Synthesis, characterization, and biomedical evaluation of ethylene-bridged tetra-NHC Pd(ii), Pt(ii) and Au(iii) complexes, with apoptosis-inducing properties in cisplatin-resistant neuroblastoma cells

Synthesis and characterization of the first two cyclic ethylene-bridged tetradentate NHC ligands, with an unsaturated (imidazole) and saturated backbone (2-imidazoline), are described. Complexes of both ligands containing palladium(ii) have been obtained. For platinum(ii) and gold(iii), only the unsaturated tetracarbene complexes could be isolated. The attempts to synthesize a methylene-bridged 2-imidazoline macrocycle are also described. Furthermore, a novel bisimidazolinium ligand precursor and its open-chain PdII and PtII tetracarbene complexes are obtained. Finally, it is shown that the unsaturated gold(iii) tetracarbene is able to induce apoptosis in malignant SK-N-AS neuroblastoma cells via the mitochondrial and ROS pathway and overcomes resistance to cisplatin in vitro.


Introduction
N-heterocyclic carbenes (NHCs), rst described in 1991, 1 have found many applications. 2There are several structural features that allow the tuning of their electronic properties.Ring size, the adjacent heteroatoms, N-substituents, and the backbone can be modied.Changing one or more structural properties of a NHC ligand can lead to signicantly different reactivities and stabilities of the resulting complexes. 3Oen several NHC units are combined in multidentate ligands, making use of the chelating effect, and a plethora of multidentate NHC metal complexes has been reported. 4,5r group has developed several bidentate and cyclic tetradentate NHC ligands.The respective transition metal complexes have been applied e.g. in medicinal chemistry 6,7 and epoxidation catalysis. 3While the bidentate ligands can form open-chain tetracarbene complexes, 8,9 the tetradentate ligands give cyclic tetracarbene compounds.Most commonly applied in our recent examinations is the calix [4]imidazolium ligand precursor (a, Fig. 1). 10 Its iron complex (c) can be used as olen epoxidation catalyst achieving unprecedented activity. 3Coinage metal tetracarbene complexes (and metal NHC complexes in general [11][12][13][14][15][16][17] ) have been investigated regarding their antiproliferative activity and selectivity against cancer cells (b, d-f, Fig. 1). 6,7g. 1 Tetracarbene ligand precursor a and derived transition metal complexes b-f.
In this study, the scope of multidentate NHC ligands is extended with an ethylene-bridged bisimidazolinium ligand precursor and two cyclic ethylene-bridged tetradentate NHC ligands, with an unsaturated (imidazole) and saturated backbone (2-imidazoline).Pd II , Pt II and Au III tetracarbene complexes containing the novel ligands are synthesized, characterized and applied in preliminary medicinal studies regarding their activity in inducing apoptosis in malignant cells.Finally, the synthetic attempts to a calix [4]imidazolinium macrocycle (structurally analog to c but with a saturated backbone) are described, because there is an increasing demand for reliable training data, including data on negative outcomes, for machine learning systems in chemistry. 18specially the two new macrocyclic ligand precursors are intended to lay the foundation for electronic comparisons induced by the different backbone in future studies.0][21] A saturated backbone, in turn, can lead to higher basicity because the electron density is more concentrated on the C2 carbene carbon atom due to the lack of p-interactions. 22

Results and discussion
The synthesis of a saturated macrocyclic ligand precursor similar to c, but containing 2-imidazoline moieties instead of imidazole, calix [4]imidazolinium, was pursued parallel to the synthesis of the other ligand precursors.However, the synthesis was not successful with the chosen synthetic approaches as described in the ESI.†

Synthesis and characterization of H 2 L3
H 2 L3 is based on the literature known ethylene-bridged imidazoline moiety (1). 23Alkylation of 1 with MeI in MeCN at 82 °C, followed by an anion exchange with NH 4 PF 6 in water, gives H 2 L3 in 91% yield (Fig. 2).

