Ru–N(2) 93.06(10) 92.59(16) 93.22(15) 89.06(17)S(2)–Ru–S(3) 93.80(4) 95.85(7) 90.57(7) 92.22(9)S(2)–Ru–Cl(1) 89.16(4) 89.58(7) 92.40(8) 92.62(9)S(2)–Ru–N(1) 94.29(9) 93.12(15) 93.81(17) 91.77(19)S(2)–Ru–N(2) 172.71(10) 172.69(16) 172.94(16) 175.4(2)S(3)–Ru–Cl(1) 173.71(4) 173.37(7) 174.78(8) 172.09(10)S(3)–Ru–N(1) 90.72(11) 91.43(15) 88.94(16) 87.0(2)S(3)–Ru–N(2) 89.71(10) 87.27(15) 92.16(17) 90.9(2)Cl(1)–Ru–N(1) 83.51(11) 84.46(15) 86.58(16) 86.6(2)Cl(1)–Ru–N(2) 86.74(10) 86.91(15) 84.41(17) 83.9(2)N(1)–Ru–N(2) 79.27(13) 80.2(2) 79.7(2) 85.1(3)

Fig. 7 ORTEP drawing (thermal ellipsoids at 35% probability level) ofthe cation of fac-[RuCl(dmso-S)3(en)][PF6] (4).

structural analysis of each complex confirms the substitution ofthe dmso-O ligands of P2 with the N donor ligands en, dach and 2NH3, respectively. In 5 two very similar but independent complexeswere found in the unit cell (labeled A and B in Table 3). In allcomplexes the Ru–S bond lengths have closely comparable values,in the range 2.277(3)–2.291(2) A. The Ru–Cl distance averages to2.415(1) A, whereas the Ru–N distances show small but significantvariations in the range from 2.125(5) to 2.174(6) A. The N–Ru–N coordination angle is close to 80◦ in complexes 4 and 5, andslightly wider in the bis(ammonia) derivative 6 (85.1(3)◦). Thelatter complex has bond distances (e.g. Ru–Cl and Ru–N) andangles (e.g. N–Ru–N) similar to those found for the organometallicanalogue [RuCl(h6-p-cymene)(NH3)2][PF6],30 indicating that thepresence of either an aromatic ring or a fac-Ru(dmso-S)3 fragment

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Fig. 8 ORTEP drawing (thermal ellipsoids at 35% probabilitylevel) of one (labeled as A) of the two independent cations offac-[RuCl(dmso-S)3(dach)][PF6] (5). The structure of compound 5B isdepicted in Fig. S1.†

Fig. 9 ORTEP drawing (thermal ellipsoids at 35% probability level) ofthe cation of fac-[RuCl(dmso-S)3(NH3)2][PF6] (6).

does not influence dramatically the coordination sphere of the Rucenter. The two independent complexes of 5 show small differencesin the bond angles as well as in the Ru–N distances that can bereasonably ascribed to packing requirements. Since compounds4 and 5 crystallize in centrosymmetric space groups, in eachcase both conformational isomers of the five-membered Ru(NH2–CH2–CH2–NH2) rings are present in the crystals (Fig. 10).

For comparison, the coordination geometries of 4–7 agree withthat of the cation of complex [RuCl([9]aneS3)(en)][CF3SO3] (8),which is the [9]aneS3 counterpart of 4, although the steric demandof the three dmso-S ligands (compared to [9]aneS3) should begreater.9

The 1H NMR spectra of complexes 4–6 in D2O are consistentwith the structures found in the solid state. In the spectra of 4 and6 the three facial dmso-S ligands give three singlets (6H each).In the case of 4, this spectral feature is consistent with a rapid

Fig. 10 Perspective view of the two possible conformational isomers ofthe five-membered Ru(NH2–CH2–CH2–NH2) ring formed by the chelatingligands en and dach on the Ru centre.

motion in solution of the –(CH2)2– backbone of en that averagesthis ligand to a planar conformation: as a consequence, the planebisecting the N–Ru–N angle becomes a plane of symmetry for thecomplex. In both spectra the resonances of the NH protons arenot visible, most likely because they are rapidly exchanged withdeuterium from D2O; consistently, their resonances are clearlyvisible in CD3NO2. It is worth noting that in the NMR spectrum inD2O of [RuCl([9]aneS3)(en)][CF3SO3] (8) the NH resonances areclearly visible, also in aged solutions. This observation suggeststhat the NH protons are more acidic in 4 than in 8 (and thusexchanged more rapidly), in agreement with the fact that [9]aneS3

is a significantly weaker p-acceptor than the three facial dmso-Sligands.

