TrxR inhibition and antiproliferative activities of structurally diverse gold N-heterocyclic carbene complexes †

Institute of Medicinal and Pharmaceut Braunschweig, Beethovenstraße 55, 38106 ott@tu-bs.de; Tel: +49-531-391-2743 Institute of Inorganic Chemistry, Bergische 42103 Wuppertal, Germany Chair of Inorganic Chemistry I – Bioinorga Universitätstraße 150, 44787 Bochum, Germ Institute of Inorganic Chemistry, Georg-Aug 4, 37077 Göttingen, Germany † Electronic supplementary information and crystallographic data in CIF or 10.1039/c3md00076a Cite this:Med. Chem. Commun., 2013, 4, 942


Introduction
2][3][4] Complexes like aurothiomalate, aurothiosulfate or auranon were recognized to be effective drugs for the treatment of rheumatoid arthritis. 5he cellular pathophysiology of rheumatoid arthritis is related to the malfunction of biochemical pathways, which are also relevant to cancer development (e.g.7][8] Consequently, gold(I) complexes have also been intensively investigated as new anticancer drugs and nowadays bioinorganic medicinal chemists have a large number of bioactive structures at their disposal. 2,3,9,10Among them, N-heterocyclic carbene (NHC) complexes of the Arduengo type represent a very promising class of molecules. 11This was in part motivated by their high stability that is comparable (if not better) than those of phosphane ligands.][18][19][20] Among the numerous hypotheses concerning the molecular mode of drug action, the inhibition of the enzyme thioredoxin reductase (TrxR) seems to play a central role in the pharmacology of gold complexes. 2,3TrxR is a ubiquitous avoprotein in charge of regeneration of the functionality of small molecules (e.g.thioredoxin, glutathione), which are oxidized by different xenobiotics or enzymes belonging to the antioxidant network, and responsible for controlling the cellular redox homeostasis.The catalytic activity of TrxR is carried out by the so-called Trxmotif present in the active site of the enzyme.This characteristic structure consists of a cysteine-AA-AA-cysteine peptidic sequence in the N-terminal motif and a selenocysteine-AA-AAcysteine peptidic sequence at the C-terminal of the protein (where AA stands for any amino acid other than cysteine). 21,22old is a so Lewis acid that can be expected to display a good affinity to so donor atoms like selenium.It has been also reported that, because of their increased metabolism, cancer cells over-express TrxR. 23,24Consequently, the selective inhibition of this key enzyme represents a valuable parameter for the development of new gold-based anticancer drugs.Several a Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany.E-mail: ingo.ott@tu-bs.de;Tel: +49-531-391-2743 effective anticancer agents were shown to inhibit TrxR efficiently.Gold complexes resulted to be the strongest and most selective inhibitors for this enzyme discovered nowadays, in many cases with IC 50 values in the nanomolar range. 23,25,26espite their effective biological prole and an increasing pool of active derivatives, reports that aim at establishing structure-activity relationships for gold NHC complexes remain rather rare.We report here a screening of 20 structurally diverse gold NHC complexes (1a-7) with the aim of identifying correlations between TrxR inhibition, cytotoxicity and structural features of the compounds.The selected examples consist of compounds with a gold(I) or gold(III) central atom with coordinated halides, thiolates as well as structurally diverse NHC ligands and thus they form a library of structurally diverse gold NHC complexes that is suitable to identify structure-activityrelationships (see Fig. 1).

Synthesis
The preparation and characterization of complexes 2-7 have been reported recently. 27,28he thiolato complexes 1a-f were prepared by the reaction of the (NHC)Au(I)Cl complexes with the in situ generated sodium salt of the appropriate thiol.The resulting (NHC)Au(I)thiolato complexes were obtained in good yields as colorless, beige, yellow or grey solids which are soluble in halogenated solvents, acetone and methanol.The new compounds were characterized by 1 H and 13 C NMR spectroscopy and elemental analysis.In one case, the solid-state structure was determined by X-ray diffraction.
The most diagnostic feature in the 13 C-NMR spectra of the (NHC)Au(I)thiolato compounds is the chemical shi of the carbene carbon atom.In the halide complexes the signals are observed at around 170 ppm, while those of the thiolato compounds are observed at approximately 180 ppm.
The structure of the 1-naphthylthiolato derivative 1f was solved by single crystal X-ray crystallography (see Fig. 2 for an ORTEP plot and Table S1 † for full geometrical parameters) and was found to have dimeric structure in the solid state.The angle about the metal centre is close to linearity (175.1 ) and the Au-C bond length is in the expected range for carbene gold complexes.The compound forms dimers through an intermolecular aurophilic interaction [Au(1)/Au(1*) ¼ 3.27 Å], a feature which is oen observed in the solid-state structures of gold(I) complexes.