Synthesis and characterization of H 4 L5/6 and H 4 L8/L9
For the preparation of H 4 L5/6 and H 4 L8/9, a slightly modied literature procedure for similar macrocycles was used (Fig. 3). 10 Ring closure to form the macrocyclic imidazolium salt a is commonly achieved with CH 2 (OTf) 2 , 3 but also CH 2 Br 2 is reported. 24Here the ethylene-bridged imidazoline 1 (ref.23) and the ethylene-bridged imidazole 7 (ref.25) are reacted with ethylene bistriate (4) under dry conditions at −45 °C over a period of 5 h in dry MeCN for H 4 L5 and H 4 L8 (see ESI †).
The synthesis of H 4 L5 and H 4 L8 yields a mixture of a 20membered macrocycle (87% H 4 L5 and 90% H 4 L8, as determined by NMR), consisting of four imidazole (C [4]) units, and a 30-membered macrocycle (13% H 4 L5, 10% H 4 L8) consisting of six imidazole (C [6]) units (see ESI †).Separation attempts of C [4]  and C [6] via column chromatography, precipitation or sublimation were not successful.However, by increasing the cooling period during the addition of the ethylene bistriate at −45 °C to a total of 5 h, the purity of the kinetically preferred C [4] unit could be easily increased up to 98% C [4] for H 4 L5 and in case of H 4 L8 an increase up to 100% (ESI †).Due to the absence of similar macrocyclic imidazolinium compounds, H 4 L5 is compared to a and H 4 L8 in the following. 3elative to a, all signals of H 4 L5 and H 4 L8 are upeld shied, indicating a higher electronic density due to the +I effect of the ethylene bridge leading to an increased shielding effect in the NMR. 10,26The higher upeld shi of H 4 L5 compared to H 4 L8 can be explained by the electronic inducing effect of the saturated bond. 20,26,27nlike in 1 H-NMR, each individual 13 C signal of H 4 L8 in DMSO-d 6 is in the same range as the signals obtained for the macrocyclic compound a. 10 However, in case of H 4 L5, opposite to the 1 H-NMR, the C2 carbon resonance at 159.16 ppm is downeld shied compared to H 4 L8 and a (H 4 L8, Dd # 22.08 ppm, a, Dd # 22.0 ppm), thus contradicting expectations.According to literature and as described by H. V. Huynh, the hypothetical free carbene of the imidazoline ligand H 4 L5 should be a stronger s-donor than H 4 L8, so an enhanced upeld shi of the C2 signal of H 4 L5 should have been detectable. 20,28,29Interestingly this expectation is not met here, and apparently other factors play a role.Every other resonance in the 13 C-NMR is upeld shied. 10alt metathesis of the formed macrocyclic salts can be performed with NH 4 PF 6 to increase the solubility in organic solvents and as additional purication step. 3,30 Synthesis and characterization of complexes (Pd/PtL3, PdL5/6, Pd/PtL8, Pd/AuL9) A well-established route to obtain NHC complexes is to convert the corresponding imidazolium salts with group 10 metal acetates.2][33] An alternative route is via a silver transmetalation. 34n the rst approaches, attempts were made to synthesize the respective Ag I complex with H 2 L3 to obtain a dinuclear structure similar to already published open chain bis-NHCcomplexes. 