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Rhodium(I) N-Heterocyclic Carbene Bioorganometallics as in Vitro Antiproliferative Agents with Distinct Effects on Cellular Signaling.
Article
pubs.acs.org/jmc
‑
Rhodium(I) N Heterocyclic Carbene Bioorganometallics as in Vitro
ff
Antiproliferative Agents with Distinct E ects on Cellular Signaling
† † ‡ § ‡
Luciano Oehninger, Sarah Spreckelmeyer, Pavlo Holenya, Samuel M. Meier, Suzan Can,
Hamed Alborzinia,
‡
Julia Schur,
†
Bernhard K. Keppler,
∥
Stefan Wo
̈
l
fl
,
‡
and Ingo Ott
*,†
† Institute of Medicinal and Pharmaceutical Chemistry, Technische Universitaẗ Braunschweig, Beethovenstraße 55, D-38106
Braunschweig, Germany
‡ Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-Universitaẗ Heidelberg, Im Neuenheimer Feld 364, D-69120
Heidelberg, Germany
§
Department of Analytical Chemistry, University of Vienna, Waehringer Straße 38, 1090 Vienna, Austria
∥
Institute of Inorganic Chemistry, University of Vienna, Waehringer Straße 42, 1090 Vienna, Austria
*
S Supporting Information
ABSTRACT: OrganometallicswithN-heterocycliccarbene(NHC)ligandshavetriggeredmajorinterestininorganicmedicinal
chemistry. Complexes of the type Rh(I)(NHC)(COD)X (where X is Cl or I, COD is cyclooctadiene, and NHC is a
dimethylbenzimidazolylidene)representapromisingtypeofnewmetallodrugsthathavebeenexploredbyadvancedbiomedical
methodsonlyrecently.Inthiswork,wehavesynthesizedandcharacterizedseveralcomplexesofthistype.Asobservedbymass
spectrometry,thesecomplexesremainedstableoveratleast3hinaqueoussolution,afterwhichhydrolysisofthehalidoligands
occurredandreleaseoftheNHCligandwasevident.Effectsagainstmitochondriaandgeneralcelltumormetabolismwerenoted
athigherconcentrations,whereasphosphorylationofHSP27,p38,ERK1/2,FAK,andp70S6Kwasinducedsubstantiallyalready
at lower exposure levels. Regarding the antiproliferative activity in tumor cells, a clear preference for iodido over chlorido
secondary ligands was noted, as well as effects of the substituents of the NHC ligand.
■
INTRODUCTION
Organometallic complexes have been emerging as promising
new metallodrug candidates and hold great promise in
particular for cancer
chemotherapy.1−3
Among the most
frequently studied types, N-heterocyclic carbene (NHC)
Figure 1. Examples of previously biologically studied rhodium(I)
complexes with gold and silver as central atoms have
NHC complexes.8,9,16,17
demonstrated fascinating biochemical properties as enzyme
inhibitors and antimitochondrial agents, suggesting possible
applications in the therapy of tumors or infectious
diseases.4−6
complexes with square-planar geometries. Accordingly, some
Early reports7−9 demonstrate that various metals other than analogies with the clinically used platinum drugs are evident,
silver or gold can be successfully used to generate biologically andinfact,interactionwithDNAwasfoundtobealikelymode
active metal NHC complexes; however, only during the past of drug action also for Rh(I) NHC complexes.16
few years has this particular field of bioorganometallic Rhodium(III) complexes with ligands other than NHCs
chemistrybeengraduallyextendedtodifferentmetalsincluding (e.g., polypyridyls, staurosporine) have been studied by
platinum,10−12 ruthenium,13 iridium,14,15 and rhodium.15−17 Sheldrick and co-workers,18,19 Meggers and co-workers,20
AlthoughRh(I)NHCcomplexeswereamongthefirstmetal Leung and Ma and co-workers,21,22 Che and co-workers,23
NHC complexes to be synthesized, with biological (anti-
andothers.24,25Dependingonthecoordinatedligandsforthese
bacterial) activity reported in 1996,8 it was not until 2013 that complexes, several targets were confirmed, including protein
their promising potential as anticancer metallodrugs was kinases, other enzymes (e.g., the ubiquitin−proteasome
described in more detail in three further reports by Simpson system), and DNA.
etal.,15 McAlpine andco-workers,16 andus17(see Figure1 for
examples). The use of the Rh(I) center is of special interest Received: July23, 2015
because Rh(I) is isoelectronic with Pt(II), and both ions form Published: November 23, 2015
©2015AmericanChemicalSociety 9591 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry Article
a
Scheme 1. Synthesis Procedure for the (COD)Rhodium(I) Derivatives
a(i) Formic acid, reflux, 12 h; (ii) alkylhalide, KCO, CHCN, reflux, 6 h; (iii) AgO in CHCl, 4 h; (iv) bis[halido(η2,η2-cycloocta-1,5-
2 3 3 2 2 2
diene)rhodium(I)], 2h.
In this article, we report on interesting chemical-biological
propertiesthatwehaveobservedusingRh(I)complexes1c/d−
4c/d of the type Rh(I)(NHC)(COD)X (where X is Cl or I,
COD is cyclooctadiene, and NHC is a dimethylbenzimidazo-
lylidene; see Scheme 1). The dimethylimidazolylidene NHC
fragment was identified as a useful organometallic ligand for
obtainingcytotoxiccomplexesofthisgeneraltypeinourrecent
report.17 Initially, the effects of model complexes containing a
5-nitrobenzimidazole-basedNHCligandandchloridooriodido
secondary ligands (complexes 2c and 2d) were evaluated. In
these compounds, the electron-withdrawing nitro group
reduces the donor capacity of the NHC ligand, and this
should, in turn, facilitate ligand-exchange interactions with
biomolecular targets. Compounds 2c and 2d can thus be
regardedasmodelcompoundswithhigherchemicalreactivities
and were therefore used to investigate important effects of the
compound type. In a further step, the effects of different
substituentsonthe5-positionoftheNHCligandontumorcell Figure2.1HNMRspectraof(top)4c(X=Cl)and(bottom)4d(X
p■roliferationinhibitionwereevaluated(1c/d,3c/d,and4c/d). =I)inCDCl.(a)N−CH,(b)O−CH,(c)transCHofCOD,(d)
3 3 3
cisCH ofCOD, (e )pseudoequatorial CH ofCODincisposition,
A 2
CHEMISTRY (e )pseudoequatorialCH ofCODintransposition,(f)pseudoaxial
B 2
Complexes 1c/d−4c/d were obtained starting from the CH 2 of COD.
respective substituted 1,2-diaminobenzenes or benzimidazoles
(Scheme 1). Ring closure of the diaminobenzenes with formic ligand, resulting in a greater steric hindrance toward the CH
2
acidaffordedthebenzimidazoles1a−3a.Benzimidazole4awas protons in proximity, and this difference in the electronic
commercially available. Next, benzimidazolium cations 1b−4b surroundings of the protons presumably led to the splitting of
were formed by alkylation using methyl iodide in the presence the pseudoequatorial (e and e ) signals. The same effect was
A B
of K CO . Finally, complexes 1c/d−4c/d were conveniently ■observed with the couples 1c/d−3c/d.
