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A chemical-biological evaluation of rhodium(I) N-heterocyclic carbene complexes as prospective anticancer drugs.
FULL PAPER
DOI:10.1002/chem.201302819
A Chemical–Biological Evaluation of Rhodium(I)
N-Heterocyclic Carbene Complexes as Prospective Anticancer Drugs
Luciano Oehninger,[a] Laura Nadine K(cid:2)ster,[b] Claudia Schmidt,[a]
Alvaro MuÇoz-Castro,[c] Aram Prokop,[b] and Ingo Ott*[a]
Abstract: Rhodium(I) complexes bear- plexes, along with moderate inhibitory and confirmed the transport of rhodi-
ing N-heterocyclic carbene (NHC) li- activity of thioredoxin reductase um into the nuclei. Changes in the mi-
gandshavebeenwidelyusedincatalyt- (TrxR) andefficient bindingtobiomol- tochondrial membrane potential
ic chemistry, but there are very few re- ecules (DNA, albumin). Biodistribu- (MMP) were observed as well as DNA
ports of biological properties of these tion studies showed that the presence fragmentation inwild-typeand daunor-
organometallics. A series of RhI-NHC of albumin lowered the cellular uptake ubicin- or vincristine-resistant Nalm-6
derivatives with 1,5-cyclooctadiene and leukemia cells. Overall, these studies
CO as secondary ligands were synthe- indicated that RhI-NHC fragments
Keywords: antitumor agents · car-
sized, characterized, and biologically could be used as partial structures of
benes · cytotoxicity · drug resist-
investigated as prospective antitumor new antitumor agents, in particular in
ance · rhodium · thioredoxin reduc-
drug candidates. Pronounced antiproli- those drugs designed to address resist-
tase
ferative effects were noted for all com- antmalignanttissues.
Introduction agents.[12–15] This type of complex showed among other ef-
fects a strong and selective inhibition of the enzyme thiore-
Over the last years organometallic complexes have experi- doxin reductase (TrxR), induction of apoptosis, or depolari-
enced an increasing interest not only because of their cata- zation of the mitochondrial membrane.[14,16–19] Besides gold
lytic properties but also due to their fascinating biological and silver, other metals including platinum,[20–24] palladi-
effects.[1–4] Among those, organometallics bearing N-hetero- um,[25,26] ruthenium,[27–29] or rhodium[30,31] have also been
cyclic carbenes (NHCs) as ligands have been increasingly in usedascentersofbioactiveNHCcomplexes.
the focus of inorganic medicinal chemists.[5–7] The advantag- Rhodium complexes have been widely studied as an anti-
es of NHC ligands include the fact that sufficiently stable cancer agents mainly containing RhIII coordinated to differ-
coordinative bonds can be formed to many different metals ent ligands such as polypyridyls or cyclopentadienyls (see
and that the versatile synthetic chemistry related to the use Figure1 forsomeselectedexamples).[32–40]Biologicalstudies
of NHC ligands offers a broad variety of possible structural on rhodium(I) complexes as anticancer agents are less fre-
modifications. So far, metal-NHC complexes have been quent.[31,41–44] One recent report by McAlpine and co-work-
mostly studied as new antibacterial and anticancer agents. ers described the alteration of cell migration, DNA replica-
Although research on anti-infectives has been largely fo- tion, and DNA condensation by a RhI-NHC derivative.[31]
cused on AgI-NHC complexes,[8–11] AuI-NHC derivatives are The choice of RhI as metal center appears promising be-
the most studied examples concerning new anticancer cause it is isoelectronic with platinum(II) and exhibits
a square-planar geometry like cisplatin-type tumor thera-
peutics.
[a] L.Oehninger,C.Schmidt,Prof.Dr.I.Ott
InrecentstudiesweandothersreportedonthepromisACHTUNGTRENNUNGing
InstituteofMedicinalandPharmaceuticalChemistry anticancer effects of gold(I)- and ruthenium(II)-NHC
TechnischeUniversit(cid:2)tBraunschweig,Beethovenstrasse55 ACHTUNGTRENNUNGcomplexes containing benzimidazol-2-ylidene li-
38106Braunschweig(Germany)
gands.[14,16,28,30,45–48]StartingfromthistypeofNHCligandwe
Fax:(+49)5313918456
E-mail:ingo.ott@tu-bs.de designed in this study a series of related NHC ligands with
[b] L.N.K(cid:3)ster,Dr.med.habil.Dr.rer.nat.A.Prokop different surface volumes, lipophilicities, and coordinative
DepartmentofPaedriatricOncology,ChildrensHospitalCologne donor strengths (see Scheme1). Perimidines have been de-
AmsterdamerStrasse59,50735Cologne(Germany) scribed as DNA intercalating agents and therefore NHC li-
[c] Prof.Dr.A.MuÇoz-Castro gands derived of this N-heterocyclic core were included in
DepartmentofChemicalSciences the study.[49] The target RhI complexes contain the respec-
UniversidadAndresBello
tive NHC moiety as well as chlorido, 1,5-cyclooctadiene
Republica275,Santiago(Chile)
(COD), or carbon monoxide (CO) ligands, which them-
SupportinginformationforthisarticleisavailableontheWWW
underhttp://dx.doi.org/10.1002/chem.201302819. selves exhibit different coordinative and physicochemical
Chem.Eur.J.2013,19,17871–17880 (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim 17871
a silver intermediate, followed
by a transmetalation reaction
by addition of bisACHTUNGTRENNUNG[chlorido(1,5-
cyclooctadiene)rhodium(I)]
(see Scheme1). The resulting
benzimidazolylidene-rhodi-
um(1,5-cyclooctadiene) deriva-
tives (3a–d) and perimidinyli-
dene-rhodium(1,5-cycloocta-
diene) complexes (4a and 4b)
were purified by filtration over
Celite and recrystallized from
a mixture of CHCl/n-hexane
2 2
(1:4) at 08C. In the case of 3a–
d, the reaction was complete
within4h,whereasanextended
Figure1.Examplesofantitumoractiverhodiumcomplexes. reaction period of 24h was re-
quiredinthecaseof4aand4b.
The dicarbonylrhodium(I) de-
rivatives 5a–d and 6 were ob-
tained by stirring the respective
[RhACHTUNGTRENNUNG(cod)] complex in dichloro-
methane under carbon monox-
ide atmosphere for 20min. The
volume of the solution was re-
duced, n-hexane was added,
and the solution was stored at
(cid:2)208C. After approximately
10h the corresponding com-
plexes had precipitated as pale-
yellow solids. All target com-
plexes were clearly character-
ized by MS and NMR spectros-
Scheme1.Synthesis procedures:1)alkylhalide,KCO,CHCN,heatatreflux,6h; 2)AgOinCHCl,4h; copy (see below) and their high
2 3 3 2 2 2
3)[RhClACHTUNGTRENNUNG(cod)],2–12h;4)COatmosphereinCHCl,20min. purity was confirmed by ele-
2 2 2
mentalanalysis.