9,25,35However, no product formation was observed in our case.Either no reaction took place or complex signals were observed in the aliphatic region of d = 1.9-4.4ppm in the 1 H-NMR aer purication, indicating the decomposition of . Several other conditions with different Ag I -salts and addition of sodium acetate as internal base at different temperatures were tested without success.A possible problem might be the stability of the Ag I -complex.Another issue might be hydrolysis of imidazolines under acidic and basic conditions. 36,37It has been proposed in literature that the moisture in the solvent can react with sodium acetate to generate hydroxide ions which can attack the electrophilic center of the C2 carbon and lead to ring-opening products, rather than nucleophilic attacking the acidic proton at the C2 carbon. 38Therefore, the next attempts were conducted under moisture-free reaction conditions by using dried solvents.Even the direct metalation with palladium(II) acetate or palladium(II) chloride under dry reaction conditions did not lead to the desired product.The focus was then shied to a combination of the transmetalation route using Ag 2 O in situ with the direct metalation, by applying the respective metal precursor and sodium acetate as a mild base in dry solvents (Fig. 4).
The absence of the acidic imidazolinium proton signal and appearance of characteristic carbene carbon signals conrms the successful formation of PdL3 and PtL3.Unfortunately, despite several attempts, a clean elemental analysis for PtL3 could not be obtained.Also, the 1 H-NMR of PtL3 shows some impurities, which could not be identied and no 195 Pt isotope coupling phenomena was observed in the 13 C-NMR.
The carbene carbon signal of PdL3 at 194.29 ppm in DMSOd 6 [PtL3; 188.38 ppm in CD 3 CN], is surprisingly downeld shied compared to other Pd(II) bis-NHCs reported in literature. 39,40Due to the theoretically stronger s-donation of the imidazolinylidene ligand H 2 L3 compared to its unsaturated analog, an upeld shi of the 13 C NHC signal was expected.Literature indicates that the signicant downeld shi of the carbene carbon resonance from imidazolium to imidazolinium compounds is a general phenomenon. 38,41,42Another interesting fact is that the analytic data, including HR-ESI-MS and elemental analysis, are not supporting a dinuclear complex or a mono-carbene complex as expected, but indicate that PdL3 has rather a [Pd(L3) 2 ](PF 6 ) 2 structure similar to e.This is further conrmed by single-crystal X-ray diffraction (SC-XRD).The PdL3 complex displays a distorted square planar structure.Two L3 ligands coordinate to the Pd center, resulting in an openchain tetracarbene complex of similar geometry like the cyclic complex e. 34 The Pd-C (2.039 Å, 2.038 Å) distances are in good accord with palladium(II) NHC complexes reported in literature. 34,39The alkyl groups of the ligand L3 adopt a syn conformation in the solid state, while the imidazole rings are tilted by 55.30°out of the palladium square plane (Fig. 5).
The PtL3 complex exhibits a similarly distorted square planar structure compared to PdL3.4][45][46][47] The alkyl groups of L3 also adopt a syn conformation, while the imidazole rings are tilted by 50.53°out of the palladium square plane as in PdL3 (Fig. 6).

