DMSO-Mediated Ligand Dissociation: Renaissance for Biological Activity ofN-Heterocyclic-[Ru ACHTUNGTRENNUNG(h6-arene)Cl2] Drug Candidates

Malay Patra,*[a] Tanmaya Joshi,[a] Vanessa Pierroz,[a, b] Katrin Ingram,[c] Marcel Kaiser,[c]

Stefano Ferrari,[b] Bernhard Spingler,[a] Jennifer Keiser,[c] and Gilles Gasser*[a]

� 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2013, 19, 14768 – 1477214768

DOI: 10.1002/chem.201303341

The swiftly progressing field of metal-based drug researchhas, in recent years, seen active interest in the developmentof non-platinated anticancer compounds. As an outcome ofthis shifting momentum, several new classes of metal com-plexes have been inducted into the library of potential alter-natives.[1] With KP1019 and NAMI-A (both in phase II clini-cal trial) as two standout candidates, ruthenium (Ru) com-plexes continue to dominate the research in this direction.[2]

One of the popularly studied class of compounds from theruthenium family are the RuII organometallic complexeswith a general formula [Ru ACHTUNGTRENNUNG(h6-arene)(X)(Y)(Z)], in whichthe arene units and the ligands (X, Y and Z) resemble theseat and legs of the piano-stool, respectively (Figure 1).[3]

Also referred to as half sandwich “piano-stool” complexes,these have shown promising in vitro as well as in vivo anti-cancer activity.[2,3]

With a view to further improve the efficacy of such“piano-stool” systems, various biologically active ligands,like ethacrynic acid derivatives (GST inhibitor), phenoxa-zine derivatives (P-glycoprotein inhibitor), flavone deriva-tives (topoisomerase inhibitor),[4] and organic anticancerdrugs (e.g., metronidazole (MTZ)[5] and lonidamine[6] deriv-atives), have been incorporated into the RuACHTUNGTRENNUNG(h6-arene)framework with the hope that they might interact with mul-tiple biological targets in a synergetic manner. Beyond anti-cancer research, [Ru ACHTUNGTRENNUNG(h6-arene)] half-sandwich complexeshave also been studied for their antiparasitic activities.[7]

In most cases, the biologically relevant ligands (L) havebeen linked to the ruthenium centre via N-coordination ofa nitrogen heterocycle.[6,8] Due to the limited aqueous solu-bility of such [Ru ACHTUNGTRENNUNG(h6-arene)Cl2(L)] complexes, their stocksolutions in DMSO are usually employed for in vitro biolog-

ical activity determination.[6–9]

This has become a more or lessstandard experimental protocol.However, we noticed, visuallyand by means of NMR spec-troscopy, an immediate liganddissociation when a similarruthenium complex of a well-known anthelmintic drug, me-bendazole (MBZ), [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2ACHTUNGTRENNUNG(MBZ)] (1, Figure 1,cym= cymene), was dissolvedin DMSO (vide infra). Much toour surprise, literature surveyrevealed the in-solution dis-placement of nitrogen heterocy-clic or amine-based ligandsfrom the metal coordinationsphere in RuACHTUNGTRENNUNG(h6-arene) “piano-stool” complexes has, in gener-al, itself been touched upononly briefly in previous re-ports.[8c,10] Metal-based drugsare attracting the attention ofan increasing number of majorinternational research groups.

Thus, such significantly rapid dissociation of ligand inDMSO (as observed for 1), which is a popular medium forpreparing stock solutions for biological screening, if also oc-curring in other similar complexes, demands serious consid-eration. It can indeed have a crucial impact on the biologicalactivity outcomes.