2 3
obtained by activation of the respective benzimidazolium
cations with Ag O and subsequent transmetalation using MASS SPECTROMETRY: HYDROLYTIC STABILITY
2
bis[halido(η2,η 2-cycloocta-1,5-diene)rhodium(I)]. The target Electrospray ionization ion-trap mass spectrometry (ESI-IT
compounds were purified by filtration over Celite and MS)wasusedtostudythestabilityandreactivityof2cand2d.
recrystallized from a mixture of CH Cl and n-hexane. Nuclear For this purpose, the compounds were dissolved in dimethyl
2 2
magnetic resonance (NMR) and mass spectrometry (MS) sulfoxide (DMSO) or dimethylformamide (DMF) (10 mM
spectra confirmed the suggested structures, and elemental concentration),dilutedto100μMwithwater,andincubatedat
analyses indicated their high purities. 37 °C in the dark, and aliquots withdrawn after different
The target complexes give well-resolved NMR spectra, as incubation periods were investigated.
discussedherefor4cand4dasexamples(seeFigure2).Inthe Compound 2c is stable for 3 h, irrespective of whether the
1H NMR spectra of 4c and 4d, the shifts of the signals of the solventisDMSOandDMF,whichsuggeststhatthesesolvents
respective methoxy groups (b in Figure 2) show no difference, do not influence the stability of 2c. After 6 and 24 h, the mass
whereas the N−CH protons (a in Figure 2) of the iodido spectra in positive-ion mode of 2c dissolved in DMSO and
3
derivative 4d are shifted slightly upfield compared to 4c. This DMF were very similar. As evidenced by the mass signal
phenomenon can be explained by the difference in the corresponding to [LRh(COD)(OH) − H]+, where L is the
electronic densities of the halido ligands. The cis- (c in Figure nitro-NHC ligand and COD is cyclooctadiene, the compound
2) and trans- (d in Figure 2) positioned olefinic signals (cis/ hydrolyzes the Rh−Cl bond and forms either aqua or
trans relative to X) of the COD moiety are shifted slightly hydroxido complexes. Loss of the NHC ligand was observed
downfield in the iodido derivative 4d. However, the most and seemed to become more pronounced at longer incubation
obviouseffectobservedinthespectraofthesederivativesisthe times, whereas the COD ligand remained coordinated. The
splittingofsomeoftheCH signals(einFigure2)incomplex protonated NHC ligand was detected at m/z 192.05 (m =
2 theor
4d. The iodido ligand has a larger volume than the chlorido 192.08). Moreover, nonselective solvent adducts with the
9592 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry Article
organometallic compound were observed for both DMF and (DMSO) − H]+ were observed in positive-ion mode.
DMSO.Increasingthedrytemperature intheESIsourcefrom Generally, the iodido analogue 2d undergoes very similar
180 to 300 °C did not reduce either the DMSO or DMF hydrolysis processes, giving mass signals virtually identical to
adducts, but led to enhanced loss of the NHC ligand. those of 2c (Scheme 2). The compound hydrolyzes slightly
Nonselective solvent adducts are indicated by broad signals in faster and forms aqua or hydroxido complexes again
themassspectrumcomparedtothesharpcoordinatedadducts; corresponding to [LRh(COD)(OH) − H]+. However, release
for example, Figure 3 shows the broad nonselective solvent of the NHC ligands seems slightly more pronounced for 2d.
The mass spectra of both the DMSO and DMF solutions in
negative-ion mode revealed two major signals at m/z 480.82
and 605.05. MS/MSexperiments ofthese mass signals did not
reveal any additional information on their identity. Further-
− −
more, mass signals corresponding to I and I were detected,
3
supporting the hypothesis of the hydrolysis pathways.
Ofnoteisthemasssignalatm/z509.96,whichwasfoundin
thepositive-ionmassspectraof2cand2dwithDMSOafter>6
h of incubation. The identity of the parent mass signal is
unclear, but MS/MS experiments revealed a fragment
corresponding to [LRh(OH)(DMSO) − H + Fu]+, where
2
Fu is an unknown neutral fragment with m/z 44. This mass
signalincreasedovertimeforbothcompounds,suggestingthat
COD release mightalso represent apossiblereaction pathway.
Further details on MS measurements are provided in the
S■upporting Information.
EFFECTS ON MITOCHONDRIAL AND CELL
METABOLISM
Figure3.SectionsoftheESI-ITmassspectrainpositive-ionmodeof
2cshowingthedifferenttypesofsolventadductswith(A)DMSOand I
s
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es
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we
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ll
(B) DMF. Broad mass signals indicate nonselective adducts between
metabolism.13,27,28 Analogous experiments with 2c and 2d
thesolventandmetal,andsharppeaksindicatesolventcoordinationto
the metal. showed some comparable trends (e.g., a decrease of the
mitochondrial membrane potential with both compounds,
adduct of hydrolyzed 2c at m/z 480.06 and the respective
inhibitionofcellrespirationwith2d);however,theeffectswere
complex giving a sharp mass signal at m/z 402.06. The less evident and appeared significant only at much higher
negative-ionmodespectraof2cdissolvedinDMSOandDMF dosages (e.g., 50 μM). A more detailed discussion of these
feature a single mass signal that increases over time and r■esults is presented in the Supporting Information.
corresponds to a dimeric Rh complex that has undergone
carbene release, namely, [Rh (COD) (μ-Cl) (OH) − 2H] − ELISA MICROARRAYS
2 2 3 2
(Figure S1B, Supporting Information). Rhodium has a strong Toanalyzetheeffectsofcomplexes2cand2doncellsignaling,
affinity for chloride, and a similar type of Rh-chlorido dimer enzyme-linked immunosorbant assay (ELISA) microarrays
was reported that was also accompanied by ligand release.26 were applied to detect the absolute levels of a selection of
Compound 2d is also stable for 3 h in DMF and DMSO, keysignalingproteinsintheirphosphorylatedstates.Toobtain
after which increases in free carbene and [Rh(COD)(OH)- time-resolvedimagesofsignalingmodulation,HT-29cellswere
− −
Scheme 2. Proposed Hydrolysis Pathway of 2c (X = Cl ) and 2d (X = I ) in Aqueous Solution
9593 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry Article
Figure4.ELISAmicroarrayswith2cand2d:(A)phospho-HSP27S78/S82,(B)phospho-p38T180/Y182(nolegend),(C)phospho-ERK1T202/Y204,(D)
phospho-ERK2T185/Y187, (E) phospho-FAKY397,(F) phospho-p70S6T421/S424,(G) phospho-WNK1T60, (H) phospho-GSK-3βS9.