Complex PdL5/6 and PdL8/9
Since H 4 L5 and H 4 L8 are quite similar to other macrocycles (a), it seemed suitable to synthesize PdL5 and PdL8 according to alike compounds via the direct metalation route. 32Therefore, H 4 L8 was rst converted with Pd(OAc) 2 in a mixture of dry   DMSO/MeCN (1 : 1) at 40 °C for 16 h. 34However, no product formation was observed aer work-up.Several other conditions such as increasing temperature and reaction time led to the absence of the imidazolium protons and the formation of new product signals in the 1 H-NMR aer 4 d at 80 °C.Still these intensities were very low, and no product could be isolated.Another approach was tried via the transmetalation route with Ag I salts, but this also led to no product formation.Finally, both PdL8 (50%, Fig. 7) and PdL5 (3%) could be obtained by applying the same reaction conditions as for the already synthesized complexes PdL3 and PtL3.The yield of the imidazolinylidene tetracarbene complex could be increased to 46%, by using H 4 L6 instead of H 4 L5, resulting in PdL6.
Again, the absence of the acidic position 2 proton signals in the 1 H-NMR and appearance of the carbene carbon peaks conrm the formation of Pd(II) carbene complexes.The observed chemical shi of PdL8 is in the typical range of Pd(II) tetra-NHC compounds and indicates the formation of a complex with similar coordination sphere as e. 6,34,45 The 1 H-NMR of PdL5 in CD 3 CN shows three signals, with two of them in a similar range to PdL8 and one upeld shied signal of the backbone protons.As already mentioned in the discussion of H 4 L5, the 13 C-NMR of PdL5 is contrary to expectations.The carbene carbon of PdL5 (191.30ppm) is surprisingly strong downeld shied compared to PdL8 (165.84 ppm) and in a similar range to the carbene carbon of PdL3 (195.6 ppm in CD 3 CN).Literature indicates that the signicant downeld shi of the carbene carbon resonance from imidazole to imidazoline compounds is a general phenomenon. 38,41,42The uncertainty of a 13 C-NMR measurement is expected to be below 0.1 ppm; by using three times the weighted standard deviation, a difference of >0.4 ppm is required for a signicant difference that exceeds the statistic uncertainty. 3,48Therefore, PdL5 (191.30ppm in CD 3 CN) and PdL8 (165.84 ppm in CD 3 CN) show a sufficiently different chemical shi to allow its discussion.In general, the normal NHC unit (without any modication) of the tetracarbene ligands is in a range of rather low to negligible pbackdonation, hence the changes in electronic properties are dominated by the s-donation of the tetracarbenes. 3,29According to literature, the imidazoline ligand L5 should be in general a stronger s-donor than L8, so an enhanced upeld shi of the carbene signal would have been detectable. 3,20,28,29Interestingly, this expectation is also not met here, and apparently other factors may play a role, as already observed with PdL3 and PtL3.Therefore, further investigations on this subject, e.g. by means of DFT calculations, have to be carried out, since only conjectures can be made with the present analytical data.The elemental analysis and HR-ESI-MS for PdL8 are in accord with a composition [Pd(L8)](OTf) 2 similar to e.It needs to be noted that no clean elemental analysis of PdL6 could be obtained.However, the elemental analysis and HR-ESI-MS of PdL6 are in accordance with the composition [PdL6](PF 6 ) 2 .Due to unsatisfying results in crystallization of PdL8, an anion exchange in water towards PF 6 − was conducted, resulting in PdL9 (41%).
Single crystals suitable for SC-XRD were obtained by slow diffusion of Et 2 O into MeCN solution of PdL9.As expected, the Pd(II) ion is coordinated in a nearly square planar fashion with C-Pd-C angles deviating from 180°by ∼8°, thus liing the metal slightly above the carbene carbon atom plane (Fig. 8).However, due to the C 2 -bridge, the ligand is strongly bent (C5-Pd1-C5_b = 98.97°) and adopts a crisp-shape, while tilting the imidazole rings 53.62°in an alternating pattern out of the palladium square plane. 49][47] In the following Table 1 the Applying the same reaction conditions and work-up methods to Pt(MeCN) 2 Cl 2 instead of Pd(OAc) 2 results in the formation of PtL8 (25%,Fig.7).The absence of acidic proton signals in the 1 H-NMR and the appearance of the carbene 13