Inhibition of enzymatic activities
The inhibition of the target enzyme TrxR was measured using an established assay, which is based on the NADPH dependent reduction of the disulde bond of the substrate 5,5 0 -dithiobis(2nitrobenzoic acid) (DTNB or Elmann's reagent) by the isolated enzyme (see Table 1). 29Glutathione reductase (GR) inhibition was studied for some of the complexes for reference purposes.Many complexes showed an effective inhibition of the target enzyme TrxR with IC 50 values in the low micromolar or submicromolar range, which is comparable or even lower as observed for "organic" inhibitors (e.g.PX12). 30Some interesting conclusions can be drawn from the obtained data.
The most effective inhibitors of TrxR with IC 50 values well below 1 mM were 1a, 1b, 1d, 1e, which contain a thiophenolate derived ligand, 5, with a peptidic side chain, as well as the pyrazole/pyridazinyl containing compounds 6a and 7.The majority of the complexes (1c, 1f, 2a-4b, 4d, 6b, 6c) displayed more moderate activity (IC 50 values in the range of 1-10 mM), and complex 4c was not active (no IC 50 values below 50 mM).In the series of 1a-f, the comparably lower activity of 1c and 1f could be the consequence of steric hindrance caused by the two methyl groups of 1c or the additional annellated benzene ring of 1f.An increased steric demand at the thiolate supposedly counteracts ligand exchange processes with the selenocysteine side chain in the TrxR binding site.Similar to the thiophenolate moiety of 1a-f, the halide or thiolate ligands of 2a-6c represent leaving groups that can undergo ligand exchange processes.Not surprisingly, these compounds were active inhibitors with 4c as the only exception.The nature of the halide (Br vs. Cl) is of minor relevance with a slight preference for chlorine.Comparison of the pairs 2a/2b, 4a/4b and 6a/6b indicates that for the chlorido complexes 2a and 6a (but not for 4a) the activity against TrxR is slightly elevated.These minor differences may be due to only small kinetic differences between the two halido ligands.The inuence of the oxidation state of the gold central atom can be evaluated by comparing 2b vs. 2c, 4b vs. 4c and 6a vs. 6c.In all cases the gold(I) derivatives were more effective TrxR inhibitors than the gold(III) complexes with strongly increased potency in the case of 4b and 6a.Compounds 2a, 2b, 3, 4a, 4b, 5, 6a, and 6b contain a halido ligand coordinated to a gold(I) center but among them they differ in the substituents at the nitrogen atoms of the NHC ligand, which enables some comparisons to be made.The most active enzyme inhibitors of this group are 5 and 6a, which carry on one NHC nitrogen atom a rather bulky and lipophilic tert-butyl or mesityl group and on the other nitrogen atom a more polar peptide moiety or a pyrazole/pyridazinyl containing side chain.Interestingly, 5 is approximately one order of magnitude higher active than 2a.These two compounds differ only in an additional leucine in the R 2 side chain of 5.It can thus be speculated that this particular position could be important for a proper interaction with the active site of TrxR.Finally, 7 represents a cationic lipophilic complex, for which anti-mitochondrial effects might play a role due to the facilitated mitochondrial targeting of such compounds. 31These data are in excellent agreement with recent published studies on other cationic NHC-Au-NHC complexes as well as NHC-Au-L species. 14,17,32R is an enzyme with a close functional and structural relationship to TrxR.The most striking difference between these two enzymes is that GR does not contain a selenocysteine in its binding site and carrys a cysteine at the respective position.Based on its higher acidity the selenocysteine exhibits a stronger affinity to the so gold(I) center and this enables the convenient design of gold based TrxR inhibitors with a strong selectivity for TrxR over GR.A number of 14 of the 20 investigated complexes was subjected to studies on GR inhibition, which clearly conrmed the expected preferential interaction with TrxR (see Table 1).

Antiproliferative effects and correlation with TrxR inhibition
The triggering of antiproliferative effects by the complexes was investigated in HT-29 colon adenocarcinoma cells, which represent tissues obtained from a highly relevant human tumor.IC 50 values were calculated and are presented in Table 1.The  ).It has to be noted that in particular 5 was a strong inhibitor of TrxR activity but not cytotoxic.In order to establish the possible correlations between TrxR inhibition and cytotoxicity, the IC 50 values obtained in the antiproliferative experiments were compared with those for TrxR inhibition (see Fig. 3).
The majority of the compounds showed a combination of low IC 50 values for both TrxR inhibition and cytotoxicity, which conrms that inhibition of TrxR can be an important contributing factor in triggering antiproliferative effects.However, an unambiguous correlation between the cytotoxicity of the complexes and the inhibition of TrxR could not be established and this may be related to the contribution of additional parameters affecting drug activity such as biodistribution and metabolisation, particularly for compounds 2a, 2c and 5. Conversely, compound 4c shows excellent antiproliferative activity at low TrxR affinity, which might point to an activation mechanism in vitro.