The NMR spectrum of complex 5 in D2O is remarkably differentfrom the others. Firstly, owing to the lower symmetry of thiscomplex, the three facial dmso-S ligands originate six singlets (3Heach). In addition, the dach ligand, that is conformationally rigid,gives four well resolved multiplets (two doublets and two triplets)for the NH protons31 and a series of multiplets – most of whichwell resolved – for the CH2 protons. With the aid of H–H and H–Ccorrelation spectra (Fig. 11) it was possible to assign most of theseresonances. Briefly, we explain our stepwise procedure: (1) whentrans-1,2-diaminocyclohexane (dach) is coordinated to a metalcenter, both NH2 groups are equatorial, regardless of their absoluteconfiguration; thus, the two adjacent CH protons – C1H and C2H– must have a fixed axial geometry. (2) According to the Karplusequation, C1H and C2H are expected to give stronger couplingswith the axial protons in the adjacent NH2 and CH2 groups. (3) Wereasonably assumed that the most downfield multiplet (2H) amongthe CH resonances belongs to the C1H and C2H protons (in thephase-sensitive HSQC spectrum this multiplet indeed correlatesto two different CH carbon resonances). (4) Consistent with thisassumption, the resonances that give the most intense cross peakswith this multiplet in the H–H COSY spectrum were assigned tothe axial N1H and N2H (two triplets at d = 4.19 and 3.64 ppm) –as well as to C3H and C6H – protons. (5) With the same reasoning,it was possible to assign the other CH resonances. (6) Protonassignments were confirmed by the HSQC spectrum: typically, ineach CH2 group the resonances of the axial and equatorial protonsare different but they correlate with the same carbon resonance.

When mono-anionic N–O chelating ligands were used in placeof the neutral N–N ligands, the neutral half sandwich typederivatives of the general formula fac-[RuCl(dmso-S)3(N–O)]could be obtained directly from compound P1. For example,treatment of P1 with picolinic acid (Hpic) in the presence oftriethylamine afforded fac-[RuCl(dmso-S)3(pic)] (7) in good yield.In this case, the anionic N–O donor ligand pic replaces the labile

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Fig. 11 1H-1H COSY (left) and 1H-13C HSQC (right) NMR spectra of complex 5 in D2O. In the COSY spectrum are indicated as dotted lines theassignment pathways starting from the equatorial N1H (red) and from the equatorial N2H (blue). In the HSQC spectrum, red cross-peaks pertain to CHand CH3 groups, blue cross-peaks pertain to CH2 groups. On top is the schematic representation of complex 5 with numbering scheme.

Table 4 Selected coordination bond distances (A) and angles (◦) forcompound 7

Ru–S(1) 2.2907(8) Ru–Cl(1) 2.4269(8)Ru–S(2) 2.2778(8) Ru–O(4) 2.0874(18)Ru–S(3) 2.2537(8) Ru–N(1) 2.122(2)S(1)–Ru–S(2) 93.18(3) S(2)–Ru–N(1) 98.56(6)S(1)–Ru–S(3) 97.44(3) S(3)–Ru–Cl(1) 174.98(3)S(1)–Ru–Cl(1) 85.97(3) S(3)–Ru–O(4) 90.48(5)S(1)–Ru–O(4) 89.60(5) S(3)–Ru–N(1) 91.72(6)S(1)–Ru–N(1) 165.10(6) Cl(1)–Ru–O(4) 85.85(5)S(2)–Ru–S(3) 90.06(3) Cl(1)–Ru–N(1) 84.20(6)S(2)–Ru–Cl(1) 93.43(3) O(4)–Ru–N(1) 78.56(8)S(2)–Ru–O(4) 177.08(5)

dmso-O ligand and one chloride ion of P1, as verified by thestructural analysis of 7 (Fig. 12, Table 4).

In complex 7 the Ru ion displays a distorted octahedralgeometry upon formation of the 5-membered ring with thechelating picolinate. The bond distances are comparable to thosefound for the cis-[Ru(dmso-S)2(pic)2] complex, described a fewyears ago.32

The 1H NMR spectrum of 7 in D2O is consistent with themolecular structure found in the solid state. In the upfield region,the spectrum exhibits six equally intense (integrating for 3H each)singlets attributed to the diastereotopic methyl groups of the threeinequivalent dmso-S molecules. Worth noting, while four methyl

Fig. 12 X-ray structure of the neutral complex fac-[RuCl(dmso-S)3(pic)](7) (thermal ellipsoids at 35% probability level).

resonances (at d = 3.58, 3.52, 3.49 and 3.45) are in the region typicalfor dmso-S, the other two (at d = 3.13 and 2.91) are remarkablyshifted upfield because the methyls fall into the shielding coneof the adjacent aromatic rings of pic. The pic ligand displays theexpected pattern of resonances in the aromatic region, that werefully assigned by the 1H–1H COSY experiment.

