Guided Antitumoural Drugs: (Imidazol‐2‐ylidene)(L)gold(I) Complexes Seeking Cellular Targets Controlled by the Nature of Ligand L

Abstract Three [1,3‐diethyl‐4‐(p‐methoxyphenyl)‐5‐(3,4,5‐trimethoxyphenyl)imidazol‐2‐ylidene](L)gold(I) complexes, 4 a (L=Cl), 5 a (L=PPh3), and 6 a (L=same N‐heterocyclic carbene (NHC)), and their fluorescent [4‐(anthracen‐9‐yl)‐1,3‐diethyl‐5‐phenylimidazol‐2‐ylidene](L)gold(I) analogues, 4 b, 5 b, and 6 b, respectively, were studied for their localisation and effects in cancer cells. Despite their identical NHC ligands, the last three accumulated in different compartments of melanoma cells, namely, the nucleus (4 b), mitochondria (5 b), or lysosomes (6 b). Ligand L was also more decisive for the site of accumulation than the NHC ligand because the couples 4 a/4 b, 5 a/5 b, and 6 a/6 b, carrying different NHC ligands, afforded similar results in cytotoxicity tests, and tests on targets typically found at their sites of accumulation, such as DNA in nuclei, reactive oxygen species and thioredoxin reductase in mitochondria, and lysosomal membranes. Regardless of the site of accumulation, cancer cell apoptosis was eventually induced. The concept of guiding a bioactive complex fragment to a particular subcellular target by secondary ligand L could reduce unwanted side effects.


Introduction
Although N-heterocyclic carbene( NHC) complexes have been much used as catalysts, their medicinal relevance was recognised surprisingly late, given their chemicals tability under physiological conditions and their structuralf lexibility. [1,2] Unlike cisplatin (CDDP) and relatedp latinum coordination complexes,w hich all lead to DNA adducts, resulting in an inhibition of the cancer cell cycle and eventually in apoptotic cancer cell death, [3] NHC complexeso fv arious metals may address ab roader array of molecular targets.C omplexes with the charactero fd elocalised lipophilicc ations( DLCs) were found to selectively accumulate in mitochondria, whichc an be explained by their negative innert ransmembranep otential. [4,5] Because cancercellshave amore hyperpolarised mitochondrial membrane potential (MMP) than normalc ells, the selectivea ccumulation of metal-carbene complexes with DLC character in cancer cells can be expected. [5,6] With the detection of antitu-mour activity of the antirheumatic gold(I)c ompound auranofin, (2,3,4,6-tetra-O-acetyl-1-thio-b-d-glucopyranosato)(triethylphosphane)gold, gold complexes came to the fore as potential anticancer drug candidates. [7] Auranofin mainly acts through the inhibitiono fm itochondrial thioredoxin reductase (TrxR) and by enhancing the mitochondrialp ermeability. [8,9] Through the inhibition of TrxR activity,t he intracellularl evels of reactive oxygen species( ROS) rise, which damages predominantly cancerc ells because of their elevated ROS levels compared with healthyc ells. [10] As ar esult, cytochromec is released into the cytosol, triggering apoptotic cell death. [11] Due to their stability,N HC ligands can also be annulated and substituted in multifarious ways, allowing the mimicking or combinatorial attachment of pharmacophores to afford pleiotropicd rugs. [12] Herein, we report on NHC gold(I) complexes 4a-6a,c arryinga 1,3-diethyl-4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazol-2-ylidene ligand, akin to the natural antimitotic combre-tastatinA 4( CA-4), and differing only in the second ligand on the gold atom (Scheme 1). Preliminary studies had shown strongc ytotoxicity against cancerc ells with IC 50 values in the low triple-to double-digitn anomolar rangef or complex 6a, but its actual mechanism of actionr emained unclear. [13] A seconds eries of complexes 4b-6b,b earingt he same "second ligands L",y et ab etter detectable fluorescent1 ,3-diethyl-4-(anthracen-9-yl)-5-phenylimidazol-2-ylidene ligand, were synthesised and studied for their intracellular accumulation and their modes of anticancer action. The aim of this study was to find out whether ligand Lc ould be used to set the site of accumulation,a nd thus, the targets and nature of antitumour effects of gold complexes with identical or closely relatedN HC li-gands.T his wasp articularly tempting because similar cis-[{bis(1,3-dibenzylimidazol-2-ylidene)Cl(L)}Pt II ]c omplexes were previously shown by us to always accumulate in mitochondria, regardless of the chargeo ft he complex and nature of ligandsL . [14] Likewise, Ott et al. reported at riad of (1,3diethylbenzimidazol-2-ylidene)(L)gold(I) complexes with the same ligands (L = Cl, PPh 3 ,N HC), whicha ll localised in the mitochondria, albeit to different degrees. [15] Results and Discussion