incubated in time-course experiments (0−24 h) with two phospho-ERK1 (T202/Y204), phospho-ERK2 (T185/Y187),
concentrations of each compound, namely, 2.5 and 5.0 μM and phospho-p38α (T180/Y182) increased 2−10-fold upon
(Figure 4), and cell lysates collected at indicated time points treatmentwith5.0μM2ccomparedtomock-treated(0.1%v/v
were incubated with the microarrays. DMF) samples. The same concentration of2dshowed slightly
We observed a significant time-dependent activation of weaker effects on MAPK activation. ERK1/2 and p38 are
mitogen-activated protein kinases (MAPKs). The levels of generallyknowntobeactivatedinresponsetotheintracellular
9594 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry Article
redox state and oxidative stress and potentially contribute to MDA-MB-231 cells over HT-29 cells. Established anticancer
influencing cell survival or cell death.29 drugs such as cisplatin and 5-fluorouracil trigger comparable
Our analysis also revealed a strong increase of chaperone cytotoxiceffectsintherangeofapproximately1−10μMinthis
HSP27 phosphorylation. This protein mediates the protection assay.35,36 Regarding the substituents on the NHC ligand, the
properties of cells in response to cytotoxic stress (e.g., loss of methyl group of 3c/d triggered the lowest IC values in both
50
protein functionality upon binding of organometallic frag- celllines,andregardingthesecondaryhalidoligands,theiodido
ments), growth arrest, or receptor-mediated apoptosis.30 derivatives 1d−4d were in most cases more active than the
Interestingly, phospho-FAK (Y397), knowntoregulate focal respective chlorido complexes 1c−4c.
■
contacts and cell detachment,31 exhibited a transient activation
profilewithbothcompounds:5μM2cand2dinducedaslight
CONCLUSIONS
increaseinitsphosphorylationduring2hoftreatmentfollowed
byasustaineddecrease.Thelevelsofphospho-p70S6 kinase,a Complexes of the type Rh(I)(NHC)(COD)X (where X = Cl
key downstream effector of mTORC1, were also increased or I) can be conveniently prepared following an established
upon treatment, indicating changes in cellular protein syn- method that is based on the reaction of the precursor
thesis.32Severalsignalingproteins,amongthemthekeyprotein benzimidazolium cations with Ag
2
O and a subsequent trans-
ofPI3kinasesignaling,Akt1;theimportanttranscriptionfactor
metalationreactionusingbis[halido(η2,η2-cycloocta-1,5-diene)-
CREB; and the DNA damage checkpoint Chk2 showed only rhodium(I)]. 1H NMR spectra confirmed differing electronic
minor or insignificant changes in phosphorylation (see effectsofthechloridoandiodidoligands,whichmightindicate
Supporting Information). Finally, we observed a decrease in different kinetic reactivities. The two organometallic Rh
phosphorylated ERK5 signaling upstream modulator WNK1, compounds 2c and 2d were stable in aqueous solution
implicated in antiapoptotic signaling.33 GSK-3β was also containing 1% DMF or 1% DMSO for at least 3 h. After 6 h,
dephosphorylated (activated) upon treatment. This enzyme is however, the nitro-NHC ligand was released to a significant
a key regulator of numerous signaling pathways, including degree and might provide a route of decomposition. In
cellular responses to Wnt, receptor tyrosine kinases, and G contrast, the COD ligand remained largely coordinated
protein-coupledreceptorsandisinvolvedglycogenmetabolism throughout the incubation period, although the mass signal at
and cell cycle regulation and proliferation.34 m/z 509.97 was indicative of some COD release. The mass
spectra of the DMSO and DMF solutions were very similar,
Table 1. Antiproliferative Effects against HT-29 and MDA- featuringseveralselectiveandnonselectivesolventadductswith
MB-231 Cells the organometallics. Compound 2c eventually forms a dimeric
compound corresponding to [Rh (COD) (μ-Cl) (OH) −
IC 50 HT-29(μM) IC 50 MDA-MB-231(μM) 2H] − ,whereasunambiguousdecomp 2 osition 2 product 3 sof2d 2 are
1a >100 >100 still elusive.
2a >100 >100 Of note, in a previous study, we observed rapid cellular
3a >100 >100 uptakeofthestructurallyrelatedcomplexRh(I)(NHC)(COD)
4a >100 >100 Cl (see Figure 1) within 1−4 h.17 Accordingly, it can be
1b >100 >100 speculatedthatthecomplexesreachthecellularenvironmentin
2b >100 >100 an intact form and that the mentioned ligand-exchange
3b >100 >100 processes occur mainly inside the cells, leading to bioactive
4b >100 >100 metabolites. Importantly, the observed products of hydrolysis
1c 12.1±1.5 9.0±0.9 are of a cationic nature, and this indicates some analogies with
1d 5.1±0.2 2.7±1.2 the biochemistry of cisplatin, which forms cationic aqua
2c 10.2±2.2 5.2±0.1 complexes upon intracellular hydrolysis that represent the
2d 8.6±0.6 6.4±1.2 species attacking DNA.37 Also regarding cellular metabolism,
3c 5.2±1.6 2.4±0.6 interactions with molecular targets have to be considered (e.g.,
3d 1.5±0.3 1.5±0.1 glutathione or other cellular thiols).
4c 9.5±0.4 4.1±0.6 The above-noted differences between the iodido and
4d 6.6±0.6 3.5±0.3 chlorido ligands also were reflected in certain differences in
■ their biological activities. Whereas the chlorido derivative 2c
was a stronger uncoupling-like agent in mitochondria, the
ANTIPROLIFERATIVE EFFECTS IN TUMOR CELLS iodido complex 2d triggered stronger activity against general
To evaluate the effects on proliferation inhibition of different cellular metabolism (impedance, respiration, acidification; see
substituents on the NHC ligand, we screened complexes theSupportingInformationfordetails).However,theobserved
containing different donor/acceptor groups against tumor cell activitiesrequiredratherhighconcentrations andcantherefore
lines. Complexes 1c/d−4c/d along with their respective be considered as secondary effects.
benzimidazole and benzimidazolium iodide precursors were Clear effects at lower concentrations were observed on
investigated on two tumor cell lines (namely, HT-29 colon cellular signaling determined by ELISA microarrays that had
carcinoma and MDA-MB-231 human breast adenocarcinoma). previously been developed to study the activation of major
The precursor [Rh(I)Cl(COD)] without NHC ligand is pathways controlling stress response and cellular prolifer-
2
inactive in terms of cytotoxicity, as reported earlier.17 Whereas ation.38,39 In these experiments, the two complexes triggered
themetalfreebenzimidazolesandbenzimidazoliumcations1a/ comparably strong phosphorylations of HSP27, and MAPKs
b−4a/b were not active, the rhodium complexes 1c/d−4c/d such as p38 and ERK1/2 were also significantly activated,
exhibitedhalf-maximalinhibitoryconcentration(IC )valuesin reflecting a general cellular stress response. In the case of
50
the low micromolar range (1.5−12.1 μM) with preference for MAPK phosphorylation, the chlorido complex 2c showed
9595 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry ■ Article
higher efficiency. Other important signaling targets that were EXPERIMENTAL SECTION
modulated included FAK and p70S6.