properties and could hence be used to modulate the charac- In the case of CO-containing complexes, IR spectroscopic
teristicsofthetargetcomplexes. analyses were also performed. In particular, the CO com-
The synthesis and characterization of these RhI-NHC de- plexes were included in this study to compare the donor
rivatives as wellastheir biologicalevaluation asprospective propertiesoftheNHC ligands among eachotherbyobserv-
anticancer drugs are reported here. The antiproliferative ef- ing the IR band of the CO ligand (v (A)).[54,55] Ligands
CO 1
fectsofthecomplexeswereevaluated,alongwithstudieson that are positioned trans to a CO ligand can have strong
possible molecular mechanisms of drug action (TrxR inhibi- effect on the ability of the CO ligand to effectively p-back-
tion, DNA binding, effects on mitochondrial membrane po- bond to the metal and this influences the IR-stretching fre-
tential)andeffectsindrug-resistantleukemiacells. quencies of the CO ligand. Free CO shows a band at
2155cm(cid:2)1; a strong donating ligand will shift the CO band
to lower frequencies.[56] This shift was observed for all CO
Results and Discussion complexes but between the single complexes no strong var-
iation could be noted in the v (A) bands, which were in
CO 1
Synthesis and characterization: The complexes were pre- therangeof2074–2086cm(cid:2)1.The1,3-diisopropylbenzymida-
pared according to procedures depicted in Scheme1. Com- zolylidene ligand of 5c had the weakest donor properties of
plexes3a–cand5b,chavebeendescribedpreviously.[50–53] the used NHC ligands with v (A)=2086cm(cid:2)1, whereas
CO 1
The benzimidazolium and perimidinium halides 1a–d and the methyl derivative 5a had strongest one (v (A)=
CO 1
2a,b were synthesized by heating benzimidazole or perimi- 2074cm(cid:2)1). The v (A) bands of the N-ethyl complexes5b
CO 1
dine at reflux with an excess of an alkyl halide in the pres- (2077cm(cid:2)1) and 6 (2079cm(cid:2)1) indicated that the s-donation
enceofKCO.Next,compounds1a–dand2a,bweretreat- of the benzimidazolylidene- and perimidinylidene-NHC li-
2 3
ed with a half equivalent of AgO in CHCl to generate gandswassimilar.
2 2 2
17872 www.chemeurj.org (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Chem.Eur.J.2013,19,17871–17880
Rhodium(I)N-HeterocyclicCarbeneComplexes FULL PAPER
NMR spectroscopy: In the 1HNMR spectra of the benzi- tive labilization of the coordinative bond between Rh and
midazolium and perimidinium halides, 1a,b and 2a,b, re- the respective trans-positioned alkene fragment is reflected
spectively (see FigureS1, the Supporting Information) the in smaller coupling constants (trans to NHC: 1J =6.6–
RhC
protonsoftheC2positionin1a,b(d=9.65and9.80ppm,re- 6.8Hz,cistoNHC:14.2–14.6Hz).[57]
spectively[16]) are shifted to higher ppm values compared ExchangeoftheCODwithtwoCOligandscausedanup-
with those of 2a,b (d=9.01 and 8.95ppm, respectively). field shift of the C2 signals of around d=11–12ppm in the
This difference is reflected in a higher acidity and hence re- 13CNMR spectra. This indicates a weaker influence of the
activity of 1ab with silver oxide and explains the longer re- rhodium atom over the C2 carbon of the NHC ligand in the
action time necessary to obtain the [Rh- CO derivatives compared with the COD complexes. An in-
ACHTUNGTRENNUNG(cod)(perimidinylidene)]complexes(seeabove). creaseintheNHC(cid:2)Rhdistancecanbeassumed bycompar-
In the 1HNMR spectra of the COD derivatives 3b and ingthecouplingconstantsbetweentheNHC(cid:2)C2carbonand
3d the signal of the N(cid:2)CH(cid:2)R protons is split into two sig- Rh(e.g.,J=51Hzfor3band43Hzfor5b,respectively).In
2
nals (Figure2, (a) signals), which is the consequence of an the 13CNMR spectra of the perimidinylidene derivative 6,
the NHC(cid:2)C2 is shifted to lower ppm values compared with
the benzimidazolylidene complexes 5a,b. Taken together,
the signals for the cis- (1J (cid:3)74Hz) and trans- (1J
RhC RhC
(cid:3)55Hz) CO ligands can also be distinguished in the
13CNMR spectra according to their coupling constants (see
Figure3).
Figure2.1HNMR spectra of 3b (top) and 3d (bottom) in CDCl. N(cid:2)
3
CH(cid:2)R (a);trans-CH of COD (b);cis-CH ofCOD (c); equatorial-CH
2 2
ofCOD(d);axial-CH COD(e).
2
Figure3.Rh(cid:2)Ccouplingsin13CNMRspectra(CDCl)of5b(top)and6
3
(bottom).
asymmetrical disposition of the N-residues regarding the
[RhACHTUNGTRENNUNG(cod)] moiety. The same effect was also observed for
complex 4b. However, in this case the N(cid:2)CH(cid:2)R signals Theoreticalcalculations:Thegeometry,bonding,andmolec-
2
were not well-defined and appeared as broad multiplets. Of ular properties of the studied compounds have been ana-
note, similar phenomena can also be observed with RuII- lyzedthroughrelativisticdensityfunctionalcalculations.The
NHCderivatives.[28] COD moiety exhibits a nearly h2,h2 coordination mode, in
The [RhClACHTUNGTRENNUNG(cod)] moiety shows a characteristic pattern in which the Rh(cid:2)COD bond lengths trans to the NHC ligand
the 1HNMR spectrum, in which two signals are observed areslightlyelongated((cid:3)0.1(cid:5))inrelationtotheotherRh(cid:2)
that correspond to the cis- and trans-olefin protons regard- COD distances (TableS1 and FigureS3, the Supporting In-
ing the NHC ligand (signals (c) and (b) in Figure2, respec- formation).Thisisingoodagreementwiththeresultsofthe
tively), as well as two signals from the equatorial and axial 1HNMR measurements (see above). Similarly, the Rh(cid:2)CO
CH protons (signals (d) and (e) in Figure2, respectively). distancesinthedicarbonylcounterpartsaredifferent,butto
2
This signal distribution was corroborated by an exemplary a small extent of about 0.64(cid:5). The Rh(cid:2)NHC and Rh(cid:2)Cl
2D NMR spectrum of 3a (see FigureS2, the Supporting In- distances are similar within the studied series, displaying an
formation). averaged value of (2.07(cid:4)0.05) and (2.40(cid:4)0.02)(cid:5), respec-
In complex 3d the equatorial and axial proton signals tively, which are in the range of other similar compounds.[58]
become unfolded due to the steric influence of the benzyl Moreover, calculated 13CNMR spectroscopy and v are in
CO
residues of the NHC moiety over the COD ligand (see good agreement with the available experimental data (see
Figure2,bottom,signals(d)and(e)). theSupportingInformation).
The olefinic CH groups cis and trans to the NHC ligands The calculated bond-dissociation energies (BDE) of the
show Rh(cid:2)C coupling constants in the 13CNMR spectra of NHC(cid:2)Rh bond display quite similar values for the studied
3a–d and 4a,b. The NHC moiety has a stronger trans effect series (TableS2, the Supporting Information), denoting
in comparison to the chlorido ligand and the resulting rela- a more stabilizing situation in the dicarbonyl counterparts
Chem.Eur.J.2013,19,17871–17880 (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemeurj.org 17873
I.Ottetal.
(av: 60.18kcalmol(cid:2)1) in relation to the COD derivatives wellasthedirhodiumRhIsynthesisreagentbisACHTUNGTRENNUNG[chlorido(1,5-
(av: 51.38kcalmol(cid:2)1). A deeper understanding into the cyclooctadiene)rhodium(I)] ([RhClACHTUNGTRENNUNG(cod)]) were inactive
2
terms that contribute to the BDE in the framework of Mo- (IC values>100mm,seeTable1).Incontrast,all[RhACHTUNGTRENNUNG(cod)-
50
rokuma–Ziegler energy decomposition analysis[59] reveals
that the main difference is attributable to the term that ac- Table1. IC valuesofantiproliferativeeffectsinMCF-7andHT-29cells
50
counts for Pauli (steric) repulsion, which is more destabiliz- and inhibition of TrxR; the results are expressed as means ((cid:4)error) of
ing in the COD derivatives (av: 17kcalmol(cid:2)1) due to the repeatedexperiments.
shorter NHC(cid:2)Rh distance (TableS1, the Supporting Infor- Complex IC 50 [mm]
MCF-7 HT-29 TrxR
mation). In contrast, the term that accounts for the stabiliz-
ing covalent character of the NHC(cid:2)Rh interaction due to 1a >100 >100 >100
ACHTUNGTRENNUNG[RhClACHTUNGTRENNUNG(cod)] >100 >100 n.d.