Judging from the NMR spectroscopy examination of [Ru-ACHTUNGTRENNUNG(h6-arene)Cl2(L)] type complexes (L= N-heterocyclic ligandnamely, benzimidazoles, imidazoles, pyridine and quinoline,and their derivatives; Figure 1 and Scheme S1 in the Sup-porting Information) conducted in our laboratories, theDMSO-mediated ligand dissociation indeed takes place.This raises questions about the reported interpretations andconclusions on the activity of these and other similar com-plexes, which could have been misled in the past.[6–9] In fact,

[a] Dr. M. Patra, Dr. T. Joshi, V. Pierroz, Priv.-Doz. Dr. B. Spingler,Prof. Dr. G. GasserInstitute of Inorganic Chemistry, University of ZurichWinterthurerstrasse 190, 8057 Zurich (Switzerland)E-mail : malay.patra@uzh.ch

gilles.gasser@aci.uzh.ch

[b] V. Pierroz, Priv.-Doz. Dr. S. FerrariInstitute of Molecular Cancer Research, University of ZurichWinterthurerstrasse 190, 8057 Zurich (Switzerland)

[c] Dr. K. Ingram, M. Kaiser, Prof. Dr. J. KeiserDepartment of Medical Parasitology and Infection BiologySwiss Tropical and Public Health Institute, Socinstrasse 574002 Basel, Switzerland & University of BaselPetersplatz 1, 4003 Basel (Switzerland)

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201303341.

Figure 1. Representative examples of ruthenium half-sandwich complexes examined in this work (seeScheme S1 in the Supporting Information for the structures of other complexes examined).

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COMMUNICATION

the data we obtained from biological studies strongly sup-ports our initial speculation that the extent of this dissocia-tion has an influence on activity. Beyond this, it also revealsthat mixing the otherwise inactive [Ru ACHTUNGTRENNUNG(h6-arene)Cl2-ACHTUNGTRENNUNG(DMSO)] complex (6, Figure 1) and respective biologicallyactive ligand in equimolar ratio is in itself sufficient for en-hancing the activity of a particular potent ligand.For thenon-symmetrical benzimidazole ligands, MBZ, albendazole(ABZ), albendazole sulfoxide (ABZ-SO) and fenbendazole(FBZ), formation of about 1:1 mixture of two regioisomersfor their respective [RuACHTUNGTRENNUNG(h6-p-cym)Cl2] complexes is expectedexpected (1 a and 1 b in Figure 1, and 7–9 in Scheme S1 inthe Supporting Information). However, instead of two setsof unique signals being observed, the 1H NMR spectrum ofthe complex in [D6]DMSO shows only one set of signals foreach type of proton (see Figure 2 for representative exam-ple on complex 1). Of particular relevance here is the factthat we did observe unique signals for each type of proton

and carbon when the 1H and 13C NMR spectra were record-ed in CDCl3. At this stage, we speculated that this was dueto a possible ligand-exchange reaction between the rutheni-um centre and [D6]DMSO.[8c] As far as ligand dissociation isconcerned, although it was established that such compoundscan undergo the Cl-ligand exchange reaction readily inaqueous media,[11] the possibility for exchange of the organicligand with coordinating solvents was not seriously takeninto account when reporting the biological activities forDMSO stock solutions. If the individual 1H NMR spectraare overlaid together, it can be seen that the spectrum of

1 is an exact sum of the individual 1H NMR spectra of 6 andMBZ in [D6]DMSO, which confirms the dissociation(Figure 2).

Moreover, the intensities of the respective signals increaseas the concentration of 6 or MBZ is increased in the[D6]DMSO solution of 1 (Figure S1 in the Supporting Infor-mation). These spectral changes allowed us to conclude thatthe MBZ ligand in 1 (likewise for other complexes 7–10, seeScheme S1 and Figures S2 and S3 in the Supporting Infor-mation) no longer remains intact to the ruthenium centre ina DMSO solution of the complex. In case of 1, due to thepoor solubility of MBZ in chloroform, ligand dissociationcould also be confirmed visually (Figure S13 in the Support-ing Information). Addition of a few drops of DMSO toa concentrated orange-colour solution of 1 in chloroform re-sulted in immediate formation of a white precipitate, whichwas spectroscopically characterised as the free MBZ ligand.Similar studies on known pyridine or quinoline coordinated

RuACHTUNGTRENNUNG(h6-arene) complexes 4,[12]

5,[8e] and 14[7d,8e, 13] (Scheme S1in the Supporting Information)showed them to also undergoan immediate and complete dis-sociation of the N-heterocyclicligand from the metal centrewhen dissolved in [D6]DMSO(Figures S9–S11 in the Support-ing Information).