Complex AuL9
For the synthesis of AuL9 (Fig. 7), the same reaction conditions were applied as reported in the literature for similar complexes. 51Therefore, H 4 L8 was converted with KAuCl

Biological evaluation
Induction of apoptosis as cell death type PdL3, PdL8, AuL9 and their respective protonated ligand precursors were tested for their apoptotic effects on Nalm-6 cells (human B cell precursor leukemia cell line) and SK-N-AS cells (human neuroblastoma cell line) at different concentrations and quantied by the nuclear DNA fragmentation by ow cytometry analysis.PdL3 and PdL8 as well as the ligand precursors do not show any apoptosis inducing effects in Nalm-6 cells and SK-N-AS cells (see ESI †).AuL9 shows no apoptotic effect in Nalm-6 cells, but signicant apoptosis induction by AuL9 is detected in SK-N-AS cells (Fig. 9A); therefore, the effect of AuL9 in SK-N-AS cells was further characterized.
To exclude necrotic effects of AuL9, lactate dehydrogenase (LDH) leakage from SK-N-AS cells aer 2 h incubation with AuL9 was measured.LDH is released from the cell in case of necrosis and can be detected in the cell culture medium in case of loss of cell integrity and thus serves as a necrosis indicator. 53AuL9 shows no signicant non-specic cytotoxic effects on SK-N-AS cells in the relevant concentration range up to 100 mM (Fig. 9B).
In addition to apoptosis induction, it was tested whether AuL9 can inhibit the proliferation of malignant cells.For this purpose, SK-N-AS cells were incubated with different concentrations of AuL9 for 48 hours.The proliferation inhibition was determined by comparing the total cell number of vital cells of the DMSO control with the total cell number of vital cells of the treated cells.The results show that AuL9 inhibits cell proliferation of SK-N-AS cells in a dose-dependent manner (Fig. 9C).A concentration of 50 mM AuL9 causes nearly 100% inhibition of proliferation, indicating G1 arrest.For the investigation of the mechanism of action of AuL9, the mitochondrial membrane potential of SK-N-AS cells was measured aer 48 h incubation with AuL9.It was shown that the mitochondrion and thus the intrinsic apoptosis pathway plays at least a partial role in the effect of AuL9 (Fig. 10A).
To further characterize the role of mitochondria in AuL9induced apoptosis, the apoptosis pathway mediated by reactive oxygen species (ROS) was investigated.Therefore, N-acetylcysteine (NAC) as a known ROS inhibitor and H 2 O 2 , which belongs to the ROS, as a positive control was investigated.It was shown that apoptosis induction could be signicantly inhibited by NAC.It can therefore be concluded that the generation of ROS plays a role in the AuL9-induced apoptosis (Fig. 10B).However, it is not possible in the present state to be sure how the ROS are generated and whether AuL9 directly leads to an increased ROS production or triggers pathways that result in the generation of ROS.

Overcoming cisplatin resistance
Cisplatin is a well-known chemotherapeutic agent for the treatment of many different types of cancer. 54The development of resistance in tumor cells is a major problem in therapy and is usually the limiting factor in the cure of cancer patients. 55erefore, it is of great importance for drug development that new agents are able to overcome cytostatic drug resistance.In addition to SK-N-AS cells, AuL9 was tested on cisplatin resistant SK-N-AS cells and cisplatin resistance overcoming could be demonstrated (Fig. 11).In a previous characterization of the cisplatin resistant SK-N-AS cells, procaspase-8 under expression was shown. 56The cisplatin resistance overcoming of SK-N-AS cells indicates that procaspase-8 has a minor role in AuL9induced apoptosis.

Conclusion and outlook
A synthetic approach to a calix [4]imidazolinium macrocycle as saturated analog to a is presented.The synthesis of two new macrocyclic ligand systems, being bridged by ethylene groups and containing imidazoline (H 4 L5/6) and imidazole moieties (H 4 L8/9) are discussed.In addition, a novel bisimidazolinium ligand precursor (H 2 L3) is described.All complexes (Pd/PtL3, PdL5/6, Pd/PtL8, Au/PdL9) with their respective ligands synthesized in this work are not accessible via the direct metalation of the respective ligand, due to irreproducible or unreliable results, except for AuL9.Even the route via the silver salt transmetalation does not lead to reliable results.The silver complexes of the respective ligands could not be isolated, probably due to instability of the respective complexes.Therefore, a modied synthetic method has been established.Here, in situ transmetalation with silver oxide is used in combination with the metal precursor and an excess of sodium acetate as a mild base, resulting in the corresponding complexes.Furthermore, the complexes PdL3, PdL8, AuL9 and their respective ligands were tested for their ability to induce apoptosis on Naml-6 and SK-N-AS cells.According to the experiments performed, the data suggest that AuL9 is capable of inducing apoptosis in malignant cells via the mitochondrial and ROS pathway.However, so far, an effect could only be observed on SK-N-AS neuroblastoma cells.In addition, a relatively high dose of AuL9 is required to induce apoptosis in neuroblastoma cells, which could be challenging for clinical applicability.AuL9 is able to overcome resistance to cisplatin in  Further characterization experiments would be required to determine the exact mechanism of action of AuL9, for example identication of molecular targets that are involved in the AuL9 induced apoptosis, as well as the selectivity for cancer cells.