General. All reagents and solvents were used as received from
The observed phosphorylation profiles differ significantly
Sigma,Aldrich,orAcros.5-Methoxybenzimidazole(4a)wasobtained
from results obtained for cisplatin and camptothecin, which from Acros. 1H NMR and 13C NMR spectra were recorded on a
directly target DNA or replication. In comparable assays, Bruker DRX-400 AS NMR System; MS spectra were recorded on a
although under slightly different conditions and with another FinniganMAT4515. The purities of the target compounds (>95%)
cancer cell line in the case of cisplatin, these compounds did were confirmed by elemental analysis (Flash EA112, Thermo Quest
not lead to ERK1/2 phosphorylation.40 Within 8−11 h, Italia). For all compounds undergoing biological evaluation, the
cisplatin also did not affect Akt1 and GSK-3β phosphorylation experimental values differed by less than 0.5% from the calculated
ones.
levelsbutshowedadecreaseafter24h.40Complexes2cand2d
5-Chlorobenzimidazole (1a).44 4-Chloro-1,2-phenylendiamine
initially decreased GSK-3β phosphorylation, but levels
(1.891 g, 13.26 mmol) and 40 mL of formic acid were added to a
increased after longer incubation. Akt1 was not strongly 100 mL flask and refluxed at 110 °C for 4 h, giving a deep blue
influenced by 2c and 2d (small decrease of phosphorylation). solution. The mixture was cooled to 0 °C, and the product was
These results indicate that the mode of action of rhodium(I) precipitated by neutralization with concentrated ammonia. 1a was
NHC complexes differs significantly from the activities of filteredoff,washedtwicewithwater,anddriedat50°C.Yield:1.911g
cisplatinorcamptothecin.Thisisfurtherconfirmedbytheonly (12.53 mmol, 95%), purple powder. 1H NMR (CDCl
3
, ppm): 10.43
(s,1H,NH),8.20(s,1H,ArH2),7.65(d,1H,4J=2.0Hz,ArH4),7.55
weak activation of Chk2 that is activated in response to DNA
(d, 1H, 3J = 8.8 Hz, ArH7), 7.27 (dd, 1H, 4J = 2.0 Hz, 3J = 8.8 Hz,
damage and replication stress.38 Regarding a comparison with
ArH6). 13C NMR (CDCl): 141.3 (ArC-2), 138.2 (ArC-Cl), 136.3
recently studied gold species, complexes 2c and 2d showed a 3
(ArC-7a),128.8(ArC-3a),123.7(ArC-6),116.4(ArC-4),115.4(ArC-
pattern similar to that of previously studied phosphane- 7). Elemental analysis for CHClN (% calcd/found): C (55.10/
containing complexes (e.g., auranofin) that also triggered 54.20), H(3.30/3.30), N (18. 7 36 5 /17.8 2 7).
ERK, p38, and HSP27 activation but differing from that of a 5-Nitrobenzimidazole (2a).44 4-Nitro-1,2-phenylendiamine (1.784
biscarbene gold complex, which, again, did not activate ERK g, 11.65 mmol) and 40 mL of formic acid were added to a 100 mL
phosphorylation.41,42 flask and refluxed at 110 °C for 4 h, giving a green solution. The
mixture was cooled to 0 °C, and the product was precipitated by
Screening of an extended series of Rh(I)(NHC)(COD)X
complexes for their antiproliferative effects against two cancer t n w eu ic t e ra w li i z t a h ti w on ate w r i , th an c d o d n r c i e e n d tr a a t t 5 ed 0° a C m . m Y o ie n l i d a : . 1 2 . a 62 w 1 as g fi (9 lt . e 9 r 4 ed m o m ff o , l w ,8 a 5 sh % e ) d ,
lines showed, in general, a certain preference for iodido over light green powder. 1HNMR (DMSO-d,ppm): 13.10 (s, 1H, NH),
6
chlorido ligands. However, the couple 2c/2d represented an 8.56(s,1H,ArH2),8.52(dd,1H,5J=0.5Hz,4J=2.3Hz,ArH4),8.15
exception where no clear trend could be noted in this respect. (dd,1H,4J=2.3Hz,3J=8.8Hz,ArH6),7,77(dd,1H,5J=0.5Hz,3J
Advantages of switching from chlorido to iodido complexes = 8.8 Hz, ArH7). 13C NMR (DMSO-d): 146.8 (ArC-2), 142.6
6
were previously reported by Sadler and co-workers for (ArCC-NO ), 117.6 (ArC-6), 114.9 (ArC-4), 112.8 (ArC-7).
2
organometallic complexes of the type [M(p-cymene)(azo/ Elemental analysis for C 7 H 5 O 2 N 3 (% calcd/found): C (51.54/
imino-pyridine)X]+, where M was Ru or Os and X was Cl or 51.48), H(3.09/2.97), N (25.76/25.65).
I.43 In that case, the iodido complexes were also more potent 5-Methylbenzimidazole (3a).45 4-Methyl-1,2-phenylendiamine
and differed in other anticancer-related properties such as p53 ( 1 0 0 . 0 57 m 9 L g, fl 4 a . s 7 k 4 a m nd mo re l) flu a x n e d d 2 a 0 t 1 m 1 L 0 ° o C f fo fo rm r i 4 c h a , cid giv w in e g re a ad re d d e - d bro to wn a
dependency for activity. solution. The mixture was cooled to 0 °C and neutralized with
Further conclusions regarding structure−activity relation- concentrated ammonia, and the solvent was removed under reduced
ships for cytotoxicity indicated a preference for the methyl pressure.3awasextractedwithdichloromethane,whichwasremoved
group (see results for 3c/d in Table 1), which exhibits a by reduced pressure. The product was highly hydroscopic brown oil.
positiveinductiveeffectthatmightenhancethedonorcapacity Yield:0.510g(3.86mmol,81%),brownoil.1HNMR(CDCl
3
,ppm):
of the NHC ligand, or the unsubstituted NHC ligand, which 12.73 (s, 1H, NH), 8.26 (s, 1H, ArH2), 7.54 (d, 1H, 3J = 8.3 Hz,
afforded higher cytotoxicity in our previous study.17 Taken ArH7),7.44(s,1H,ArH4),7.13(d,1H,3J=8.3,ArH6),2.4(s,3H,
−CH).13CNMR(CDCl):139.7(ArC-2),135.6(ArC−CH),134.1
together, future lead-optimization strategies for Rh(I) NHC 3 3 3
(ArC-3a),133.9(ArC-7a),125.4(ArC-7),114.8(ArC-4),114.5(ArC-
complexes should further address the coordinated halide 6),21.5(−CH).ElementalanalysisforCHN (%calcd/found):C
ligands as well as the fine-tuning of donor properties of the (72.70/69.78), 3 H(6.10/5.95), N (21.20/1 8 9.8 8 4). 2
NHC ligand system. Regarding our previous report, solubility General Procedure for Synthesis of the Benzimidazolium
plays a deciding role, and therefore, rather low-lipophilicity
Iodides1b−4b.Oneequivalentofsubstitutedbenzimidazole,1equiv
NHC ligands should be preferred in future studies. ofK 2 CO 3 ,andanexcessofmethyliodidewereheatedunderrefluxin
acetonitrile/toluene (1:1) for 12 h. The solvent was removed under
Replacement of the chlorido ligand with CO has already
reduced pressure, and the resultant solid was resuspended in
beeninvestigated, butitturned out tobe problematic interms dichloromethaneandfilteredtoremovetheformedpotassiumiodide
of lipophilicity and stability in solution.17 along with the remaining KCO. The solvent of the filtrate was
2 3
Insummary,ourstudyshowsthatthebiologicalpropertiesof removed under reduced pressure, and the product resuspended in
rhodium(I)NHCcomplexescanbemodulatedbychangingthe tetrahydrofuran to remove the excess methyl iodide and unreacted
substituents on the NHC ligands and the nature of the substituted benzimidazole.
secondary halide ligand. Regarding their mode of drug action, 5-Chloro-1,3-dimethylbenzimidazolium Iodide (1b). 1a (0.391 g,
effects on cellular signaling of certain targets such as p38, 2.56mmol),K 2 CO 3 (0.354g,2.56mmol)andmethyliodide(0.7mL,
10.25 mmol) were dissolved in 20 mL of acetonitrile/toluene (1:1)
ERK1, and ERK2 have to be taken into account.