Complete fall-off of the in-troduced organic ligand, howev-er, is not warranted in all cases.Our studies on complexes [Ru-ACHTUNGTRENNUNG(h6-p-cym)Cl2ACHTUNGTRENNUNG(BIZ)] (2),[8a] [Ru-ACHTUNGTRENNUNG(h6-p-cym)Cl2ACHTUNGTRENNUNG(CTZ)] (3),[7a]

[Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2 ACHTUNGTRENNUNG(MCZ)] (12),[Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2 ACHTUNGTRENNUNG(KCZ)] (13)(BIZ= benzimidazole; CTZ =

clotrimazole; MCZ= micona-zole; KCZ=ketoconazole, seeFigure 1 and Scheme S1 in theSupporting Information forcomplex structures) confirmedthat the situation can be a bitmore complex. Instead ofa complete dissociation of theligand from the complex unit in

DMSO solution, formation of a mixture of different specieswas noticed; [D6]6 and free ligand were found to be the twomajor species present (see the Supporting Information Fig-ures S4, S5, S7 and S8 for details). In contrast, a completeand instant dissociation of the metronidazole (MTZ) ligandfrom the metal centre was observed for the [RuACHTUNGTRENNUNG(h6-p-cym)Cl2ACHTUNGTRENNUNG(MTZ)] (11)[5] complex in [D6]DMSO (Figure S6 inthe Supporting Information).

Of particular relevance, similar metal complexes of imida-zole-, pyridine- or quinoline-based ligands and their deriva-tives have already been reported to show different magni-

Figure 2. Complete dissociation of MBZ ligand from the original framework of complex 1 in DMSO, as con-firmed from stacked 1H NMR spectra of: a) [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2ACHTUNGTRENNUNG(MBZ)] (1) in CDCl3; b) 1 in [D6]DMSO;c) MBZ in [D6]DMSO; and d) [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2 ACHTUNGTRENNUNG(DMSO)] (6) in [D6]DMSO. Signals marked with blacksquares and black stars in spectrum (b) belong to free MBZ ligand and complex 6, respectively. NMR spectrawere measured immediately after dissolving the compounds in [D6]DMSO.

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M. Patra, G. Gasser et al.

tudes of antiprotozoan, antibacterial and anticancer activi-ty.[5–8, 14]

At this stage, 1H NMR spectroscopic studies unequivocal-ly indicate that the ligand exchange with DMSO does readi-ly occur in the piano-stool complexes with a general formula[Ru ACHTUNGTRENNUNG(h6-arene)Cl2(L)], where L= different N-heterocyclic li-gands). We can also firmly say that when dissolved inDMSO, the complete or partial dissociation of [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2(L)] depends entirely on the nature of the coordi-nated organic moiety. We believe that for complexes thatpartially dissociate in DMSO, an in-solution equilibrium be-tween the coordinated and uncoordinated ligand is estab-lished, and the kinetics for the equilibrium is speculated tobe mainly ligand dependent (Scheme 1). Furthermore, this