General procedures and analytical methods
Unless otherwise stated, all manipulations were performed under normal atmosphere without dried and degassed chemicals.All syntheses regarding the complexes were conducted under the exclusion of light.Every work-up was performed under normal atmosphere without dried and degassed chemicals; the complexes' work-ups were conducted in addition under the exclusion of light unless otherwise stated.Purication, in case of the Pt and Pd complexes, is achieved by dissolving the crude product in MeCN and ltering it through basic aluminum oxide to remove impurities.Acidic aluminum oxide promotes the decomposition of the complexes while pH-neutral aluminum oxide leads in smaller yields. 6All obtained complexes are air-and water stable; however, PtL3, PtL8 and AuL9 decompose aer extensive exposition to light.Solvents were obtained water-free from a MBraun solvent purication system and stored over molecular sieves (3 Å).The procedures for novel compounds obtained during the synthetic approaches to the saturated macrocyclic ligand precursor, containing 2imidazoline moieties instead of imidazole, calix [4]imidazolinium, (2-imidazoline, N-benzyl-2-imidazoline, 3,3 0methylenebis(1-benzyl-2-imidazolinium)dibromide, N 1 ,N 1 ,N 2 ,N 2 -tetrabenzylethane-1,2-diamine, tert-butyl (2-aminoethyl)carbamate, tert-butyl 2-imidazoline-1-carboxylate) are stated in the ESI.† N-Benzylethylenediamine (12), [57][58][59] ethylenebis(triuoromethanesulfonate) (4), 60 1,1 0 -ethylene-di-2imidazoline (1) 23 and 1,1 0 -ethylenebis-1H-imidazolyl (7) 25,61 were synthesized according to literature procedures.All other reagents were purchased from commercial suppliers and used without further purication.NMR spectra were recorded on a Bruker Avance DPX 400 ( 1 H-NMR, 400 MHz; 13 C-NMR, 100 MHz; 19 F-NMR, 376 MHz) and chemical shis are given in d values in ppm (parts per million) relative to TMS (tetramethylsilane) and reported relative to the residual signal of the deuterated solvent. 62Elemental analysis (C/H/N) were obtained by the Microanalytical Laboratory at Technische Universität München.Electrospray ionization mass spectrometry (ESI-MS) data were acquired on a Thermo Fisher Ultimate 3000 and with higher resolution (HR-ESI-MS) on Exactive Plus Orbitrap from Thermo Fisher.
The resulting reaction mixture is heated to 80 °C for 3 d and is ltered, aer cooling to ambient temperature, over a short plug of basic aluminum oxide.The lter column is eluted with MeCN (100 mL) and all volatile compounds are removed in vacuo.The resulting oily solution (still approx.6  ) is added to a solution of H 4 L8 (320 mg, 307 mmol, 1.00 eq.) and NaOAc (202 mg, 2.46 mmol, 4.00 eq.) in dry MeCN (15 mL) and stirred for 1 h at ambient temperature, followed by the addition of Pd(OAc) 2 (72.4 mg, 322 mmol, 1.05 eq.).The resulting reaction mixture is heated to 80 °C for 4 d.Aer cooling to ambient temperature, the reaction mixture is ltered over a short plug of basic aluminum oxide.The lter column is eluted with MeCN (50 mL) and all volatile compounds are removed in vacuo.The resulting crude material is resuspended in MeCN (5 mL) and centrifuged.Upon the addition of Et 2 O (20 mL) to the supernatant, a white solid is precipitated.The crude material is collected via centrifugation, washed with Et 2 O (3 × 5 mL) and redissolved in MeCN (5 mL).Aer the precipitation with Et 2 O (15 mL) and drying in vacuo, the titled compound PdL8 is obtained as an off-white solid (119 mg, 153 mmol, 50%).