Taken together with previous
reports,8,9,15−17
this study
and refluxed for 12 h. Yield: 0.354 g (1.95 mmol, 76%), purple
powder.1HNMR(CDCl,ppm):9.69(s,1H,ArH2),8.28(d,1H,4J
shows that the Rh(I)(NHC)(COD) fragment might represent 3
=2.0Hz,ArH4),8.06(d,1H,3J=8.8Hz,ArH7),7.78(dd,1H,4J=
auseful organometallic pharmacophore for the design ofnovel 2.0 Hz, 3J = 8.8 Hz, ArH6), 4.08 (s, 3H, N−CH), 3.98 (s, 3H, N−
3
anticancer and antibacterial agents that interfere with MAPK CH). 13C NMR (CDCl): 156.2 (ArC-Cl), 144.3 (ArC-2), 132.5
signaling and other yet-to-be-identified targets. (ArC 3 -7a),131.1(ArC-3a), 3 126.7(ArC-6),115.1(ArC-4),113.7(ArC-
9596 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry Article
7), 34.3 (N−CH ), 34.1 (N−CH ). Elemental analysis for (0.042 g, 0.18 mmol), bis[iodido(η2,η2-cycloocta-1,5-diene)rhodium-
3 3
CH ClNI (% calcd/found): C (35.03/36.45), H (3.27/3.39), N (I)](0.085g,0.12mmol),2h.Yield:0.044g(0.09mmol,28%),light
9 10 2
(9.08/8.50) brownpowder.1HNMR(CDCl,ppm):7.28(dd,1H,5J=0.6Hz,4J
3
1,3-Dimethyl-5-nitrobenzimidazolium Iodide (2b). 2a (0.313 g, =1.7Hz,ArH4),7.20(dd,1H,4J=1.7Hz,3J=8.5,ArH6),7.18(dd,
1.92mmol),KCO (0.300g,1.92mmol),andmethyliodide(0.5mL, 1H,5J=0.6Hz,3J=8.5,ArH7),5.37(s,2H,CH−COD),4.18(s,3H,
2 3
7.67mmol)weredissolvedin20mLofacetonitrile/toluene(1:1)and N−CH), 4.17 (s, 3H, N−CH), 3.52 (s, 2H, CH−COD), 2.38 (m,
3 3
refluxed for 12 h. Yield: 0.338 g (1.75 mmol, 91%), light brown 4H, CH−COD), 2.05 (m, 2H, CH−COD), 1.88 (m, 2H, CH−
2 2 2
powder. 1H NMR (DMSO-d, ppm): 9.94 (s, 1H, ArH2), 9.08 (dd, COD). 13C NMR (CDCl): 198.9 (d, 1J = 49.5 Hz, NHC), 136.1
6 3
1H,5J=0.5Hz,4J=2.3Hz,ArH4),8.57(dd,1H,4J=2.3Hz,3J=9.0 (ArC-Cl), 134.2 (ArC-7a), 128.5 (ArC-3a), 122.5 (ArC-4), 109.8
Hz,ArH6),8.27(dd,1H,5J=0.5Hz,3J=9.0Hz,ArH7),4.19(s,3H, (ArC-6),109.5(ArC-7),98.6(d,1J=5.2Hz,CH−COD),98.5(d,1J
N−CH),4.15(s,3H,N−CH).13CNMR(DMSO-d):147.4(ArC- =5.2Hz,CH−COD),71.9(d,1J=14.0Hz,CH−COD),71.8(d,1J=
3 3 6
2),145.5(ArC-NO),135.3(ArC-7a),131.5(ArC-3a),121.4(ArC-6), 14.0 Hz, CH−COD), 34.9 (N−CH), 34.8 (N−CH), 32.3 (CH−
2 3 3 2
114.8(ArC-4),110.8(ArC-7),33.8(2×N−CH).Elementalanalysis COD), 32.3 (CH−COD), 29.5 (CH−COD), 29.5 (CH−COD);
3 2 2 2
forCH ONI(%calcd/found):C(33.88/33.97),H(3.16/3.27),N MS(EI): 518 (M+). Elemental analysisfor C H NClIRh(%calcd/
9 10 2 3 17 21 2
(13.17/11.94). found): C(39.37/40.67), H(4.08/4.05), N (5.40/5.25).
1,3,5-Trimethylbenzimidazolium Iodide (3b).46 3a (0.510 g, 3.86 Chlorido(η2,η2-cycloocta-1,5-diene)(5-nitro-1,3-dimethylbenzimi-
mmol), KCO (0.540 g, 3.86 mmol), and methyl iodide (1.0 mL, dazol-2-ylidene)rhodium(I) (2c). 2b (0.102 g, 0.32 mmol), AgO
15.44 mm 2 ol) w 3 ere dissolved in 20 mL of acetonitrile/toluene (1:1) (0.044g,0.19mmol),bis[chlorido(η2,η2-cycloocta-1,5-diene)rhodiu 2 m-
and refluxed for 12 h. Yield: 0.611 g (2.12 mmol, 54.9%), white (I)] (0.089 g, 0.16 mmol), 2 h. Yield: 0.117 g (0.27 mmol, 84%),
powder.1HNMR(CDCl,ppm):11.03(s,1H,ArH2),7.57(d,1H,3J yellowpowder.1HNMR(CDCl,ppm):8.24(dd,1H,4J=2.1Hz,3J
3 3
=8.6Hz,ArH7),7.51(dd,1H,4J=1.0Hz,3J=8.6Hz,ArH6),7.48 =8.6Hz,ArH6),8.22(d,1H,4J=2.1Hz,ArH4),7.35(d,1H,3J=8.6
(d, 1H, 4J = 1.0 Hz, ArH4), 4.24 (s, 3H, N−CH), 4.23 (s, 3H, N− Hz,ArH7), 5.24(s,2H,CH−COD);4.40(s,3H, N−CH),4.39(s,
CH),2.61(s,3H,−CH).13CNMR(CDCl):1 3 42.3(ArC-2),138.5 3H, N−CH), 3.40 (s, 2H, CH−COD), 2.50 (m, 4H, CH 3 −COD),
(ArC 3 −CH), 132.1 (ArC 3 -3a), 130.0 (ArC-7a 3 ), 129.1 (ArC-7), 112.2 2.09 (m, 4H 3 , CH−COD). 13C NMR (CDCl): 204.5 (d, 2 1J = 51.0
3 2 3
(ArC-6), 112.2 (ArC-4), 33.8 (N−CH), 33.7 (N−CH), 21.9 Hz,NHC),143.4(ArC-NO),138.7(ArC-7a),134.9(ArC-3a),118.7
3 3 2
(−CH). Elemental analysis for C H NI (% calcd/found): C (ArC-6), 109.0 (ArC-4), 105.5 (ArC-7), 101.9 (d, 1J = 6.5 Hz, CH−
3 10 13 2
(41.69/41.46), H(4.55/4.50), N(9.72/9.72).