Synthesis
The new gold(I) NHC complexesw ere prepared from imidazolium salts 3a and 3b (Scheme1). Compound 3b was synthesised analogously to known compound 3a by the van Leusen reactiono ft oluenesulfonylmethyl isocyanide (TosMIC) reagent 1 with 9-formylanthracene, followed by N-alkylationa nd anion exchange of the resulting imidazole 2.R eactions of 3a and 3b with Ag 2 Oa nd transmetalation of the corresponding silver carbene complexesw ith different amounts of [AuCl(SMe 2 )] afforded mono-a nd bis-carbene gold(I) complexes 4a/b and 6a/b analogously to literature procedures. [12,13] New cationic complex 5a was prepared by the reaction of complex 4a with triphenylphosphane. Complex 5b wasobtained by diprotonation of 3b and reactiono ft he free carbene with [AuCl(PPh 3 )].T he stabilityo fa ll complexes 4-6 in aqueous solution was ascertained by 1 HNMR spectroscopicm onitoring over ap eriod of 72 h( see the Supporting Information).

Intracellular localisation
The fluorescent complexes 4b, 5b and 6b were synthesised as easy-to-track analogueso fc omplexes 4a, 5a and 6a,r espectively.W ell-observable, flat 518A2 melanoma cells weret reated with the b complexes, then counterstained with dyes specifically accumulatingi np articularc ancer-relevantc ellular organelles, and eventually fixed and examined through confocal microscopy (Figure1). By counterstaining with NuclearG reen, neutralc hloride complex 4b could be localised in the area of the nucleusa nd to am inor degree in the cytoplasm. This is in line with reports on the nuclear accumulationo fn eutral gold(I) complexes bearing an aryl-substituted NHC ligand. [19,20] Many established first-line anticancer drugs target cancerc ell nuclei, [21] yet suffer from therapeutic shortcomings,i ncluding off-target side effects and an early onset of resistance, owing to insufficient nuclear accumulation. [22] Against this background, the enrichment of new (NHC)Au I Cl complex 4b predominantly in cancerc ell nuclei is remarkable. Cationic phosphane complex 5b accumulatedi nt he mitochondria, as demonstrated by counterstaining of treated 518A2 cells with red mitochondria-selectiveM itoTracker ( Figure 1). Apparently,t he DLC character of this complex favours accumulation in the negativelyc harged mitochondrial compartments overa ny potential DNA intercalation of the planara nthracene residue. Mitochondria are considered to be promising targets for cancer therapy.Adistinct disruption of the MMP typicallyr esults in the induction of apoptosis. One of the pro-apoptotic stimuli is an increased mitochondrial ROS production, which,i nt urn, causes disruption of the MMP. [23] Cationic bis-NHC complex 6b accumulated mainly in lysosomes within the cytoplasm. It should be noted that Gust et al. found an accumulation of all three [1,3-diethyl-4,5-di(p-fluorophenyl)imidazol-2-ylidene](L)gold(I) analogues of complexes 4a, 5a and 6a in the nuclei of MCF-7 and HT-29 cells upon a2 4h long exposure. [24] So, the organelle-selective accumulationo fo ur a complexes after only 30 min might be ak inetic effect. The bottom row of Figure 1 shows confocal fluorescencem icroscopy images of 518A2 melanoma cells treated with complex 6b and lysotropic acridine orange, as well as the good match of the blue fluorescenceo f 6b (UV) with the orange fluorescence of the counterstained lysosomes. Lysosomesa re the recycling centres of the cell and are involved in cellular digestion processes, such as autophagy, endocytosis and phagocytosis. Moreover,t he release of lysosomal hydrolases, so called cathepsins, is involved in the induction of cell death. [25,26] Cathepsins mediate caspase-andm itochondrion-independent cell death, especially in cancer cells with mutationsi ng enes involved in the classic apoptotic pathway,f or example, the TP53 tumour suppressor gene. [27] Induction of cancer cell apoptosis The majority of p53 mutations are missense mutations, as in the case of 518A2 melanoma cells, [28] leading