the formation of bonds (sharing of electrons) is more favor- 2
2a >100 >100 >100
able in this series (about of av. (cid:2)4kcalmol(cid:2)1) supporting
3a 0.6(cid:4)0.2 0.9(cid:4)0.0 1.8(cid:4)0.2
the observations made by 13CNMR spectroscopy (see 3b 2.9(cid:4)0.8 4.2(cid:4)0.5 1.5(cid:4)0.1
above). 3c 2.2(cid:4)1.3 2.9(cid:4)0.7 1.5(cid:4)0.4
3d 2.3(cid:4)0.3 2.7(cid:4)0.1 2.0(cid:4)0.2
The COD ligand exhibits a BDE of about 88.03kcal
4a 2.4(cid:4)0.6 6.6(cid:4)1.3 1.7(cid:4)0.3
mol(cid:2)1, and the CO ligand exhibits values of 38.68kcalmol(cid:2)1
4b 3.0(cid:4)0.8 5.6(cid:4)0.7 1.9(cid:4)0.1
(transtotheNHCmoiety)and57.03kcalmol(cid:2)1(cis).Incon- 5a[a] 7.6(cid:4)0.1 10.2(cid:4)2.3 n.d.
trast, the chlorido ligand exhibits a higher BDE ((cid:3)124kcal 5b[a] 5.0(cid:4)2.3 10.8(cid:4)3.5 n.d.
mol(cid:2)1 for COD derivatives, and (cid:3)145kcalmol(cid:2)1 for the di- 5c >100 97.2(cid:4)0.6 2.3(cid:4)0.4
5d 47.5(cid:4)4.0 43.6(cid:4)1.4 1.2(cid:4)0.3
carbonyl counterparts, however, denoting a more polarized
situationthatpromptsitsabstractioninpolarsolvents.Thus, [a]Obvious decomposition during the assay procedure. n.d.: not deter-
mined.
the differences observed for the chemical behavior of sys-
tems involving the methyl- and benzyl side chains, namely,
compounds 3a, 5a and 3d, 5d, concerning their behavior in ACHTUNGTRENNUNG(NHC)] derivatives (3a–d and 4ab) were active at low mi-
the biochemical assays (see below) and in solution (DMF cromolar concentrations against both cancer cell lines with
and HO), are ascribed to steric-hindrance effects, which IC valuesinthe0.6–3.0mmrangeinMCF-7cellsandinthe
2 50
hinder the access towards the rhodium center for external 0.9–6.6mm range in HT-29 cells. The most active [RhIACHTUNGTRENNUNG(cod)]
groups or molecules. Lastly, we evaluate the rotation of the complexwas3a,whichtriggeredIC valuesbelow1.0mmin
50
benzylgroup(Figure4),which seems toprotectthemetallic bothcelllines.Compound6experiencedsolubilityproblems
center from solvent molecules, in relation to the methylat- under the assay conditions and was not studied further.
ed-NHCligandof3a. Whereasthecarbonylcomplexes5aand5bshowedantipro-
liferative effects in the low micromolar range (IC =5–
50
Biological screening as antiproliferative agents and TrxR in- 11mm), complexes 5d and 5c were low-active or inactive
hibitors: Motivated by previous results on metal-NHC com- (IC values>40mm). However, for solutions of complexes
50
plexes the initial biological screening as prospective anti- 5a,b a color change and the formation of a precipitate from
cancerdrugsconsistedofmeasuringthecellgrowthinhibito- theaqueoussolutionwasnotedduringthefirsthourofincu-
ry activities in cultured cancer cells as well as the inhibitory bation. This clearly indicated the formation of decomposi-
effectsagainsttheenzymeTrxR.[12,14–18,28,60] tionproductsthatmighthaveinfluencedtheoutcomeofthe
The triggering of antiproliferative effects by the rhodiu- biologicaltests.Thecolorchangewasnotobservedforcom-
m(I) derivatives was investigated in two tumor cell lines plexes5c,d and their aqueous solutions remained visually
(namely, MCF-7 human breast adenocarcinoma and HT-29 unchanged for 12h. Based on these observations complexes
colon carcinoma). The rhodium-free cations 1a and 2a, as 5a,bwereexcludedfromfurtherbiologicaltests.
Next, the ability to inhibit TrxR was investigated. The
rhodium-freeligands1aand2aexpectedlyshowednoactiv-
ityupto100mm.FortheinvestigatedNHCcomplexes3a–d,
4a,b and 5c,d, IC values were observed in a narrow range
50
around 1.5mm. Hence the activity against TrxR was largely
independent on the NHC ligand structure and can be con-
sidered as moderate compared with other metal-NHC com-
plexesinvestigatedinexactlythesameassay.[14,16,17,28]
Binding to biomolecules, cellular distribution, and effects in
resistant cell lines of 3a: For further extended studies, com-
plex 3awasselected asthestrongestantiproliferativeagent.
Metal complexes, including NHC derivatives, are known to
interact with and bind to several biomolecules such as albu-
Figure4.Qualitativeenergybarrierfortherotationofthebenzylgroup
in3d(halfoftheNHCligandhasbeenremovedforclarity). min or DNA. Binding of 3a to albumin and DNAwas pre-
17874 www.chemeurj.org (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Chem.Eur.J.2013,19,17871–17880
Rhodium(I)N-HeterocyclicCarbeneComplexes FULL PAPER
liminarily evaluated by precipitation assays and quantifica-
tion of rhodium by atomic absorption spectroscopy (see the
ExperimentalSectionfordetails).
After 2h of exposure to albumin, (67(cid:4)10)% of the total
rhodium was bound to albumin and the values remained
rather stable over an extended exposure up to 24h. Com-
plexes3b and 3c were used for comparison and showed
a very similar behavior (see the Supporting Information for
more details). Next, DNAwas exposed to 3a as well as the
platinum anticancer drug cisplatin (as a reference) at a nu-
cleotide/metal ratio of 200:1 for 4h and the percentage of
thetotalamountofmetalattachedtoDNAwasdetermined.
Under these conditions (72(cid:4)3)% of rhodium of 3a was
boundtotheDNA,whereasDNAplatinationcausedbycis-
platin was (26%(cid:4)3)%. The efficient binding to DNA is in
good agreement with the recent results by McAlpine
etal.[31] Overall, these experiments clearly indicatethat RhI-
NHC complexes can undergo strong molecular interactions
with relevant biomolecules. To understand the relevance of
these interactions in a more complex system, the uptake of
3a in whole HT-29 cells and into their nuclei was deter-
mined. The cellular uptake was measured in a comparative
manner from the serum-free cell culture medium and from
the same medium with added albumin. This experimental
setup enables the evaluation of the influence of albumin on
rhodium bioavailability. The experiments were performed
with a concentration of 3a of 1.0mm, which is related to the
IC value of the complex in this cell line. As observed in
50
Figure5a,thelevelofcellularrhodiumwashigherintheex-
periments with albumin-free medium than in those contain-
ingalbumin.Thisindicatedthatthepresenceofserumalbu-
minhasanegativeinfluenceontheuptakeprocessof3a. Figure5.a)Cellular uptake of 3a (1.0mm) from cell culture media with
and without albumin into HT-29 cells; b)Rhodium levels in nuclei ex-
For the evaluation of the rhodium uptake into the nuclei
posedtoof3a(5.0mm).
of HT-29 cells, the experimental setup was changed to re-
flect the standard cell culture conditions under which 3a
would trigger strong cytotoxic effects (experiments with 3a dencedbyatleast40%apoptoticcells.Accordingly,thecal-
(5.0mm) in serum containing cell culture medium, see Fig- culatedIC valuesfromtheseexperimentswerecomparable
50
ure5b). In these experiments, well-detectable rhodium (Nalm-6: (0.6(cid:4)0.1)mm, Nalm-6-DNR: (0.6(cid:4)0.1)mm, Nalm-
levels were reached that were higher after short exposure 6-VCR:(1.0(cid:4)0.1)mm).