Conclusions
An extended and structurally diverse set of gold(I) and gold(III) compounds with N-heterocyclic carbene ligands was investigated for the inhibition of the target enzyme TrxR and for the triggering of antiproliferative properties in the HT-29 colon carcinoma cell line.The majority of the complexes presented a strong TrxR inhibition and led to antiproliferative effects in the range of established cytotoxic drugs (e.g.cisplatin, auranon).Although a direct correlation cannot be claimed, the data strongly indicated that TrxR inhibition contributes largely to the mode of action of gold NHC complexes.
Analysis of the TrxR inhibition data helped to establish the following trends in structure-activity-relationships: (i) gold(I) NHC complexes were stronger TrxR inhibitors than gold(III) NHC complexes; (ii) thiophenolate ligands lead to strong TrxR inhibition unless they are sterically hindered; (iii) the insertion of a leucine-containing amino acid into the side chains at the NHC nitrogens resulted in strong TrxR inhibition.
Whereas the enhanced activity of thiophenolates is certainly related to kinetic reasons, the positive effect of the leucine containing residue may be related to specic binding characteristics in the enzyme active site.This clearly warrants further investigations and suggests that an optimization of the peptide side chain could lead to derivatives with increased TrxR inhibition.
The lower activity of gold(III) NHC complexes compared to gold(I) analogues, which has recently also been observed by Gust et al., might be the consequence of reduction of gold(III) upon reaction with the enzyme. 20In this context it should be noted that the formation of gold(I) species from gold(III) upon reaction with proteins has been reported by Gabbiani et al. and Messori et al. 33,34 Moreover, reduction of gold(III) NHC to gold(I) NHC complexes in cells triggered by the reaction with thiols has been clearly conrmed by Chi-Ming Che and co-workers, 35 and this could in fact be a relevant mechanism for the good cytotoxic activity of compound 4c in this study.
In summary our report clearly suggests that inhibition of TrxR activity is a major factor contributing to the biological prole of gold NHC derivatives and that subtle changes in the coordinated ligands at the gold central atom can lead to major changes in the interaction with the target enzyme.

Chemistry
Unless otherwise stated, all manipulations were carried out without taking precautions to exclude air and moisture.All chemicals and solvents (HPLC grade or better) were sourced commercially and used as received.1-Benzyl-3-(2-hydroxycyclohexyl)imidazol-2-ylidene gold chloride was prepared as described in the literature. 36 1  and 13 C NMR spectra were recorded on Bruker Avance 400 or Bruker Avance III 600 MHz spectrometers and are referenced to external TMS. IRspectra were run as KBr disks on a Bruker Tensor 27 instrument.Elemental analyses were performed by staff of the microanalytical laboratory of the University of Wuppertal.
X-ray crystallography diffraction data were collected at 150 K using an Oxford Diffraction Gemini E Ultra diffractometer, equipped with an EOS CCD area detector and a four-circle kappa goniometer.For the data collection the Mo source emitting graphite-monochromated Mo-Ka radiation (l ¼ 0.71073 Å) was used.Data integration, scaling and empirical absorption correction were carried out using the CrysAlis Pro program package (Oxford Diffraction Ltd, 2009).The structures were solved using Direct Methods or Patterson Methods and rened by Full-Matrix-Least-Squares against F 2 .The nonhydrogen atoms were rened anisotropically and hydrogen atoms were placed at idealized positions and rened using the riding model.All calculations were carried out using the program Olex2. 37Full crystallographic and renement parameters as well as tables with bond lengths and angles are included in the ESI.†

Synthesis
Complexes 2-7 were prepared as recently described. 27,28eneral procedure for the synthesis [(NHC)Au(SR)] complexes 1a-f: thiol (1.5 equiv.)and NaOMe (2 equiv.)were dissolved in MeOH (15 ml) and stirred for 10 min.The (NHC) Au(I)Cl complex (1 equiv.) was added, the mixture was stirred at room temperature for 6 h and 5 ml of 2 M KOH in MeOH was added.Aer stirring for a short time, the solvent was concentrated in a vacuum and water was added until the appearance of a precipitate.The solid was isolated by ltration and washed with water and subsequently dried in air.

Biological experiments
Enzymatic inhibition and tumor cell proliferation experiments were performed as described in more detail in recent reports. 17,29For the studies of the enzymatic inhibition a DTNB reduction assay was applied.Cell proliferation was evaluated using the crystal violet assay.Data were generally obtained in 2-3 independent experiments and mean values with errors are presented.In some cases only results from one experiment could be obtained.

Fig. 1
Fig.1Gold NHC complexes investigated in this study.

Fig. 3
Fig. 3 IC 50 values for TrxR inhibition plotted against IC 50 values for cytotoxicity; the inset represents an enlargement.