(50.0 mg, 305 mmol, 2.5 eq.) a white precipitate is collected via centrifuge and washed three times with H 2 O (5 mL, 3 mL, 3 mL) and Et 2 O (10 mL, 5 mL, 3 mL).Aer drying in vacuo, the titled compound PdL9 is obtained as an off-white solid (39 mg, 50 mmol, 41%).Single crystals suitable for SC-XRD were obtained by slow diffusion of Et 2 O into MeCN solution of PdL8.Pt[(cC Et CC Et C)OTf] (PtL8).Ag 2 O (209 mg, 900 mmol, 2.20 eq.) is added to a solution of H 4 L8 (400 mg, 410 mmol, 1.00 eq.) and NaOAc (202 mg, 2.46 mmol, 4.00 eq.) in dry MeCN (30 mL) and stirred for 1 h at ambient temperature, followed by the addition of Pt(MeCN) 2 Cl 2 (156 mg, 450 mmol, 1.10 eq.).The resulting reaction mixture is heated to 80 °C for 3 d and is ltered, aer cooling to ambient temperature, over a short plug of basic aluminum oxide.The lter column is eluted with MeCN (50 mL) and all volatile compounds are removed in vacuo.The resulting crude material is resuspended in MeCN (5 mL) and centrifuged.Upon the addition of Et 2 O (20 mL) to the supernatant, a white solid is precipitated.The crude material is collected via centrifugation, washed with Et 2 O (3 × 5 mL) and redissolved in MeCN (5 mL).Aer the precipitation with Et 2 O (15 mL) and drying in vacuo, the titled compound PtL8 is obtained as an off-white solid (90 mg, 103 mmol, 25%).Au[(cC Et CC Et C)PF 6 ] (AuL9).H 4 L8 (500 mg, 458 mmol, 1.00 eq.), KAuCl 4 × 2H 2 O (209 mg, 505 mmol, 1.05 eq.), and NaOAc (197 mg, 2.41 mmol, 5.00 eq.) are suspended in dry DMSO (5 mL).The resulting reaction mixture is stirred for 5 h at 100 °C and ltered at ambient temperature.MeCN (5 mL) is added to the ltrate.Aer the addition of Et 2 O (30 mL) to the solution, white solid precipitated.It is washed with MeCN (3 × 5 mL) and DCM (2 × 5 mL) and aer the removal of all volatiles in vacuo, the solid is dissolved in H 2 O (2 mL) and added dropwise to a solution of NH 4 PF 6 (353 mg, 2.17 mmol, 4.00 eq.) in H 2 O (5 mL).The resulting white precipitate is collected and washed with H 2 O (3 × 5 mL) and aer removal of all volatiles in vacuo, the titled compound AuL9 (230 mg, 228 mmol, 47%) is obtained as a white solid. 1
M-C carbene bond lengths [Å], the C carbene -M-C carbene angle [°], the tilt of the NCN unit [°] of the complexes Pd/PtL3 and PdL9 and additionally the C bridge -M-C bridge angle [°] for PdL9 are summarized.
4 and NaOAc in dry DMSO under exclusion of light at 100 °C for 5 h.Aer the work-up, including an ion exchange to PF 6 − as a puri-cation step, AuL9 (47%) was obtained.The absence of acidic proton signals in the 1 H-NMR and the appearance of a new 13 Cpeak at 146.03 ppm in CD 3 CN conrm the formation of the respective Au(III) complex.The chemical shi of the carbene carbon is in accord with Au(III) tetracarbene complex (e) previously reported in literature and slightly downeld shied by 1.79 ppm when compared to e. 51 Furthermore, the backbone carbons are also slightly downeld shied by 0.68 ppm.The 1 H-NMR in CD 3 CN shows similar signals compared to complex PdL8 and PtL8 with the backbone protons at 7.47 ppm and the bridge protons as two multiplets in close proximity at 4.83 and 4.71 ppm.Both elemental analysis 52 and HR-ESI-MS are in agreement with the composition [Au(L15)](PF 6 ) 3 .Although no single crystals suitable for SC-XRD were obtained, the discussed analytical data strongly support the coordination of one tetracarbene ligand similar to PdL9.