(1h) than after longer incubation (6h). However, these
amounts corresponded to only 1–2% of the total cellular
rhodium. Conclusion
The cytotoxic action and apoptosis induction of many an-
ticancer drugs is related to changes in the mitochondrial RhI-NHC complexes with COD or CO secondary ligands
membrane potential (MMP). For 3a, moderate MMP were prepared and characterized. Both spectroscopic and
changes in Nalm-6 leukemia cells were observed at concen- theoretical evaluations indicated a higher kinetic stability of
trations between 0.25 and 1.5mm (<40%), whereas drasti- the COD complexes compared with the dicarbonyl ana-
cally increased effects (>80%) were noted at a concentra- logues. This was further confirmed when the complexes
tionof2.0mm(seeFigure6a). were subjected to biological screening as some of the CO
TheDNA fragmentationby 3a asa parameter of apopto- complexes experienced major stability problems in biologi-
sisinductionwasassessedinwild-type-aswellasindaunor- cal media. Further limitations for biological applications
ubicin- or vincristine-resistant P-glycoprotein (P-gp) overex- were related to insufficient solubility in aqueous environ-
pressing Nalm-6 cells.[16] A strong DNA fragmentation was ment, especially in the case of the larger perimidine-derived
observed in all Nalm-6 cell lines in concentrations higher NHCligands.
than 0.5mm. In concentrations of 0.75mm (in daunorubicin- However, complexes of the general structure
resistant cells) or 1.0mm (in vincristine-resistant cells) and [RhICl(benzimidazolylidene)ACHTUNGTRENNUNG(cod)] did not experience the
higher, drug resistance could be clearly overcome as evi- mentioned problems, showed pronounced antiproliferative
Chem.Eur.J.2013,19,17871–17880 (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemeurj.org 17875
I.Ottetal.
Total cellular-uptake studies showed that the presence of
albumin lowered the rhodium uptake. Rhodium was well-
detectable in isolated nuclei, however, at a low percentage
of the total. Therelevance ofthe nuclear rhodium uptake is
not clear at this stage. Onthe one hand, theabsolutevalues
arelowcomparedwiththatofothermetallodrugsinvestigat-
ed under similar experimental conditions, on the other
hand, DNA targeting by the drug cisplatin also leads to
a small extent of DNA platination and the low relative
uptakeofmetalsintonucleiiscommon.[69–72]
Complex 3a also triggered considerable changes in the
mitochondrial membrane potential and led to DNA frag-
mentation,whichunderlinethepotentialofthismetallodrug
as an cytotoxic and apoptosis-inducing agent. Moreover,
complex 3a effectively overcame P-gp-related drug resist-
ance to daunorubicin and vincristine in leukemia cells,
apropertyhighlydesirableinthedesignofprospectiveanti-
cancerdrugs.
In consequence, [RhICl(benzimidazolylidene)ACHTUNGTRENNUNG(cod)] com-
plexes showed promising properties for the design of new
cancer chemotherapeutics. Further exploration of this type
of bioorganometallics is highly warranted and is the subject
ofongoingstudies.
Experimental Section
General: All reagents and the solvents were used as received from
Figure6.a)ChangesinthemitochondrialmembranepotentialofNalm-6
Sigma, Aldrich, or Fluka. 1HNMR spectra were recorded on a Bruker
cellsafter48hincubationwith3a;b)Apoptosisinductiondeterminedas
DRX-400ASNMRspectrometerand13CNMRspectraonaBrukerAV
nuclearDNAfragmentationafter72hdrugexposureinwild-typeNalm-
II-600ASNMRspectrometer,MSspectrawererecordedonaFinnigan
6 cells as well as daunorubicin- (Nalm-6-DNR) or vincristine-resistant
MAT4515.Thepurityofthetargetcompoundswasconfirmedbyelemen-
(Nalm-6-VCR)subtypes.Valuesaregivenaspercentagesofcellswithhy-
talanalysis(FlashEA112,ThermoQuestItalia).Forallcompoundsun-
podiploid DNA ((cid:4)SD (n=3)). Resistance to daunorubicin (DNR) and
dergoingbiologicalevaluation,theexperimentalvaluesdifferedlessthan
vincristine(VCR)wasconfirmedbythemissingorverylowapoptosisin-
ductionafterexposuretoDNR(52.5nm)orVCR(20nm). 0.5% from the calculated ones. Perimidine and the benzimidazolium
salts 1a–d were synthesized as described in the literature.[16,49] Cell cul-
ture: MCF-7 breast adenocarcinoma and HT-29 colon carcinoma cells
effectsinculturedtumorcells,andweremoderateinhibitors were maintained in DMEM high glucose (PAA) supplemented with
50mgL(cid:2)1gentamycinand10%(v/v)fetalcalfserum(FCS)priortouse.
of TrxR. TrxR is an enzyme that is involved in several dis-
Nalm-6leukemiacellsweremaintainedinRPMImediumsupplemented
ease-relevant pathways and has to be considered as a possi-
with1%penicillin/streptomycinand10%(v/v)FCS.
blemoleculartargetforvariousmetallodrugs.[61–68]Takento-
Synthesis of perimidine (2):[49] 1,8-Diaminonaphthalene (1.140g,
gether, this also suggests TrxR inhibition as a relevant con- 7.0mmol) was dissolved inethanol (20mL), followed byaddition ofan
tributing factor in the chemical biology of [RhIClACHTUNGTRENNUNG(cod)- excess of formic acid (1.60mL, 40mmol). The mixture was heated at
ACHTUNGTRENNUNG(NHC)] metallodrugs, although it is unlikely their major refluxfor4hundernitrogenatmosphere.Theethanolicsolutionwasdi-
lutedwithwaterandneutralizedbyadditionofNHOHat08C.Thepre-
modeofaction. 4
cipitatewascollectedandwashed4timeswithwateranddriedat508C
Because the rhodium-free cationic ligand precursors and for 12h. Yield: 1.119g (6.7mmol, 95%) light-brown powder. 1HNMR
rhodium(I) in the form of [RhClACHTUNGTRENNUNG(cod)] were devoid of any (CDCl):d=3.24(brs,1H,NH),6.47(d,2H,3J =6.8Hz,ArH ),7.11
2 3 HH 4/9
activityinthebiologicalassays,itcanbespeculatedthatthe (m,4H,ArH 5(cid:2)8 ),7.23ppm(s,1H,ArH2);elementalanalysiscalcd(%)
[RhI(benzimidazolylidene)] fragment represents a useful ve- forC 11 H 8 N 2 :C78.55,H4.79,N16.66;found:C78.58,H4.69,N16.30.
hicletodeliverRhItobiologicaltissues. Generalprocedureforsynthesisofthedialkylperimidiniumhalides:Peri-
midine(2)(1.009g,6mmol),KCO (1.037g,7.5mmol)andanexcessof
More detailed studies on the most active derivative 3a 2 3
therespectivealkylhalide(18mmol)wereheatedatrefluxinacetonitrile
showed effective binding to albumin and DNA. Albumin for8–14h.Thesolventwasremovedunderreducedpressureandthere-
contains an accessible cysteine residue (Cys-34) as most sultant solid was resuspended in dichloromethane and filtered off to
probable binding site for metal species, whereas binding to remove the formed potassium halide along with the remaining K 2 CO 3 .
Thesolventofthefiltratewasremovedunderreducedpressureandre-
DNAwasnotunexpectedbasedonthesimilarchemicalfea-
suspendedintetrahydrofuranetoremovetheexcessofalkylhalideand
tures of RhI and platinum(II) (isoelectronic, square-planar
remainingperimidine.Theobtainedwhitesolidcorrespondstothepure
geometry). desiredproduct.
17876 www.chemeurj.org (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Chem.Eur.J.2013,19,17871–17880
Rhodium(I)N-HeterocyclicCarbeneComplexes FULL PAPER
1,3-Dimethylperimidinium iodide (2a): Yield: 0.622g (1.9mmol, 32%) (1,3-Dibenzylbenzimidazol-2-ylidene)chlorido(h2,h2-cycloocta-1,5-diene)-
light-brownpowder.1HNMR([D]DMSO):d=3.52(s,6H-NCH),7.04 ACHTUNGTRENNUNGrhodium(I) (3d): Yield: 0.070g (0.12mmol, 35%) yellow powder.