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Table 5 IC50 values of compounds 4 and 5 on MDA-MB-231 cells incomparison to 8 and RM175

Compound IC50 (mM)

fac-[RuCl(dmso-S)3(en)][PF6] (4) > 300fac-[RuCl(dmso-S)3(dach)][PF6] (5) > 300[RuCl([9]aneS3)(en)][CF3SO3] (8)a 146–177[RuCl(h6-bip)(en)][PF6] (RM175)a 8.99–12,1

a from ref. 10

Chemical behavior in aqueous solution

Compounds 4–6 are very stable in aqueous solution: their NMRspectra in D2O remained unchanged for several hours afterdissolution. Also the addition of excess NaCl (20 : 1) induced nochanges in the spectra. Thus we had no experimental evidence thatchloride dissociation occurs at a measurable rate, with formationof the fac-[Ru(dmso-S)3(N)2(H2O)]2+ aqua species that is typicallyaccompanied by a downfield shift of all proton resonances. Incontrast, the corresponding half sandwich [9]aneS3 complexeswith en and dach upon dissolution in water rapidly establishan equilibrium with the aqua species [Ru([9]aneS3)(en)(H2O)]2+

and [Ru([9]aneS3)(dach)(H2O)]2+, respectively.9,12 Compound 7behaves similarly to 4–6 upon dissolution in water: no chloridedissociation was evidenced within the first hours. However, partialrelease of dmso was observed after one day, leading to theformation of several unidentified species.

Consistent with their stability in aqueous solution, compounds4–6 did not interact significantly with the DNA model bases 9-ethylguanine and 5¢-AMP at ambient temperature. No changeswere observed in the NMR spectra of 1 : 1 mixtures of eachcomplex with either ligand, also for long observation times.

Antiproliferative activity

The in vitro cytotoxic activity of compounds 4 and 5 was inves-tigated against the MDA-MB-231 human mammary carcinomacell line, in comparison with that of the previously investigatedhalf sandwich compounds 8 and [RuCl(h6-bip)(en)][PF6] (RM175,bip = biphenyl).10 The IC50 values reported in Table 5 show that,whereas the organometallic half sandwich compound RM175 isremarkably cytotoxic and 8 is moderately cytotoxic on this cellline, complexes 4 and 5 are devoid of any significant cytotoxicity.33

Conclusions

The Ru(II) complex fac-[RuCl(dmso-S)3(dmso-O)2][PF6] (P2)proved to be an excellent precursor for the facile preparationof our target half sandwich-type compounds containing thefac-Ru(dmso-S)3 fragment. Compound P2 itself is easily pre-pared in high yield from the universal Ru(II)-dmso precursorcis-[RuCl2(dmso)4] (P1) and selectively replaces – under mildconditions – the two adjacent dmso-O ligands with differentmono- and bidentate neutral N-donor ligands, affording com-pounds of the general formula fac-[RuCl(dmso-S)3(N)2][PF6]. Inparticular, in this work we reported the high-yield preparationand structural characterization of fac-[RuCl(dmso-S)3(en)][PF6](4), fac-[RuCl(dmso-S)3(dach)][PF6] (5) and fac-[RuCl(dmso-S)3(NH3)2][PF6] (6). On the other hand, P1 proved to be a

suitable precursor, by replacement of the labile dmso-O and oneCl ligand, for the preparation of half sandwich-type compoundsof the general formula fac-[RuCl(dmso-S)3(N–O)] where N–O isan anionic chelating ligand, such as in fac-[RuCl(dmso-S)3(pic)](7).

Contrary to what observed for the corresponding [9]aneS3

half sandwich compounds [RuCl([9]aneS3)(en)][CF3SO3] (8) and[RuCl([9]aneS3)(dach)][PF6], that rapidly hydrolyze the Cl ligandupon dissolution in water,9,12 compounds 4–6 are very stable andinert in aqueous solution. Thus, the nature of the face-cappinggroup or ligand has a great influence on the chemical behaviorof the compound, even when the set of donor atoms remainsunchanged (i.e. three S atoms).