Fig. 9 (
Fig. 9 (A) AuL9 induces apoptosis in SK-N-AS cells.The cells were treated with different concentrations of AuL9 and incubated for 96 h.Nuclear DNA fragmentation was analyzed.(B) To exclude unspecific cytotoxic effects, such as necrotic cell death, the viability of SK-N-AS cells was determined by measurement of LDH release into the medium after 2 h of incubation with different concentrations of AuL9.No significant LDH release could be detected in cells treated with AuL9 up to a concentration of 100 mM.Values are given as mean% of DMSO control ± SD (n = 3).(C) The inhibition of proliferation of AuL9 treated SK-N-AS cells was measured after 48 h using the CASY Cell-Counter System.A significant inhibition of cell growth was observed at concentrations as low as 10 mM.Inhibition of proliferation is given in mean% of control ± SD (n = 3); *: p < 0.05 vs. DMSO, t-test.

Fig. 10 (
Fig. 10 (A) The mitochondrial membrane potential in SK-N-AS cells was impaired by AuL15 treatment, which implicates mitochondrial pathway involvement in apoptosis induction.The mitochondrial membrane potential was measured by flow cytometric analysis in SK-N-AS cells after 48 h of incubation with different concentrations of AuL15 and staining with the cationic dye JC-1.Values are mean% of cells with low mitochondrial membrane potential ± SD (n = 3); *: p < 0.05 vs. DMSO, t-test.B The induction of apoptosis in SK-N-AS cells in response to AuL15 treatment was shown to be dependent on the ROS mediated pathway.The cells were incubated for 72 h with 50 mM H 2 O 2 as a positive control or different concentrations of AuL15 with or without pretreatment of the cells with the ROS inhibitor N-acetylcysteine (NAC, 5 mM) 1.5 h prior to substance addition.Nuclear DNA fragmentation was analyzed by flow cytometric analysis.Values are mean% of apoptotic cells ± SD (n = 3); *: p < 0.05 vs. DMSO, t-test.

Fig. 11
Fig. 11 SK-N-AS and SK-N-AS cisplatin resistant cells were treated with different concentrations of AuL15 and incubated for 96 h.It is shown that AuL9 was also effective in inducing apoptosis in cisplatin resistant cells, thus overcoming resistance.8.25 mM cisplatin has been used as a positive control to prove resistance.Nuclear DNA fragmentation was analyzed by flow cytometric analysis.Values are mean% of apoptotic cells ± SD (n = 3); *: p < 0.05 vs. DMSO, t-test.

Table 1
Summary of the M-C carbene bond lengths [Å], the C carbene -M-C carbene angle [°], the tilt of the NCN unit [°] of the complexes Pd/ PtL3 and PdL9 and the C bridge -M-C bridge angle [°] for PdL9 bridge -M-C bridge [°] --98.97 © 2024 The Author(s).Published by the Royal Society of Chemistry RSC Adv., 2024, 14, 10244-10254 | 10247 Paper 6,346,50t 159.39 ppm in CD 3 CN conrms the formation of the respective Pt(II) complex.The chemical shi of the carbene carbon is in accordance with Pt(II) tetra-NHC complexes previously reported in literature and is slightly shied to the upeld compared to PdL8 ( 13 C NHC in CD 3 CN at 165.84 ppm) by 6.45 ppm.43,46,50No 195 Pt isotope coupling was observed.The 1 H-NMR in CD 3 CN shows similar signals compared to PdL8, where the bridge protons also split into two multiplets at 5.01 and 4.44 ppm.In addition, HR-ESI-MS is in accordance to a similar composition as PdL8.Despite multiple attempts, no single crystals suitable for SC-XRD were obtained.However, the discussed analytical data strongly support a similar structure compared to PdL8 and similarly structured tetracarbene ligand.6,34 F-NMR (376 MHz, DMSO-d 6 ) d (ppm) = −77.74(CF 3 ).Elemental analysis for C 24 H 36 N 8 O 12 F 12 S 4 (%) anal.calc.: C 29.27; H 3.68; N 11.38; S 13.02 found: C 29.37; H 3.67; N 11.01; S 13.12.