COD),101.8(d,1J=6.5Hz,CH−COD),68.9(d,1J=14.2Hz,CH−
5-Methoxy-1,3-dimethylbenzimidazolium Iodide (4b). 5-Methox- COD),68.8(d,1J=14.2Hz,CH−COD),35.2(N−CH).35.2(N−
3
ybenzimidazole (0.706 g, 4.77 mmol), KCO (0.700 g, 4.77 mmol), CH), 33.0 (CH−COD), 33.0 (CH−COD), 28.8 (CH−COD),
2 3 3 2 2 2
andmethyliodide(1.2mL,19.06mmol)weredissolvedin20mLof 28.8 (CH−COD); MS(EI): 437 (M+). Elemental analysis for
2
acetonitrile/toluene (1:1) and refluxed for 12 h. Yield: 0.944 g (3.10 C H ONClRh (% calcd/found): C (46.65/46.85), H (4.84/
17 21 2 3
mmol,65%), brown powder.1H NMR(CDCl,ppm): 10.73 (s, 1H, 5.04), N (9.60/8.05).
ArH2),7.58(dd,1H,4J=1.6Hz,3J=7.8Hz,A 3 rH7),7.21(dd,1H,4J (η2,η2-Cycloocta-1,5-diene)iodido(5-nitro-1,3-dimethylbenzimi-
=2.3Hz,3J=7.8Hz,ArH6),7.16(d,1H,4J=2.3Hz,ArH4),4.22(s, dazol-2-ylidene)rhodium(I) (2d). 2b (0.091 g, 0.29 mmol), Ag
2
O
3H,N−CH),4.19(s,3H,N−CH),3.95(s,3H,−OCH).13CNMR (0.025 g, 0.10 mmol), bis[iodido(η2,η2-cycloocta-1,5-diene)rhodium-
3 3 3
(CDCl): 159.5 (ArC-OCH), 153.8 (ArC-2), 141.6 (ArC-3a), 132.8 (I)] (0.050 g, 0.07 mmol), 2 h. Yield: 0.044 g (0.011 mmol, 38%),
3 3
(ArC-7a), 116.9 (ArC-7), 113.3 (ArC-4), 95.5 (ArC-6), 56.8 lightbrownpowder.1HNMR(CDCl 3 ,ppm):8.23(dd,1H,5J=0.6
(−OCH), 33.4 (N−CH), 33.1 (N−CH). Elemental analysis for Hz,4J=2.1Hz,ArH4),8.21(dd,1H,4J=2.1Hz,3J=8.5Hz,ArH6),
C H O 3 NI (% calcd/fou 3 nd): C (39.49/4 3 0.26), H (4.31/4.31), N 7.36 (dd, 1H, 5J = 0.6 Hz, 3J = 8.5 Hz, ArH7), 5.42 (s, 2H, CH−
(9 1 . 0 21/ 13 9.04) 2 . COD), 4.29 (s, 3H, N−CH 3 ), 4.28 (s, 3H, N−CH 3 ), 3.56 (s, 2H,
General Procedure for Synthesis of Rhodium NHC Com- CH−COD), 2.42 (m, 4H, CH−COD), 2.14 (m, 2H, CH−COD),
2 2
plexes 1c/d−4c/d. One equivalent of the respective benzimidazo- 1.91 (m, 2H, CH−COD). 13C NMR (CDCl): 205.3 (d, 1J = 48.6
2 3
lium iodide 1b−4b and 0.5 equiv of AgO were added to a dried Hz,NHC),143.2(ArC-NO),138.2(ArC-7a),135.1(ArC-3a),118.7
2 2
Schlenktube.Themixturewasback-flashedthreetimeswithN,and (ArC-6), 108.8 (ArC-4), 105.3 (ArC-7), 99.6 (d, 1J = 6.2 Hz, CH−
2
then15mLofdryCHCl wereadded.Theflaskwasclosed,andthe COD),99.5(d,1J=6.2Hz,CH−COD),72.3(d,1J=14.2Hz,CH−
2 2
mixturewasstirredfor4hinthedark.Asolutioncontaining0.5equiv COD),72.2(d,1J=14.2Hz,CH−COD),35.3(N−CH),35.2(N−
3
of bis[halido(η 2,η2-cycloocta-1,5-diene)rhodium(I)] in CHCl was CH), 32.3 (CH−COD), 32.3 (CH−COD), 29.4 (CH−COD),
2 2 3 2 2 2
added(10mL),andthesolutionwasstirredfor2hinthedark.The 29.4 (CH−COD); MS(EI): 529 (M+). Elemental analysis for
2
obtained suspension was filtered over Celite (281 nm) and C H ONIRh (% calcd/found): C (38.58/39.06), H (4.00/4.10),
17 21 2 3
concentrated in a vacuum. The yellow residue was recrystallized N (7.94/7.78).