in-solution equilibrium in DMSO, between [Ru ACHTUNGTRENNUNG(h6-are-ne)Cl2(L)] and free ligand, L, can be comprehensively ach-ieved by simply mixing equimolar quantities of [Ru ACHTUNGTRENNUNG(h6-arene)Cl2ACHTUNGTRENNUNG(DMSO)] (6) and free L. For example, as can begleaned from Figure S5a and b in the Supporting Informa-tion, the 1H NMR spectrum of a mixture of 6 and CTZ (1:1,mol/mol) is exactly identical to the spectrum of complex 3.The formation of complex 3 through ligand exchange withDMSO, in this case, could be also confirmed by ESI-MSspectrometry, which showed an intense peak centred at m/z615 corresponding to the [3-Cl]+ species. Interestingly, equi-librium could also be pushed to the left by adding excessfree ligand (Figure S12 in the Supporting Information). Forexample, the 1H NMR spectrum of a 1:3 mixture of 6 andCTZ suggested 3 and free CTZ to be the only species pres-ent in solution. Similar results were obtained when 6 wasmixed with either MCZ or KCZ; as expected the 1H NMRspectra obtained were identical to those obtained by dissolv-ing 12 and 13 in [D6]DMSO (Figures S7 and S8 in the Sup-porting Information, respectively).

Having obtained NMR spectroscopy measurements thatprovided solid evidence for ligand dissociation in DMSO,we decided to compare the in vitro biological activity ofa [Ru ACHTUNGTRENNUNG(h6-arene)Cl2(L)] complex, and an equimolar mixtureof 6 and L. Accordingly, we measured the cytotoxicities andantiparasitic activity of the selected compounds against thecervical cancer (HeLa) and rat skeletal myoblast cells (L6),and against Trypanosoma cruzi (T. cruzi) parasite thatcauses Chagas diseases in human. As for experimental pro-tocols previously reported, freshly prepared stock solutionsof complexes in DMSO were used (see the Supporting In-

formation for experimental details). We thus anticipatedthat the complex and an equimolar solution of the respec-tive ligand and [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2ACHTUNGTRENNUNG(DMSO)] (6) would showsimilar activity. The results are summarised in Table 1 andTable S2 in the Supporting Information. Compound 6 dis-played no inhibitory activity up to a concentration of 100 mm

against both mammalian cell lines. On the other hand, com-pound 1 displayed an IC50 value of 0.2 mm on HeLa cells,which is similar to the values obtained for the free ligandMBZ as well as for the equimolar mixture of MBZ and 6.Similar results were obtained for complexes 8 and 9(Table S2 in the Supporting Information). Considering thefacts that: 1) the complexes decompose to form a 1:1 mix-ture of 6 and the free ligand in DMSO, and 2) 6 is inactive,these results are not surprising. For the partially dissociatingcomplex 3, as expected, IC50 values similar to those for com-plex 3, were obtained when CTZ was incubated togetherwith 1 equiv of 6 [HeLa (3.1 vs. 3.5 mm) and L6 (1.6 vs.2.1 mm)]. Similar results were observed for 12 and 13. More-over, the equimolar mixtures of ligands CTZ, MCZ andKCZ with complex 6 also showed antiparasitic activity(against T. cruzi) comparable to the corresponding [RuII ACHTUNGTRENNUNG(h6-p-cym)] complexes (3, 12 and 13, respectively), and allshowed inhibitory effect even at nanomolar sample concen-trations.

The in vitro investigations performed for the DMSOstock solutions of [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2(L)] (where L=N-heterocyclic ligands) as well as the equimolar mixtures of[Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2 ACHTUNGTRENNUNG(DMSO)] (6) and free L provide substan-tial empirical evidence for this in-solution equilibrium influ-encing the activity of the complex. With the aim to searchfor alternative metal-based drugs, exploring [Ru ACHTUNGTRENNUNG(h6-are-ne)Cl2(L)]-type complexes (L=N-heterocyclic ligands) fortheir biological activity has now become a well-establishedfield. Dissociation of such complexes in DMSO, eventhough happening spontaneously, has until now mostly slip-

Scheme 1. Proposed speciation equilibrium between [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2(L)] and [Ru ACHTUNGTRENNUNG(h6-p-cym)Cl2 ACHTUNGTRENNUNG(DMSO)] in DMSO.