Compounds 4 and 6 – that are well soluble in water – proved tohave no significant cytotoxicity against the MDA-MB-231 cellline. These results, even though concerning only a single cellline, demonstrate that in half sandwich Ru(II) compounds thenature of the face-capping ligand or group has a great importancein determining the in vitro anticancer activity, primarily bytuning their activation rate, i.e. the rate of chloride hydrolysis.In substantial agreement with the results obtained by Sadler andco-workers for the organometallic half sandwich compounds,6,34

the relatively rapid availability of one coordination position onthe Ru center is apparently an essential – even though notunique – requirement for observing anticancer activity. Once thisrequirement is fulfilled, other parameters – such as the hydrogenbonding ability of the chelating ligand and the lipophilicity ofthe compound – come into play for tuning the activity. Thus ourresults indirectly confirm that the mechanism of action of thehalf sandwich of Ru(II) compounds – regardless of the natureof the face-capping ligand – involves their coordination to atarget molecule (presumably DNA). However, the following pointsshould be carefully considered: (i) the antiproliferative tests shouldbe extended to other cancer cell lines before any serious assessmentof the in vitro activity of these compounds can be made; (ii) it istypically assumed that in vitro cytotoxicity against cancer cell linesis a prerequisite for observing in vivo activity. However, the historyof metal anticancer compounds has already shown that most oftenin vitro cytotoxicity has not been paralleled by in vivo therapeuticactivity, and only a few of the many metal compounds investigatedin vitro have shown some realistic follow-up. On the other hand,there are examples of compounds that are basically devoid ofin vitro cytotoxic activity but showed remarkable in vivo activity,the most significant being the antimetastatic compound NAMI-A. (iii) Finally, it should be noted that inert and coordinativelysaturated metal complexes, i.e. compounds that are unable to bindcoordinatively to biological targets and that we defined structuralcompounds,35 not necessarily are devoid of any biological activity.On the contrary, the number of examples of structural compoundsthat have relevant activity is rapidly increasing.36,37

Acknowledgements

Regione FVG (Project “Nuove Terapie e Farmaci Antitumorali”),Fondazione CRTrieste, Fondo Trieste, Fondazione BeneficentiaStiftung for financial support and BASF Italia Srl for a generousdonation of hydrated ruthenium chloride are gratefully acknowl-edged. Fondazione CRTrieste is also gratefully acknowledged forthe munificent donation of a Varian 500 NMR spectrometer to

9542 | Dalton Trans., 2011, 40, 9533–9543 This journal is © The Royal Society of Chemistry 2011

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the Department of Chemical Sciences. This study was performedwithin the frame of COST Action D39.

References

1 I. Bratsos, T. Gianferrara, E. Alessio, C. G. Hartinger, M. A. Jakupecand B. K. Keppler, in Bioinorganic Medicinal Chemistry, ed. E. Alessio,Wiley-VCH, Weinheim, 2011, pp. 151–174.

2 I. Bratsos, S. Jedner, T. Gianferrara and E. Alessio, Chimia, 2007, 61,692–697.

3 C. G. Hartinger, M. A. Jakupec, S. Zorbas-Seifried, M. Groessl, A.Egger, W. Berger, H. Zorbas, P. J. Dyson and B. K. Keppler, Chem.Biodiversity, 2008, 5, 2140–2155.

4 A. Levina, A. Mitra and P. A. Lay, Metallomics, 2009, 1, 458–470.5 G. Suss-Fink, Dalton Trans., 2010, 39, 1673–1688.6 F. Wang, A. Habtemariam, E. P. L. Van Der Geer, R. Fernandez,

M. Melchart, R. J. Deeth, R. Aird, S. Guichard, F. P. A. Fabbiani,P. Lozano-Casal, I. D. H. Oswald, D. I. Jodrell, S. Parsons and P. J.Sadler, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 18269–18274.

7 H. Chen, J. A. Parkinson, R. E. Morris and P. J. Sadler, J. Am. Chem.Soc., 2003, 125, 173–186.

8 S. J. Dougan and P. J. Sadler, Chimia, 2007, 61, 704–715.9 B. Serli, E. Zangrando, T. Gianferrara, C. Scolaro, P. J. Dyson, A.

Bergamo and E. Alessio, Eur. J. Inorg. Chem., 2005, 3423–3434.10 I. Bratsos, S. Jedner, A. Bergamo, G. Sava, T. Gianferrara, E.