fromCHCl/n-hexane (10mL/40 mL) at 4°C. Chlorido(η2,η2-cycloocta-1,5-diene)(1,3,5-trimethylbenzimidazol-
Chlorid 2 o(5 2 -chloro-1,3-dimethylbenzimidazol-2-ylidene)(η2,η2-cy- 2-ylidene)rhodium(I) (3c). 3b (0.092 g, 0.32 mmol), AgO (0.053 g,
2
cloocta-1,5-diene)rhodium(I) (1c). 1b (0.096 g, 0.31 mmol), AgO 0.23 mmol), bis[chlorido(η2,η2-cycloocta-1,5-diene)rhodium(I)]
2
(0.042g,0.18mmol),bis[chlorido(η2,η2-cycloocta-1,5-diene)rhodium- (0.082 g, 0.15 mmol), 2 h. Yield: 0.082 g (0.20 mmol, 63%), yellow
(I)] (0.078 g, 0.14 mmol), 2 h. Yield: 0.054 g (0.13 mmol, 41%), powder. 1H NMR (CDCl, ppm): 7.14 (d, 1H, 3J = 8.2 Hz, ArH7),
3
yellowpowder.1HNMR(CDCl,ppm):7.28(dd,1H,5J=0.5Hz,4J 7.07(t,1H,5J=0.8Hz,ArH4),7.03(ddd,1H,5J=0.8Hz,3J=8.2
3
=1.7Hz,ArH4),7.21(dd,1H,4J=1.7Hz,3J=8.5Hz,ArH6),7.18 Hz, ArH6), 5.15 (s, 2H, CH−COD),4.27 (s, 3H, N−CH), 4.26 (s,
3
(dd, 1H, 5J = 0.5 Hz, 3J = 8.5 Hz, ArH7), 5.17 (s, 2H, CH−COD), 3H, N−CH), 3.35 (s, 2H, CH−COD), 2.46 (m, 4H, CH−COD),
3 2
4.29(s,3H,N−CH),4.28(s,3H,N−CH),3.36(s,2H,CH−COD), 2.46 (s, 3H, CH), 2.01 (m, 4H, CH−COD). 13C NMR (CDCl):
3 3 3 2 3
2.47 (m, 4H, CH−COD), 2.02 (m, 4H, CH−COD). 13C NMR 195.2 (d, 1J = 50.6 Hz, ArC-2), 135.6 (ArC−CH3), 133.5 (ArC-3a),
2 2
(CDCl):198.3(d,1J=50.9Hz,ArC-2),135.9(ArC-Cl),134.0(ArC- 132.5(ArC-7a), 123.8(ArC-7),109.5(ArC-4),108.8(ArC-6),100.1
3
7a), 128.7 (ArC-3a), 122.7 (ArC-6), 109.9 (ArC-4), 109.6 (ArC-7), (d,1J=2.3Hz,CH−COD),100.0(d,1J=2.3Hz,CH−COD),68.3
100.8(d,1J=6.6Hz,CH−COD),100.7(d,1J=6.6Hz,CH−COD), (d,1J=14.5Hz,CH−COD),68.2(d,1J=14.5Hz,CH−COD),34.6
68,5(d,1J=14.6,CH−COD),68.4(d,1J=14.6,CH−COD),34.8(s, (N−CH3), 34.5 (N−CH3), 32.9 (CH2-COD), 32.9 (CH2-COD),
N−CH), 34.8 (s, N−CH), 32.9 (CH−COD), 32.9 (CH−COD), 28.9 (CH−COD), 28.9 (CH−COD), 21.8 (−CH); MS(EI): 406
3 3 2 2 2 2 3
28.8(CH−COD),28.8(CH−COD);MS(EI):426(M+).Elemental (M+). Elemental analysis for C H NClRh (% calcd/found): C
2 2 18 24 2
analysis for C H N2ClRh (% calcd/found): C (47.80/47.93), H (53.15/53.11), H(5.95/5.87), N (6.89/6.53).
(4.96/5.55), N
17
(6
2
.5
1
6/5.05
2
).
(η2,η2-Cycloocta-1,5-diene)iodido(1,3,5-trimethylbenzimidazol-2-
(5-Chloro-1,3-dimethylbenzimidazol-2-ylidene)(η2,η2-cycloocta- ylidene)rhodium(I) (3d). 3b (0.077 g, 0.27 mmol), AgO (0.029 g,
2
1,5-diene)iodidorhodium(I) (1d). 1b (0.093 g, 0.31 mmol), AgO 0.13 mmol), bis[iodido(η2,η2-cycloocta-1,5-diene)rhodium(I)] (0.098
2
9597 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry Article
g,0.13mmol).Yield:0.012g(0.02mmol,9%),lightbrownpowder. addedtocellswithoutmediumreplacement(finalDMFconcentration
1HNMR(CDCl,ppm):7.13(d,1H,3J=8.2Hz,ArH7),7.07(t,1H, =0.1%v/v).Formocktreatment,cellswereincubatedwith0.1%(v/
3
5J=0.9Hz,ArH4),7.04(dd,1H,5J=0.9Hz,3J=8.2,ArH6),5.32(s, v)DMFonly.Atindicatedtimepoints,cellswerewashedthreetimes
2H,CH−COD),4.16(s,6H,N−CH),3.51(s,2H,CH−COD),2.46 withice-coldD-PBS(Invitrogen)andlysedin100μLlysisbuffer(6M
(s,3H,−CH),2.37(m,4H,CH−C 3 OD),2.03(m,2H,CH−COD), urea, 1 mM EDTA, 0.5% Triton X-100, 5 mM NaF, 10 μg/mL
1.85 (m, 2H 3 , CH−COD). 13C 2 NMR (CDCl): 192.4 (d, 2 1J = 53.6 leupeptin,10μg/mLpepstatin,100μMPMSF,3μg/mLaprotinin,2.5
2 3
H (A z r , C N -7 H ) C , ) 1 , 0 1 9 3 .9 5.3 (A ( r A C r - C 4 − ), C 1 H 09 3 ) .4 ,1 ( 3 A 3 r . C 6 - ( 6 A ) r , C 9 - 1 3 . a 2 ), ( 1 d 3 , 3 1 . J 2 = (A 7 r . C 2 - H 7a z ) , , C 12 H 4 − .4 m ce M ntri N fu a g 4 e P d 2 O fo 7 r , 1 1 5 m m M ina N t4 a 3 ° V C O a 4 n i d n 1 D 3. - 0 P 0 B 0 S r ) p . m C , o a l n le d ct s e u d pe s r a n m at p a l n e t s s w w e e r r e e
COD),91.1(d,1J=7.2Hz,CH−COD),35.9(d,1J=15.4Hz,CH− frozen at −80 °C for further analysis.
COD),35.8(d,1J=15.4Hz,CH−COD),33.8(N−CH),33.8(N− Total Protein Concentration. Total protein concentration was
3
CH), 30.8 (CH−COD), 30.8 (CH−COD), 30.7 (CH−COD), determinedusingtheBCAProteinAssay(PierceBiotechnology)ina
3 2 2 2
30.7 (CH−COD), 21.4 (−CH); MS(EI): 498 (M+). Elemental 96-well-plate format.
analysis fo 2 r C H NIRh (% c 3 alcd/found): C (43.39/43.69), H ELISA Microarray Protocol. Proteins were quantified using
18 23 2
(4.86/4.70), N (5.62/4.98). sandwich ELISA microarrays. The microarrays were based on the
Chlorido(η2,η2-cycloocta-1,5-diene)(1,3-dimethyl-5-methoxy- ArrayStripTM platform (Alere Technologies GmbH). A detailed
benzimidazol-2-ylidene)rhodium(I) (4c). 4b (0.085 g, 0.28 mmol), descriptionoftheassayprotocol,informationonthereagentsforthis
AgO (0.050 g, 0.21 mmol), bis[chlorido(η2,η2-cycloocta-1,5-diene)- assay, and a list of the currently available targets were previously
rho 2 dium(I)](0.079g,0.14mmol).Yield:0.030g(0.07mmol,25%), reported.39 In brief, cellular samples were diluted 1:6 with dilution
yellow powder. 1H NMR (CDCl, ppm): 7.15 (d, 1H, 3J = 8.7 Hz, buffer(1mMEDTA,0.5%TritonX-100,5mMsodiumfluoride,1M
ArH7),6.83(dd,1H,4J=2.3Hz
3
,3J=8.7Hz,ArH6),6.75(d,1H,4J
ureainbufferedsaline,pH7.2)andincubatedwithmicroarraysfor60
= 2.3 Hz, ArH4), 5.14 (s, 2H, CH−COD), 4.26 (s, 6H, N−CH3), min. A detection cocktail of 15 biotin-labeled phospho-specific
3.85(s,3H,−OCH),3.35(s,2H,CH−COD),2.46(m,4H,CH− detectionantibodies(R&DSystems)wasused,withtheconcentration
3 2
COD),2.01(m,4H,CH−COD).13CNMR(CDCl):195.3(d,1J= ofeachantibodyat18ng/mL.Colorimetricsignalsweredetectedby
2 3
50.6Hz,NHC),156.4(ArC-OCH),136.1(ArC-3a),130.0(ArC-7a), transmission measurements with the ArraymateTM reader (Alere
3
110.3(ArC-7),109.7(ArC-6),100.0(d,1J=6.5Hz,CH−COD),94.0 Technology GmbH). Kinetic microarray data were analyzed with
(ArC-4) 68.3 (d, 1J = 14.5 Hz, CH−COD), 68.2 (d, 1J = 14.5 Hz, KOMA software.30 Total protein concentrations were used for signal
CH−COD), 56.0 (−OCH), 35.0 (N−CH), 35.0 (N−CH), 32.9 normalization.Thefinalresultswerecalculatedfromtwoindependent
3 3 3
(CH−COD), 32.9 (CH−COD), 28.4 (CH−COD); MS(EI): 422 experiments and are expressed as mean values with errors (standard
2 2 2
(M+). Elemental analysis for C H ONClRh (% calcd/found): C error of the mean, SEM).