Table 1. Comparative summary of IC50 values against HeLa, L6 cells andparasite T. cruzi Tulahuen C4 (amastigotes).[a]

Compounds IC50 values [mm]HeLa L6 T. cruzi

6 >100 >100 58.70�2.651 0.20�0.03 N.D.[b] N.D.[b]

MBZ 0.20�0.10 N.D.[b] N.D.[b]

MBZ +6 (1:1, mol/mol) 0.20�0.01 N.D.[b] N.D.[b]

3 3.1�1.1 1.6�0.6 0.008�0.004CTZ 15.7�0.8 38.9�2.5 0.010�0.006CTZ +6 (1:1, mol/mol) 3.5�1.6 2.1�1.1 0.017�0.01012 11.2�0.5 14.4�0.9 0.042�0.036MCZ 9.7�1.3 17.2�2.7 0.123�0.050MCZ +6 (1:1, mol/mol) 13.2�1.6 12.1�2.8 0.073�0.03513 20.1�1.6 16.3�0.6 0.006�0.003KCZ 39.5�0.3 22.7�5.0 0.006�0.005KCZ+6 (1:1, mol/mol) 20.0�0.4 26.0�4.1 0.003�0.001

[a] Freshly prepared stock solutions of complex in DMSO were used; seeTable S2 in the Supporting Information for an extended version. [b] Notdetermined.

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ped under the radar when the biological activities were de-termined using prepared stock solutions in DMSO. Thisstudy does not just concern the investigated class of com-plexes, but in general aims at instigating a different andrather cautious outlook to interpreting and deriving conclu-sions on the activity of in-solution labile metal complexes.Based on our studies, it is crucial that DMSO-mediated dis-sociation kinetics (or other coordinating solvents as well asbiological media) are taken into account by researchersbefore reporting on the biological activity of [Ru ACHTUNGTRENNUNG(h6-are-ne)Cl2(L)]-type (where L=N-heterocyclic ligands) com-plexes measured from the respective stock solutions.Beyond this, our study also reveals that mixing the [Ru ACHTUNGTRENNUNG(h6-arene)Cl2ACHTUNGTRENNUNG(DMSO)] complex and respective biologicallyactive N-heterocyclic ligand in equimolar ratio can be suffi-cient for enhancing the activity of a particular potent ligand.This revelation introduces a unique and rapid “mix-and-screen” approach to the field of [Ru ACHTUNGTRENNUNG(h6-arene)] organome-tallic-based drug discovery, which bypasses the otherwise re-quired meticulous synthetic preparation of the respective[Ru ACHTUNGTRENNUNG(h6-arene)Cl2(L)] (where L = N-heterocyclic ligands)complexes, to which a lot of attention has been dedicatedover the past years.

Acknowledgements

This work was financially supported by the Swiss National Science Foun-dation (SNSF Professorships PP00P2_133568 to G.G. and PP00P3-135170to J.K.), the Scientific and Technological Cooperation Programme, Swit-zerland–Russia (J.K., K.I.), the University of Zurich (G.G., S.F.), the Stif-tung f�r Wissenschaftliche Forschung of the University of Zurich (G.G.,S.F.), the Stiftung zur Krebsbek�mpfung (S.F.), the Huggenberger–Bis-choff Stiftung (S.F.) and the University of Zurich Priority Program (S.F.).We thank M. Cal, P. Schildknecht and R. Adelfio (Swiss TPH) for assis-tance with parasitic assays, and Dr. Thomas Fox for help with NMR spec-troscopy experiments.

Keywords: bioorganometallic chemistry · dimethylsulfoxide · ligand exchange · N-heterocycles · ruthenium

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Received: August 26, 2013Published online: October 2, 2013

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M. Patra, G. Gasser et al.