6 3
(d, 2H, ArH, 3J =7.6Hz), 7.54 (dd, 2H, ArH, 3J =7.6Hz, 3J = 1HNMR (CDCl): d=1.76–1.87 (m, 2H, CH COD), 1.87–1.98 (m,2H,
HH HH HH 3 2
8.4Hz), 7.65 (d, 2H, ArH, 3J =8.4Hz), 9.01ppm (s, 1H, ArH2); CH COD), 2.06–2.24 (m, 2H, CH COD), 2.26–2.41 (m, 2H, CH
HH 2 2 2
13CNMR([D]DMSO):d=38.67(-NCH),107.64(ArC),120.35(quater- COD),3.25–3.43(m,2H,CHCOD),5.10–5.26(m,2H,CHCOD),6.20
6 3
nary ArC), 123.53 and 128.36 (ArC), 132.84 and 133.84 (quaternary (d, 2H, -NCHAr, 2J =15.8Hz), 6.31 (d, 2H, -NCHAr, 2J =
2 HH 2 HH
ArC),153.32ppm(ArC2);elementalanalysiscalcd(%)forC H NI:C 15.8Hz),6.98(m,2H,ArH ,4J =3.0Hz,3J =6.3Hz),7.02(dd,2H,
13 13 2 4/7 HH HH
48.17,H4.04,N8.64;found:C47.98,H4.31,N8.50. ArH , 4J =3.3Hz, 3J =5.9Hz), 7.29–7.33 (m, 2H, -NCHArH),
5/6 HH HH 2
1,3-Diethylperimidinium iodide (2b): Yield: 0.622g (1.9mmol, 32%) 7.34–7.38 (m, 4H, -NCH 2 ArH), 7.38–7.42ppm (m, 4H, -NCH 2 ArH);
light-brownpowder.1HNMR([D 6 ]DMSO):d=1.41(t,6H,-NCH 2 CH 3 , 13CNMR (CDCl 3 ): d=28.65 (CH 2 COD), 32.74 (CH 2 COD), 52.96
3 3 2 J J H H H H H , = = 7 7 A . . 1 7 rH H H , z z ) ) , , 3 4 7 J . H . 0 5 H 3 2 = ( ( q 8 d , . d 3 4 , H H 2 z , H ) - , , N A C 8 r H . H 9 2 5 C , p 3 H J p H 3 m , H 3 = J H 7 ( H . s 7 , = H 7. 1 z 1 , H H 3 , J z H ) H , A = 7 r .1 8 H 2 .3 2) ( H ; d, z) 2 1 , 3 H C 7 , .6 N A 2 M r ( H d R , , ( C ( 1 - O N 8C C D , H , A 1 2 J A r R C h r C ) ) , , = 1 6 6 9 9 . 7 . 6 1 .8 9 H 3 ( z p d ), p , 1 m c 1 is 0 ( - . d C 9 , 3 H A ,1 r C 2 C O 2 2 . , 4 D 1 8 J , , R 1 1 h J C 2 R 7 = hC .0 5 = 8 1 , . 1 0 1 4 2 H .3 7 z . H 8 ) 2 ; z , M ) 1 , 2 S 1 8 ( 0 E . 0 9 . I 0 5 ) , 3 : 1 5 ( 3 4 d 5 4 , .0 [ t 1 r M a , n 1 + s 3 ] - 6 ; C . e 1 H l 5 -
([D 6 ]DMSO): d=11.82 (-NCH 2 CH 3 ), 46.72 (-NCH 2 CH 3 ), 107.65 (ArC), emental analysis calcd (%) for C 29 H 30 N 2 ClRh: C 63.92, H 5.55, N 5.14;
121.13 (quaternary ArC), 123.39 and 128.31 (ArC), 131.43 and 134.43 found:C64.14,H5.78,N5.26.
(quaternary ArC), 152.36ppm (s, ArC2); elemental analysis calcd (%) Chlorido(h2,h2-cycloocta-1,5-diene)(1,3-dimethylperimidin-2-ylidene)-
for C H NI: C 51.15, H 4.86, N 7.95; found: C 51.08, H 4.49, N 7.55. ACHTUNGTRENNUNGrhodium(I) (4a): Yield: 0.084g (0.19mmol, 61%) yellow powder.
15 17 2
Generalprocedureforsynthesisofthe[RhACHTUNGTRENNUNG(cod)ACHTUNGTRENNUNG(NHC)]derivatives:The 1HNMR (CDCl
3
): d=1.94–2.04 (m, 4H, CH
2
COD), 2.44–2.48 (m,4H,
respective benzimidazolium- or perimidinium halide (0.32mmol) and CH 2 COD),3.40(m,2H,CHCOD),4.54(s,6H,-NCH 3 ),5.08(m,2H,
AgO (0.0371g, 0.16mmol) were added to a dried Schlenk tube. The
CHCOD),6.67(m,2H,ArH),7.30–7.40ppm(m,4H,ArH);13CNMR
2
mixture was backflashed three times with N 2 and then dry CH 2 Cl 2 was (CDCl 3 ):d=28.86(CH 2 COD),32.52(CH 2 COD),43.16(-NCH 3 ),70.06
added(15mL).Theflaskwasclosedandstirredfor4hinthedark.Aso- (d, cis-CH COD, 1J RhC =14.6Hz), 98.22 (d, trans-CH COD, 1J RhC =
lution of bisACHTUNGTRENNUNG[chlorido(h2,h2-cycloocta-1,5-diene)rhodium(I)] (0.078g, 6.8Hz), 104.35 (ArC), 119.71 (quaternary ArC), 121.04 and 127.76
2 0 – .1 1 6 2 m h m in ol) th in e C d H ar 2 k C . l 2 T w h a e s o a b d t d a e i d ne ( d 10 s m us L p ) en a s n i d on th w e a s s olu fi t l i t o e n re w d as ov s e ti r rre C d el f i o te r ( 4 A 8. r 6 C H ), z) 1 ; 34 M .17 S( a E n I d ): 13 m 4 / . z 96 : ( 4 q 4 u 2 at [ e M rn + a ] r ; y e A le rC m ) e , n 2 t 1 a 1 l .6 a 3 n p al p y m sis (d c , a A lc r d C2 ( , % 1J ) RhC fo = r
(281nm)andconcentratedinavacuum.Theyellowresiduewasrecrys- C 21 H 24 N 2 ClRh:C51.99,H5.65,N7.13;found:C51.68,H5.59,N6.97.
tallizedfromCHCl/n-hexane(10/40mL)at48C. Chlorido(h2,h2-cycloocta-1,5-diene)(1,3-diethylperimidin-2-ylidene)-
2 2
Chlorido(h2,h2-cycloocta-1,5-diene)(1,3-dimethylbenzimidazol-2-ylidene)- ACHTUNGTRENNUNGrhodium(I) (4b): Yield: 0.072g (0.15mmol, 48%) brown powder.