to the expression of dysfunctional p53 proteins with oncogenic activities intensifying malignant properties of cancer cells, such as clinicald rug resistance. [29] Because the p53-independentinduction of cancer cell apoptosis hadb een reported for auranofin [17,30] and for (1,3-diethylbenzimidazol-2-ylidene)gold(I) complexes, [18] we investigated if complexes 4-6 also lead to an activation of apoptosis ( Figure 2). Upon treatment of 518A2 melanoma cells with these complexes, the activation of effector caspases-3 and -7 was observed, which we assumed to be p53 independent, given the results from our cytotoxicity studies. The treated cells showed the typical morphological signs of apoptosis,a s well as translocalisation of phosphatidylserines to the outer leaflet of the plasma membrane, which indicated early rather than late apoptosis or necrosis (see the Supporting Informa-tion). Because about 50 %o fa ll human tumours bear p53 mutations, drugst hat inducep 53-independent programmedc ell death are of particular interest. [31,32]

Mechanism of action of complexes 4a and 4b in the nucleus
The antiproliferativee ffect of CDDP and other platinum complexesi sb ased mainly on their interaction with cellular DNA. [3b, 33] Because of the localisationo fn eutralc omplex 4b in the nuclear area, ap otential DNA interaction of 4b and its close structural analogue 4a was examined by ethidium bromide (EtdBr) saturation assays ( Figure 3) and electrophoretic mobility shift assays (EMSAs; Figure 4).
Addition of complexes 4a or 4b to linear,d ouble-stranded salmons perm DNA led to ad istinct concentration-dependent displacement, andt hus, to ar eduction of the fluorescenceo f intercalated EtdBr,e xceeding that caused by CDDP by far.T his suggestsastrongi nteraction of both complexes 4 with this DNA form, possibly associated with an alteration of the DNA morphology.A uranofin showed no such effect (Figure 3). In the EMSAwith circularp lasmidD NA,aslight relaxation, that is, despiralisation, of the covalently closed circular (ccc) DNA form for the benefit of the open circular (oc) form was observed after incubation with complex 4a,a nd as trongerr elaxation after treatment with complex 4b (Figure 4). In contrast to CDDP,g old NHC complexes are known to bind non-covalently to DNA, which may be the reason for their weaker effects in the EMSA. [34] Although auranofin had previously been reported to interact neither with linear DNA nor with circular plasmidD NA, [35] various other gold(I) complexes with readily displaceable ligands (e.g.,C l À )h ad shown affinity to different types of DNA. [35,36] Irreparable DNA damage induces apoptosis, normally triggered by the tumour suppressor protein p53. However,a poptosis as ac onsequence of DNA damage caused by metal complexes had also been reported to proceed independently of p53, [37,38] through the mitogen-activated protein kinase (MAPK) signal- Figure 2. Induction of effector caspase-3/-7 activity in 518A2 melanomacells after treatment with 5 mm 4-6 for 6h,m easured by means of the Apo-ONE Homogenous Caspase-3/7 Assay Kit (Promega).C DDP was used as ap ositive control. The vitality of cells was simultaneously tested by MTT assays and was > 80 %f or all experiments, except for complex 6a (70 %). All experiments wereperformed in triplicate and resultsq uoteda sm eans AE SD. The solvent-treated negative controlw as set to 100 %. ling pathway involving JNK, p38 and ERK1/2. [37,18] Whether complexes 4,which we have found to inducec ancer cell apoptosis and to be cytotoxic independently of functional p53, operate by as imilarm echanism remains to be shown. At present, we cannot excludet hat their reactions with further biologically relevant macromolecules might also play ar ole. [39] Mechanism of action of complexes 5a and 5b in mitochondria Because cationic triphenylphosphane complex 5b was localised in the mitochondria of 518A2 melanoma cells, we anticipated am itochondria-associated mode of action for 5b and closely relatedc omplex 5a.T he anticancer effect of auranofin, and several other gold(I) complexes,m ainly relies on the inhibition of TrxR. [40,41] TrxRs,w hich catalyse the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductiono ft he redox protein thioredoxin (Trx) and other compounds,a re key enzymes