18 24 2 Antiproliferative Effects in HT-29 and MDA-MB-231 Cells.
(51.14/51.54), H(5.72/5.63), N(6.63/6.56).
(η2,η2-Cycloocta-1,5-diene)iodido(1,3-dimethyl-5-methoxy-benzi- MCF-7breastadenocarcinomaandHT-29coloncarcinomacellswere
midazol-2-ylidene)rhodium(I) (4d). 4b (0.079 g, 0.26 mmol), AgO maintained in DMEM High Glucose (PAA) supplemented with 50
2
(0.047 g, 0.20 mmol), bis[iodido(η2,η2-cycloocta-1,5-diene)rhodium- mg/Lgentamycinand10%(v/v)fetalcalfserum(FCS)priortouse.
(I)] (0.095 g, 0.13 mmol). Yield: 0.032 g (0.06 mmol, 24%), light
Theantiproliferativeeffectsofthecompoundsweredeterminedbythe
brown powder. 1H NMR (CDCl, ppm): 7.13 (d, 1H, 3J = 8.2 Hz, crystalvioletassayfollowinganestablishedprocedurethatwasapplied
ArH7),6.82(dd,1H,4J=3.0Hz,3
3
J=8.2Hz,ArH6),6.76(d,1H,4J= in a number of previous
studies.47−50
The results were calculated as
( 3 s .0 ,3 H H z , , − A O rH C 4 H ), ) 5 , . 3 3 . 2 52 (s ( , s 2 , H 2H , , C C H H − − C C O O D D ), ), 4 2 .1 .3 6 7 ( ( s m ,6 , H 4H ,N ,C − H CH − 3 C ), O 3 D .8 ) 5 , a■ IC re 50 ex v p al r u e e ss s e o d b a ta s in m e e d an fro v m alu t e w s o ± to SE th M re . eindependentexperimentsand
3 2
2.04 (m, 2H, CH−COD), 1.86 (m, 2H, CH−COD). 13C NMR
2 2
(CDCl): 175.5 (d, 1J = 48.3 Hz, NHC), 156.3 (ArC-OCH), 136.3 ASSOCIATED CONTENT
3 3
(ArC-3a),130.3(ArC-7a),110.1(ArC-7),109.6(ArC-4),97.9(d,1J= *
6.3Hz,CH−COD),94.0(ArC-6),71.7(d,1J=14.1Hz,CH−COD), S Supporting Information
71.7 (d, 1J = 14.1 Hz, CH−COD), 56.0 (−OCH), 35.9 (N−CH), The Supporting Information is available free of charge on the
3 3
35.6(N−CH), 30.8(CH−COD), 30.8(CH−COD), 27.2 (CH− ACS Publications website at DOI: 10.1021/acs.jmed-
3 2 2 2
COD);MS(EI):514(M+).ElementalanalysisforC H ONIRh(% chem.5b01159.
18 24 2
calcd/found): C(42.02/42.25), H(4.70/4.79), N (5.45/4.95). More details on mass spectrometry, effects on
MassSpectrometry.Thecompoundsweredissolvedat10mMin
mitochondria and cell metabolism, additional ELISA
dimethyl sulfoxide (DMSO, Sigma) and N,N-dimethylformamide
(DMF, Acros), and the solutions were diluted with unbuffered water array data (PDF)
■
(MilliPore)to100μMandincubatedinthedarkat37°C.ESImass
spectrainpositive-andnegative-ionmodeswererecordedafter10,60,
AUTHOR INFORMATION
120, 180, and 360 min and after 24 h. Aliquots were then further
diluted to 5−10 μM prior to injection into the mass spectrometer. Corresponding Author
Mass spectra were recorded on a Bruker AmaZon SL electrospray *E-mail: ingo.ott@tu-bs.de. Tel.: +49 531 3912743.
ionizationion-trap(ESI-IT)massspectrometer,andtheresultingdata
fileswereprocessed usingBrukerCompass1.3andDataAnalysis4.0. Notes
The compounds were injected by direct infusion into the mass
T■he authors declare no competing financial interest.
spectrometerat5μL/min,andtypicalexperimentalconditionswereas
follows:±4.5 kVcapillary voltage,63%RFlevel,55.2trapdrive,180 ACKNOWLEDGMENTS
°C dry temperature, 8 psi nebulizer, 6 L/min dry gas. The average
The support by COST action CM1105 (Functional metal
accumulation time in positive-ion mode was approximately 1 ms.
Experimentalmasssignalsincludeastandarddeviationofm/z±0.02. complexes that bind to biomolecules) and the technical
Cell Culture, Time-Course Experiments, and Lysate Prep- assistance of Filip Groznica for performing some of the ESI-
aration for Microarray ELISA. Colon cancer cell line HT-29 M■S measurements are gratefully acknowledged.
(ATCC) was maintained in DMEM High Glucose containing 10%
fetalcalfserum(bothfromPAALaboratories)at37°Cwith5%CO 2 . ABBREVIATIONS USED
Cells were seeded insix-well plates (35mm) at adensity of 3×105
cells/well and were grown for 24 h under standard cell culture BCA, bicinchoninic acid; COD, η2,η2-cycloocta-1,5-diene;
conditions to 60−70% confluence. For treatment, a stock solution of CREB, cAMP response element-binding protein; ERK,
2c or 2d in dimethylformamide (DMF) was freshly prepared and extracellular-signal-regulated kinase; FCS, fetal calf serum;
9598 DOI:10.1021/acs.jmedchem.5b01159
J.Med.Chem.2015,58,9591−9600
Journal of Medicinal Chemistry Article
FAK,focaladhesionkinase;HSP,heat-shockprotein;NHC,N- rhodium(III) and iridium(III) complexes containing methyl-substi-
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(19)Geldmacher,Y.;Splith,K.;Kitanovic,I.;Alborzinia,H.;Can,S.;
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