ACHTUNGTRENNUNGrhodium(I) (3a): The preparation of this compound has been reported 1HNMR (CDCl 3 ): d=1.59 (t, 6H, -NCH 2 CH 3 , 3J HH =7.0Hz), 1.86–2.10
previously.[50] Yield: 0.084g (0.21mmol, 69%) yellow powder. 1HNMR (m, 4H, CH 2 COD), 2.33–2.57 (m, 4H, CH 2 COD), 3.32 (m, 2H, CH
(CDCl 3 ): d=2.01–2.12 (m, 4H, CH 2 COD), 2.44–2.57 (m, 4H, CH 2 COD),4.68(m,2H,-NCH 2 CH 3 ),5.06(m,2H,CHCOD),6.29(m,2H,
COD),3.40(m, 2H, CHCOD), 4.35(s, 6H, -NCH 3 ), 5.20(m,2H,CH -NCH 2 CH 3 ), 6.67 (dd, 2H, ArH, 4J HH =1.2Hz, 3J HH =7.3Hz), 7.27–
COD), 7.23 (m, 2H, ArH 4/7 ), 7.28ppm (m, 2H, ArH 5/6 ); 13CNMR 7.35ppm(m,4H,ArH);13CNMR(CDCl 3 ):d=11.59(-NCH 2 CH 3 ),28.85
( ( d C , D c C is l 3 - ) C : H d= C 2 O 8. D 85 , ( 1 C J R H hC 2 = C 1 O 4 D .9 ) H ,3 z 2 ), .97 10 ( 0 C .3 H 5 2 C (d O , D tr ) a , n 3 s 4 -C .6 H 4(- C N O C D H , 3 ) 1 , J 6 R 8 hC .3 = 5 C (C O H D 2 , C 1J O Rh D C ) = , 1 3 4 2 .6 .5 H 1 z ( ) C , H 97 2 .7 C 3 O ( D d, ), tr 4 a 9 n . s 9 - 6 CH (-N C C O H D 2 C , H 1J 3 R ) h , C = 70 6 .4 .8 7 H ( z d ) , , c 1 is 0 - 5 C .1 H 1
6.8Hz), 109.25 and 122.38ppm (ArC), 135.32 (quaternary ArC), (ArC), 120.75 (quaternary ArC), 120.94 and 127.63 (ArC), 133.25 and
1 a 9 n 6 a . l 1 y 4 sis pp c m alc ( d d, (% Ar ) C f 2 o , r 1J C Rh 1 C 7 H = 22 5 N 0. 2 6 C H l 2 R z) h ; : M C S 5 ( 1 E .9 I 9 ): , m H /z 5 : .6 3 5 9 , 2 N [M 7. + 1 ] 3 ; ; e f l o e u m n e d n : ta C l m 13 / 4 z . : 85 47 ( 0 qu [M ate + r ] n ; a e r l y em A e r n C t ) a , l 2 a 1 n 1 a .6 ly 5 s p is p c m alc (d d , ( A % r ) C2 f , or 1J C Rh 2 C 3 H = 2 4 8 8 N .5 2 C H lR z) h ; : M C S 5 ( 8 E . I 6 ) 7 :
51.68,H5.59,N6.97. H5.99,N5.95;found:C58.41,H5.69,N5.70.
Chlorido(h2,h2-cycloocta-1,5-diene)(1,3-diethylbenzimidazol-2-ylidene)- General procedure for the synthesis of rhodium dicarbonyl derivatives:
ACHTUNGTRENNUNGrhodium(I) (3b): The preparation of this compound has been reported Therespective[RhACHTUNGTRENNUNG(cod)] derivativewas added toadried Schlenktube.
previously.[16] Yield: 0.107g (0.22mmol, 72%) yellow powder. 1HNMR The complex was back flashed three times with carbon monoxide and
(CDCl 3 ): d=1.63 (t, 6H, -NCH 2 CH 3 , 3J HH =7.3Hz), 1.96–2.06 (m, 4H, then dry CH 2 Cl 2 was added (15mL). The solution was stirred under
CH COD),3.41–3.51(m,4H,CH COD),3.37(m,2H,CHCOD),4.81 a constant flux of CO for 30min. The solution was concentrated in
2 2
(dq, 2H, -NCHCH, 3J =7.3Hz, 2J =14.4Hz), 5.05 (dq, 2H vacuum((cid:3)5mL)andprecipitatedbyadditionofn-hexane((cid:3)30mL)at
2 3 HH HH
-NCHCH, 3J =7.3Hz, 2J =14.4Hz), 5.14 (m, 2H, CH COD), 7.21 48C.
2 3 HH HH
(dd,2H,ArH ,4J =3.1Hz,3J =6.0Hz),7.32ppm(dd, 2H,ArH , Dicarbonylchlorido(1,3-dimethylbenzimidazol-2-ylidene)ACHTUNGTRENNUNGrhodium(I) (5a):
4/7 HH HH 5/6
4J =3.1Hz, 3J =6.0Hz); 13CNMR (CDCl): d=14.96 (-NCHCH), Yield: 0.021g (0.06mmol, 48%) yellow powder. 1HNMR (CDCl): d=
HH HH 3 2 3 3
28.79 (CH COD), 32.90 (CH COD), 43.60 (-NCHCH), 68.58 (d, cis- 4.14 (s, 6H, -NCH), 7.40 (m, 4H, ArH), 13CNMR (CDCl): d=35.22
2 2 2 3 3 3
CH COD, 1J =14.2Hz), 99.93 (d, trans-CH COD, 1J =6.7Hz), (-NCH), 110.40 and 123.69 (ArC), 134.85 (quaternary ArC), 182.40 (d,
RhC RhC 3
109.88and122.11(ArC),134.44(quaternaryArC),195.23ppm(d,ArC2, cis-CO, 1J =74.2Hz), 185.21, 185.30 (d, trans-CO, 1J =53.6Hz),
RhC RhC
1J =50.6Hz); MS (EI): m/z: 420 [M+]; elemental analysis calcd (%) 185.36ppm (d, NHC, 1J =43.1Hz); FTIR: n˜=2074 (n˜(CO) ),
RhC RhC sym
for C H NClRh: C 52.50, H 5.87, N 5.83; found: C 52.64, H 5.72, N 1989cm(cid:2)1(v˜(CO) );MS(EI):m/z:318[M+(cid:2)CO];elementalanalysis
19 26 2 asym
5.65. calcd(%)forC H ClNORh:C38.79,H2.96,N8.23;found:C39.20,
11 10 2 2
Chlorido(h2,h2-cycloocta-1,5-diene)(1,3-diisopropylbenzimidazol-2- H3.10,N7.89.
ylidene)ACHTUNGTRENNUNGrhodium(I)(3c):Thepreparationofthiscompoundhasbeenre- Dicarbonylchlorido(1,3-diethylbenzimidazol-2-ylidene)ACHTUNGTRENNUNGrhodium(I) (5b):
ported previously.[53] Yield: 0.087g (0.17mmol, 55%) yellow powder. The preparation of this compound has previously been reported.[52]
1HNMR (CDCl): d=1.70 (d, 6H, -NCHACHTUNGTRENNUNG(CH) 3J =7.1Hz), 1.79 (d, Yield: 0.016g (0.04mmol, 33%) yellow powder. 1HNMR (CDCl): d=
3 32, HH 3
6H,-NCHACHTUNGTRENNUNG(CH) 3J =7.1Hz),1.94–2.04(m,4H,CH COD),2.39–2.49 1.58 (t, 6H, 3J =7.3Hz, -NCHCH), 4.62 (dq, 2H, -NCHCH, 3J =
32, HH 2 K 2 3 2 3 HH
(m,4H,CH COD),3.46 (m,2H,CHCOD),5.10 (m,2H,CHCOD), 7.3Hz, 2J =14.4Hz), 4.75 (dq, 2H, -NCHCH, 3J =7.3Hz, 2J =
2 HH 2 3 HH HH
6.49(hept,2H,-NCHACHTUNGTRENNUNG(CH),3J =7.1Hz),7.14(dd,2H,ArH ,4J = 14.4Hz),7.36(dd,2H,ArH ,4J =3.1Hz,3J =6.1Hz),7.45(dd,2H,
32 HH 4/7 HH 4/7 HH HH
3.1Hz, 3J =6.1Hz), 7.50ppm (dd, 2H, ArH , 4J =3.1Hz, 3J = ArH , 4J =3.1Hz, 3J =6.1Hz); 13CNMR (CDCl): d=14.79
HH 5/6 HH HH 5/6 HH HH 3
6.1Hz); 13CNMR (CDCl): d=20.88 and 21.88 (-NCHACHTUNGTRENNUNG(CH)), 28.76 (-NCHCH),43.89(-NCHCH),110.77and123.47 (ArC),134.02(qua-
3 32 2 3 2 3
(CH COD), 32.88 (CH COD), 53.87 (-NCHACHTUNGTRENNUNG(CH)), 67.81 (d, cis-CH ternaryArC),182.61(d,cis-CO,1J =72.2Hz),184.26(d,NHC,1J =
2 2 32 RhC RhC
COD, 1J =14.5Hz), 99.17 (d, trans-CH COD, 1J =6.7Hz), 112.08 42.5Hz), 185.30ppm (d, trans-CO, 1J =53.8Hz); FTIR: 2077
RhC RhC RhC
and121.51(ArC).133.64(quaternaryArC),194.00ppm(d,ArC2,1J = (v(CO) ), 1991cm(cid:2)1 (v(CO) ); MS (EI): m/z: 340 [M+(cid:2)CO]; ele-
RhC sym asym
50.3Hz); MS (EI): m/z: 448 [M+]; elemental analysis calcd (%) for mentalanalysiscalcd(%)forC H ClNORh:C42.36,H3.83,N7.60;
13 14 2 2
C H NClRh:C54.33,H6.34,N5.51;found:C54.14,H6.33,N5.23. found:C41.98,H3.55,N7.53.