for cellular protection against oxidative stress. [42] To date, three different isoforms of TrxR are known:c ytosolicT rxR1, mitochondrial TrxR2 and testis-specific TrxR3. [43] Gold complexes, such as auranofin, are thought to inhibit TrxRs by releasing monovalent Au I species, which bind to selenocysteine residues in the active site of the enzyme. [44] This is in line with reports that mono-NHC gold(I) complexes with good leaving groups,s uch as halideso rp hosphanes, are better TrxR inhibitors than bis-NHCc omplexes. [45] For instance, sub-micromolar IC 50 values were reported by Gust et al. for donor-substituted (1,3-diethyl-4,5-diarylimidazol-2-ylide-ne)(PPh 3 )gold(I)c omplexes, [24] and by Ott et al. for benzimidazol-2-ylidene analogues, [15,46] whereas few inhibitory bis(1,3-diarylimidazol-2-ylidene)c omplexes have been reported, to date. [45,47] If applied in low sub-micromolar concentrations, complexes 5a and 5b strongly inhibited the panTrxR activityi nc olorimetric TrxR microplate assays with 5,5'-dithiobis(2-dinitrobenzoic acid (DTNB;E llman's reagent) as as ubstrate ( Figure 5). Because many tumours have elevatedT rxR levels, [43] and tumour cells are more sensitive to oxidative stress, due to their ap riori high intracellular ROS levels relative to non-malignant cells, TrxR are interesting targets for selectiveantitumour therapy.
TrxR inhibition generally leads to an accumulation of oxidised Trxa nd ROS in mitochondria, resulting in an increase of mitochondrial permeability. [40] Upon treatmento f5 18A2 melanoma cells with complexes 5a and 5b,w eo bserved ad istinct reduction of the MMP through af luorescence-basedm icroplate assay (Figure 6), exceeding that inducedb ya uranofin, which is in keeping with their strongerT rxR inhibition.
We confirmed these resultsb ya na ssessment of the intracellular ROS concentrations after treatment of 518A2 melanoma cells with auranofin, CCCP and complexes 5a and 5b using the cell permeant, fluorogenic dye 2',7'-dichlorofluorescein diacetate (DCFH-DA). After diffusion into the cells, DCFH-DA is deacetylated by cellular esterases to an on-fluorescent compound,w hich is later oxidised by hydroxyl,p eroxyl or other ROS to the intensely fluorescent 2',7'-dichlorofluorescein (DCF), detectable by fluorescence spectroscopy (Figure 7).
We conclude that the cytotoxicity of complexes 5 originates mainly from their inhibition of TrxR in the mitochondria of cancer cells and the subsequenta lteration of the intracellular ROS equilibrium. [40] Elevated concentrationso fh ydrogen peroxide and oxidised Trx2 affect further intra-mitochondrialt argets, leading to the opening of the mitochondrial permeability . EMSAs with circular pBR322 plasmid DNA after 24 htreatment with complexes 4a or 4b,asv isualised by UV radiation. CDDP was used as a positivec ontrol. Images are representative of at least two independent experiments. Figure 5. Concentration-dependenti nhibition of TrxR activityi nc ell lysates of 518A2m elanomacells by gold(I) complexes 5a and 5b,and auranofin as ap ositive control. TrxR-independent substrate reduction was accounted for by experiments in the presence and absence of the specific TrxR inhibitor aurothiomalate. All values are means AE SD of at least three independent experiments with negativecontrols set to 100 %. transition pore and/or to an increase of the permeability of the outer membrane. [9,48] As aresult, hydrogen peroxide is released into the cytosol where it oxidises cytosolic Trx1 irreversibly, due to the inhibition of TrxRs. The elevated levels of hydrogen peroxide and oxidised Trxi nt he cytosolt hen activate various signalling pathways, eventually leadingt oa poptosis, which is likely to be dependent on p38/ERK1/2,r ather than p53, as shown for auranofin. [40,49] Because cancer cells, unlike non-malignant cells, are not normally susceptible to mitochondrial membrane permeabilityt ransition, the inductiono ft his condition by mitochondria-targeting complexes, such as 5,c ould be exploited in at herapeutic context. [50] Mechanism of action of complexes 6a and 6b in lysosomes The cationic bis-NHC complex 6b was localised in the lysosomeso f5 18A2 melanoma cells. Lysosomes mediate the degradationo fm acromolecules of intracellularo rigin or those that are internalised by endocytosis