Zangrando and E. Alessio, J. Inorg. Biochem., 2008, 102, 1120–1133.11 I. Bratsos, G. Birarda, S. Jedner, E. Zangrando and E. Alessio, Dalton

Trans., 2007, 4048–4058.12 I. Bratsos, E. Mitri, E. Zangrando, A. Bergamo, G. Sava and E. Alessio,

unpublished results.13 E. Alessio, Chem. Rev., 2004, 104, 4203–4242.14 I. Bratsos and E. Alessio, Inorg. Synth., 2010, 35, 148–152.15 Z. Otwinowski and W. Minor, Methods Enzymol., 1997, 276, 307–326.16 G. M. Sheldrick, ed., SHELXL-97 Programs for Crystal Structure

Analysis (Release 97-2), University of Gottingen, Germany, 1998.17 L. J. Farrugia, J. Appl. Crystallogr., 1999, 32, 837–838.18 M. C. Alley, D. A. Scudiero, A. Monks, M. L. M. J. Hursey Czerwinski,

D. L. Fine, B. J. Abbott, J. G. Mayo, R. H. Shoemaker and M. R. Boyd,Cancer Res., 1988, 48, 589–601.

19 T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.20 As expected, trans-[RuCl2(dmso-S)2(en)] was also selectively obtained

in pure form and in high yield upon treatment of the geometrical isomerprecursor trans-[RuCl2(dmso-S)4] with en in chloroform at ambienttemperature. For related ref see: E. Alessio, E. Iengo, E. Zangrando, S.

Geremia, P. A. Marzilli and M. Calligaris, Eur. J. Inorg. Chem., 2000,2207–2219.

21 M. Henn, E. Alessio, G. Mestroni, M. Calligaris and W. M. Attia,Inorg. Chim. Acta, 1991, 187, 39–50.

22 In the experimental section the most efficient preparation of compound3 is described, in which P1 is treated with half equivalent of en. However,the same product was initially isolated, although with lower yield, usingequimolar amounts of the reactants.

23 F. H. Allen, O. Kennard and R. Taylor, Acc. Chem. Res., 1983, 16,146–153.

24 W. P. Griffith, N. T. McManus and A. C. Skapski, J. Chem. Soc., Chem.Commun., 1984, 434–435.

25 J. R. Barnes and R. J. Goodfellow, J. Chem. Res. (M), 1979, 4301.26 A. R. Davies, F. W. B. Einstein, N. P. Farrell, B. R. James and R. S.

McMillan, Inorg. Chem., 1978, 17, 1965–1969.27 E. Alessio, G. Mestroni, G. Nardin, W. M. Attia, M. Calligaris, G. Sava

and S. Zorzet, Inorg. Chem., 1988, 27, 4099–4106.28 J. D. Fotheringham, G. A. Heath, A. J. Lindsay and T. A. Stephenson,

J. Chem. Res. (S), 1986, 82–83.29 I. Bratsos, A. Bergamo, G. Sava, T. Gianferrara, E. Zangrando and E.

Alessio, J. Inorg. Biochem., 2008, 102, 606–617.30 S. Grguric-Sipka, I. N. Stepanenko, J. M. Lazic, C. Bartel, M. A.

Jakupec, V. B. Arion and B. K. Keppler, Dalton Trans., 2009, 3334–3339.

31 It is worth noting that, contrary to what just observed for 4 and 6, inthe D2O NMR spectrum of 5 the NH resonances are clearly visibleand persistent in time, implying that H/D exchange does not occurat any observable rate. This striking difference between very similarcomplexes might depend on the rigidity of coordinated dach that makesthe transition state involved in the NH/ND exchange hardly accessible.

32 A. A. Rachford, J. L. Petersen and J. J. Rack, Dalton Trans., 2007,3245–3251.

33 In this preliminary study complex 6 was not investigatedbecause the corresponding organometallic compound [Ru(h6-p-cymene)(NH3)2Cl][PF6] was found to be poorly cytotoxic, ca. twoorders of magnitude less than the active [Ru(h6-p-cymene)(en)Cl][PF6]compound against three cell lines (A549, CH1 and SW480).

34 A. M. Pizarro, A. Habtemariam and P. J. Sadler, in Topics inOrganometallic Chemistry, 2010, vol. 32, pp. 21–56.

35 T. Gianferrara, I. Bratsos and E. Alessio, Dalton Trans., 2009, 7588–7598.

36 E. Meggers, G. E. Atilla-Gokcumen, K. Grundler, C. Frias and A.Prokop, Dalton Trans., 2009, 10882–10888.

37 C. Kunick and I. Ott, Angew. Chem., Int. Ed., 2010, 49, 5226–5227.

This journal is © The Royal Society of Chemistry 2011 Dalton Trans., 2011, 40, 9533–9543 | 9543

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