21 30 2
Chem.Eur.J.2013,19,17871–17880 (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemeurj.org 17877
I.Ottetal.
Dicarbonylchlorido(1,3-diisopropylbenzimidazol-2-ylidene)ACHTUNGTRENNUNGrhodium(I) TrxR inhibition assay: To determine the inhibition of TrxR, an estab-
(5c):Thepreparationofthiscompoundhaspreviouslybeenreported.[53] lishedmicroplatereader-basedassaywasperformedwithminormodifica-
Yield: 0.016g (0.04mmol, 37%) yellow powder. 1HNMR (CDCl): d= tions.[16]Forthispurpose,commerciallyavailableratrecombinatedTrxR1
3
1.72(dd,12H,-NCHACHTUNGTRENNUNG(CH),4J =1.4Hz,3J =7.0Hz),5.77(hept,2H, (from IMCO Corporation) was used and diluted with distilled water to
32 HH HH
-NCHACHTUNGTRENNUNG(CH), 3J =7.0Hz), 7.29 (dd, 2H, ArH , 4J =3.2Hz, 3J = achieveaconcentrationof0.01UmL(cid:2)1.Thecompoundswerefreshlydis-
32 HH 4/7 HH HH
6.2Hz), 7.62 (dd, 2H, ArH , 4J =3.2Hz, 3J =6.2Hz)ppm; solved as stock solutions in DMF. Potassium phosphate buffer pH7.0
5/6 HH HH
13CNMR (CDCl): d=20.91 and 21.19 (-NCHACHTUNGTRENNUNG(CH)), 54.58 (-NCH- (25mL) containing the compounds in graded concentrations or vehicle
3 32
ACHTUNGTRENNUNG(CH)), 112.97 and 122.84 (ArC), 133.41 (quaternary ArC), 182.58 (d, (DMF) without compounds (control probe) were added to each aliquot
32
cis-CO,1J =74.4Hz),182.89(d,ArC2,1J =42.4Hz),185.52ppm(d, oftheenzymesolution(25mL)andtheresultingsolutions(finalconcen-
RhC RhC
trans-CO, 1J =53.7Hz); FTIR: n˜=2086 (v(CO) ), 1990cm(cid:2)1 tration of DMF: 0.5% v/v) were incubated with moderate shaking for
RhC sym
(v(CO)
asym
); MS (EI): 368 [M+(cid:2)CO]; elemental analysis calcd (%) for 75minat378Cina96-wellplate.Thereactionmixture(225of1000mL;
C 15 H 18 ClN 2 O 2 Rh:C45.42,H4.57,N7.06;found:C45.37,H4.49,N6.88. the reaction mixture consisted of 500mL potassium phosphate buffer
Dicarbonylchlorido(1,3-dibenzylbenzimidazol-2-ylidene)ACHTUNGTRENNUNGrhodium(I) (5d): pH7.0, 80mL 100mm EDTA solution pH7.5, 20mL BSA solution
Yield: 0.013g (0.03mmol, 28%) yellow powder. 1HNMR (CDCl): d= 0.05%,100mLof20mmNADPHsolution,and300mLofdistilledwater)
3
5.83 (d, 2H, -NCHAr, 2J =15.7Hz), 6.05 (d, 2H, -NCHAr, 2J = wasaddedtoeachwell,andthereactionstartedbyadditionofan20mm
2 HH 2 HH
15.7Hz),7.25–7.17(m,4H,ArH),7.41–7.29ppm(m,10H,-NCHArH); ethanolic 5,5’-dithiobis(2-nitrobenzoic acid) solution (25mL). After
2
13CNMR (CDCl): d=53.02 (-NCHAr), 111.67, 123.78, 128.29, 128.98, proper mixing,theformationof 5-thio-2-nitrobenzoicacid(5-TNB)was
3 2
132.55, 135.05(18C,ArC),182.04(d,cis-CO,1J =74.2Hz),185.06 (d, monitoredwithamicroplatereader(PerkinElmerVictorX4)at405nm
RhC
trans-CO, 1J =53.8Hz), 186.93ppm (d, ArC2, 1J =43.5Hz). FTIR: in 10s intervals for 6min. The increase in 5-TNB concentration over
RhC RhC
n˜=2076(v(CO) ),1994cm(cid:2)1(v(CO) );MS(EI):464[M+(cid:2)CO];ele- timefollowedalineartrend(r2(cid:5)0.99),andtheenzymaticactivitieswere
sym asym
mentalanalysiscalcd(%)forC H ClNORh:C56.06H3.68N5.69;C calculatedastheslopes(increaseinabsorbancepersecond)thereof.For
23 18 2 2
56.34,H3.75,N5.77. each tested compound, the noninterference with the assay components
Dicarbonylchlorido(1,3-diethylperimidin-2-ylidene)ACHTUNGTRENNUNGrhodium(I) (6): Yield: wasconfirmedbyanegativecontrolexperimentbyusinganenzyme-free
0.012g(0.03mmol, 27%)yellow powder.1HNMR(CDCl 3 ):d=1.51(t, solution. The IC 50 values were calculated as the concentration of com-
6H, -NCHCH, 3J =7.1Hz), 4.76 (m, 2H, -NCHCH), 5.03 (m, 2H, pounddecreasingtheenzymaticactivityoftheuntreatedcontrolby50%
2 3 HH 2 3
-NCHCH), 6.73 (dd, 2H, ArH, 4J =2.3Hz, 3J =6.3Hz), 7.40– andaregivenasthemeansanderrorofrepeatedexperiments.
2 3 HH HH
7.34ppm(m,4H,ArH);13CNMR(CDCl):d=11.06(-NCHCH),50.53 Bindingtoalbumin:Proteinbindingwasstudiedaccordingtoarecently
3 2 3
(-NCHCH),105.80(ArC),121.41(quaternaryArC),121.86and127.79 described method with minor modifications (see refs.[82] and [83]).