or phagocytosis. [51] These single-membrane acidic organelles (pH 4.5-4.8)a re involved in variousc ellular pathways and different types of cell death, and their functionality is thus inevitable for cellular homeostasis. [51] Various forms of cellular stress lead to lysosomal swelling and lysosomalm embrane permeabilisation (LMP),r esulting in the releaseo fi ntralysosomal cargo into the cytoplasm. [51] Amongst others, cathepsins Ba nd Da re released into the cytoplasm under stress, where they induce different formso fc ell death, including the p53-independent, lysosome-dependent apoptotic cell death. [26,31,51,52] To detect ap otential induction of LMP by complexes 6,w eperformed at ime-dependent staining of lysosomesi ns olvent-and complex-treated 518A2 melanoma cells ( Figure 8). Because the cytotoxicity of both complexes against 518A2 cells in MTT assays wasq uite different( IC 50 (6a) = 0.4 mm, IC 50 (6b) = 5.5 mm), we adjusted their concentrationsa ccordingly to ensure as ufficient cell viability.T he incubation with either complex 6a or 6b led to an induction of LMP.T he lysotropic orange dye used in this assay selectively accumulates in intact acidic lysosomes. If LMP occurs, the dye is released into the cytosol and the fluorescenceo fd efined lysosomal compartments disappears. As expected, complex 6a,w hich had proved to be more active in MTT assays, also led to faster lysosomal disruption after only 2hof incubation. Cells treated with 6b showed first signs of LMPo nly after 4hof treatment.

Conclusion
The [4-(anthracen-9-yl)-1,3-diethyl-5-phenylimidazol-2-ylidene] (L)gold(I)c omplexes 4b, 5b,a nd 6b accumulated quickly in different compartmentso f5 18A2 melanoma cells, that is, neutral chlorido complex 4b in the nuclei, cationic phosphane complex 5b in mitochondria and large delocalised cationic bis-NHC complex 6b in the lysosomes. The analogous a series of complexes carried as lightly different 4,5-diarylimidazol-2-ylidene ligand. The fact that all couples 4a/4b, 5a/5b and 6a/ 6b afforded similarr esults in cytotoxicity tests with cancer cells, and in tests on targets typically found at the identified sites of accumulation, supports the assumption that a complexes localise similarly to the b complexes,a nd that the nature of ligand L, whichi sr esponsible for the charge, size and lipophilicity of the complex, is decisive for the site of accumulation. However, this phenomenonmight be limited to divalent gold(I)-NHC or even to (imidazol-2-ylidene)gold(I) complexes because acomparable series of cis-[bis(1,3-dibenzylimidazol-2-ylidene)]Cl(L)Pt II [14] and (1,3-diethylbenzimidazol-2-ylidene)(L)gold(I) complexes, [15] carrying the same ligandsL(Cl, PPh 3 or the same NHC ligand), were previously shown to accumulate in mitochondria, regardless of the chargeo ft he complex and the nature of ligand L. The different distributions of DLC complexes 5 (in mitochondria) and 6 (in lysosomes) is explicable by the higherm olecular weighta nd steric demand of Figure 6. Relative MMP in 518A2 melanoma cells after treatment (45 min) with complexes 5a and 5b (10 mm each). Carbonylcyanide-m-chlorophenylhydrazone (CCCP) and auranofin (10 mm,e ach) wereu sed as positive controls and solvent-treated negative controls were setto100 %. Assaysw ere carried out in triplicate. Figure 7. Influence of gold(I) complexes 5a and 5b,and auranofin (10 mm each), as well as CCCP (10 mm)asapositive control, on the levels of ROS in 518A2 melanoma cells, as determined by fluorescence-basedDCFH-DA assays after an incubationtime of 1h.Negative controls were treatedidentically with solvent. Allv alues are meanv alues AE SD from at least four independent experiments with negativecontrols setto1 00 %. the latter,w hich are too large for embedding in the mitochondrial membrane, and thus, are dealt with by the cellular "waste-to-energy plants", the lysosomes. Once fully understood, the concept of controlling the intracellular distribution of metallodrugs by the choice of secondary ligands and charge of the complex could be exploited in rational drugd esign.
For the mode of action of new complexes 4-6,w ef ound an eventuali nduction of p53-independent apoptotic cell death, which was initiated by different effects of the three complex types at their respective sites of accumulation.