2 3
(ArC), 133.20 and 135.00 (quaternary ArC), 182.43 (d, cis-CO, 1J = Bovine serum albumin (BSA, 400mg, Sigma Aldrich) was dissolved in
RhC
75.4Hz), 185.56 (d, trans-CO, 1J =54.4Hz), 199.29ppm (d, NHC, Dulbecco’smodifiedEagle(cid:6)smedium(DMEM)highglucose(PAA)cell
RhC
1J =41.3Hz); FTIR: n˜=2079 (v(CO) ), 2000cm(cid:2)1 (v(CO) ); MS culturemedium(10.0mL)supplementedwithgentamycin(50mgL(cid:2)1).A
RhC sym asym
(EI): 390 [M+(cid:2)CO]; elemental analysis for calcd (%) for stocksolution(10mLof5.0mm)ofthecorrespondentcomplexinDMF
C H ClNORh:C48.77H3.85N6.69;found:C49.04,H3.99,N6.97. was added to the BSA-treated medium and incubated at 378C in the
17 16 2 2
Computational details: Relativistic density functional theory calcula- dark under gentle shaking. After 0, 1, 2, 4, 6, 12, and 24h, an aliquot
tions[73] were carried out by using the ADF 2012 code[74] incorporating (250mL) thereof was taken, treated with of cold ((cid:2)208C) ethanol
scalar corrections through the ZORA Hamiltonian.[75,76] Triple-x Slater (500mL)andstoredat(cid:2)208Cfor2htoallowanoptimalprecipitationof
basis set plus two polarization functions (STO-TZ2P) for valence elec- theproteinfraction.Afterwardsthesolutionwascentrifugedat400gfor
trons were employed within the generalized gradient approximation 5minat48C,analiquotofthesupernatant(400mL)wastaken andthe
(GGA) according to the Perdew–Burke–Ernzerhof (PBE) nonlocal ex- rhodium content was measured by atomic absorption spectroscopy
change-correlation functional.[77,78] The frozen core approximation was (AAS;seebelow).Theresultswereexpressedaspercentageofrhodium
appliedtothe[1s2–3d10]coreforRh,and[1s2]forC,N,andCl,leaving boundtoalbuminandaregivenasthemeansanderrorofrepeatedex-
the remaining electrons to be treated variationally. Geometry optimiza- periments.
tions were done without any symmetry restrain through the analytical BindingtoDNA:Experimentswereperformedaccordingtoapreviously
energy gradient method implemented by Verluis and Ziegler.[79] Bond- describedmethod.[69]Salmontestes DNA (Sigma)was dissolvedinPBS
dissociation energies are corrected to the basis set superposition error pH7.4 and the drugs were added as stock solutions in DMF. The final
(BSSE). solutionscontained3a(4.0mm),salmontestesDNA(247.5mgmL(cid:2)1)and
NMR properties were evaluated by employing the gauge-including 0.1%(v/v)DMF.Aftervortexing,thesolutionswereincubatedat378C
atomic orbitals (GIAO) methodology[80] as implemented in the NMR for2h.Aliquots(200mL)weremixedwith0.9msodiumacetate(100mL)
propertymoduleoftheADFcodeinconjunctiontotheGGAexchange andice-coldethanol(900mL).Sampleswerestoredat(cid:2)208Cfor30min.
expressionproposedbyHandyandCohen[81]andthecorrelationexpres- Thepelletswereisolatedbycentrifugation(2390g,10min,48C)andre-
sionproposedbyPerdew,Burke,andErnzerhof(OPBE).[77,78]Therela- suspended in 0.3m sodium acetate (300mL). Ice-cold ethanol (900mL)
tive13CNMRparameters(d=s TMS(cid:2)s calcd)werecalculatedbyusingthe was then added, and the precipitate was collected after centrifugation
C C
GIAOOPBE/ZORAleveloftheoryandreferencedtotetramethylsilane (2390g,10min,48C).Sampleswere washedtwice with ice-cold ethanol
(TMS,s TMS=191.19ppm). andstoredat(cid:2)208C.Thepelletsweredissolvedinwater(500mL,twice
C
AntiproliferativeeffectsinMCF-7andHT-29cells:Theantiproliferative distilled),andtheDNAcontentwasdeterminedbyabsorptionreadingat
effectsofthecompoundsweredeterminedfollowinganestablishedpro- 260nm in a microplate reader (PerkinElmer Victor X4). Salmon testes
cedure. In short, cells were suspended in cell culture medium (HT-29: DNAdissolvedinwaterwereusedforcalibrationpurposes.Therhodium
3000cellsmL(cid:2)1, MCF-7: 10000cellsmL(cid:2)1), and 100mL aliquots thereof from the samples was determined by AAS. The amount of rhodium
were plated in 96-well plates and incubated at 378C: 5% CO for 48h boundtoDNAwasexpressedaspercentageoftotalrhodiuminthesam-
2
(HT-29)or72h(MCF-7).Stocksolutionsoftherutheniumcomplexesin ples.Resultswerecalculatedasmeansofthreeindependentexperiments,
DMFwerefreshlypreparedanddilutedwithcellculturemediumtothe whichwereperformedwithtworeplicates.
desired concentrations (final DMF concentration: 0.1% v/v). The Uptakeintothecells:Forcellular-uptakestudiesHT-29cellsweregrown
medium in the plates was replaced with medium containing the com- untilatleast70%confluencyin75cm2cellcultureflasks.Stocksolutions
pounds in graded concentrations (six replicates, 200mL per well). After ofcomplex3ainDMFwerefreshlypreparedanddilutedwithcellcul-
furtherincubationfor72h(HT-29)or96h(MCF-7)thecellbiomasswas ture medium to the desired concentrations (final DMF concentration:
determinedbycrystalvioletstainingandtheIC valuesweredetermined 0.1%v/v,finalcomplexconcentration:1.0mm).Thecellculturemedium
50
asthoseconcentrationscausing50%inhibitionofcellproliferation.Re- of the cell culture flasks was replaced with 10mL of the cell culture
sultsarerepresentedasmeansofindependentexperiments. medium (with or without bovine serum albumin (40mg per mL)) solu-
17878 www.chemeurj.org (cid:4)2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Chem.Eur.J.2013,19,17871–17880
Rhodium(I)N-HeterocyclicCarbeneComplexes FULL PAPER
tions containing 3a and the flasks were incubated for 1, 4, and 6h at PBS2%(v/v)formaldehydeonicefor30min.Afterfixation,cellswere
378C/5%CO.Afterwardsthemediumwasremovedandthecellswere incubated with ethanol/PBS (2:1, v/v) for 15min, pelleted, and resus-
2
washed with phosphate-buffered saline pH7.4. After trypsinization, cell pended in PBS containing RNase A (40mgmL(cid:2)1). After incubation for
pelletswereisolatedbycentrifugation,resuspendedin1–5mLtwice-dis- 30minat378C,cellswerepelletedagainandfinallyresuspendedinPBS
tilled water, lysed by ultrasonication and appropriately diluted using containing propidium iodide (50mgmL(cid:2)1). Nuclear DNA fragmentation
twice-distilled water. The rhodium content of the samples was deter- was then quantified by flow cytometric determination of hypodiploid
minedbyAAS(seebelow)andtheproteincontentofseparatealiquots DNA. Data were collected and analyzed by using a FACScan (Becton
wasdeterminedbytheBradfordmethod. Dickinson, Heidelberg, Germany) equipped with the CELLQuest soft-
Uptakeintothenuclei:ThenucleiofHT-29cellswereisolatedaccording ware. Data are given in % hypoploidy (subG1), which reflects the
to previously described procedure[69,71] with some modifications: Cells numberofapoptoticcells.
were grownin 175cm2cell culture flasks untilat least 70% confluency.
The cell culture medium was removed and replaced with cell culture
medium (20mL) containing 5mm of 3a. The flasks were incubated at
378C/5% CO for 1 and 6h and the cell pellets were isolated as de-
2
scribed above. After centrifugation (923g, 5min) the pellet was resus-
pendedin0.9%NaClsolution(1.0mL)andanaliquot(200mL)wasre- Acknowledgements
moved before nuclei isolation to determine the total cellular rhodium
uptake (see above). For nuclei isolation, the cell suspension was centri- ThefinancialsupportbyDFG(granttosupporttheinitiationofinterna-
fuged at 923g for 5min and resuspendedin 300mL RSB-1 (0.01m Tris- tional collaboration), FONDECYT 11100027, FCI (Fonds der Chemi-
HCl, 0.01m NaCl, 1.5mm MgCl, pH7.4) and left for 10min in an ice schen Industrie), Dr. Kleist-Foundation, Berlin, Dr. Koch-Foundation,
2
bath. Swollen cells were centrifuged (1231g, 5min), resuspended in Berlin, as well as the excellent technical support by Davida Fernandez
300mL of RSB-2 (RSB-1 containing each 0.3% v/v Nonidet-P40 and Fernandezaregratefullyacknowledged.
sodiumdesoxycholate)andhomogenizedby10–15up/down-pushesusing
a dounce homogenizer. The resultant homogenate was centrifuged at
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948.
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