Unveiling the Potential of Innovative Gold(I) and Silver(I) Selenourea Complexes as Anticancer Agents Targeting TrxR and Cellular Redox Homeostasis

Abstract

A series of NHC‐based selenourea Ag(I) and Au(I) complexes were evaluated for their anticancer potential in vitro, on 2D and 3D human cancer cell systems. All NHC‐based selenourea complexes possess an outstanding cytotoxic potency, which was comparable or even better than that of the reference metallodrug auranofin, and were also able to overcome both platinum‐based and multi‐drug resistances. Intriguingly, their cytotoxic potency did not correlate with solution stability, partition coefficient or cellular uptake. On the other hand, mechanistic studies in cancer cells revealed their ability to strongly and selectively inhibit the redox‐regulating enzyme Thioredoxin Reductase (TrxR), being even more effective than auranofin, a well‐known TrxR inhibitor, without affecting other redox enzymes such as Glutathione Reductase (GR). The inhibition of TrxR in H157 human cancer cells caused, in turn, the disruption of cellular thiol‐redox homeostasis and of mitochondria pathophysiology, ultimately leading to cancer cell death through apoptosis.

Innovative Au(I) and Ag(I) NHC‐based selenourea complexes exhibit a prominent anticancer effect by selectively targeting TrxR in human cancer cells, thus hampering cancer cell redox machinery and inducing apoptotic cell death.

Introduction

The thioredoxin system, which is composed of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, is one of the major redox systems which regulates several essential cellular functions and plays a crucial role in maintaining the intracellular redox balance. ^[1] TrxRs are homodimeric flavoproteins catalyzing the NADPH‐dependent reduction of Trx or a wide spectrum of other cellular oxidized dithiols. Inhibition of TrxR has been shown to dysregulate the cellular redox homeostasis, ultimately leading to cell apoptosis. ^[2] TrxRs are nowadays well‐recognized and promising cancer‐specific targets for anticancer drug development since they are overexpressed in many aggressive, refractory and metastatic tumors and may contribute to drug resistance.[ ³ , ⁴ ] TrxRs are endowed with a flexible C‐terminal extension containing a highly‐nucleophilic selenocysteine (Sec) residue (pKa=5.2) that can be selectively and irreversibly targeted by different substrates and inhibitors. Among them, gold(I) and silver(I) compounds are very effective, acting at nanomolar levels, due to the high affinity of Au(I) and Ag(I) towards the nucleophilic selenolate of reduced TrxR.[ ³ , ⁵ ] The most representative example of metal‐based drugs targeting TrxR is auranofin, [2,3,4,6‐tetra‐o‐acetyl‐ L‐thio‐β‐D‐glyco‐pyranosato ‐S‐(triethyl‐phosphine) gold(I)], a lipophilic gold(I) compound in clinical use since the 1980s for the treatment of rheumatoid arthritis, which was very recently scrutinized in clinical trials for the treatment of lung (NCT01737502) and ovarian (NCT01747798) cancers.[ ⁶ , ⁷ ] However, clinical drug repositioning trials were partially unsuccessful due to its low plasma stability in vivo and consequently its limited ability to reach the tumor mass. Actually, it is well‐known that auranofin and auranofin‐like complexes are easily metabolized by thiol‐containing biomolecules, and the coordinated ligands are mostly lost before reaching the target enzyme.[ ⁶ , ⁷ ]

These achievements stimulated further research efforts aimed at obtaining gold(I) complexes endowed with better stability properties in physiological conditions and thus improved pharmacokinetic and bioavailability profiles. N‐Heterocyclic carbenes (NHCs) are a valuable class of ligands that provide a convenient way to stabilize Au(I) and Ag(I) centers via the establishment of robust metal−carbon bonds, thus preventing ligand dissociation and yielding metal complexes with an improved stability.[ ⁸ , ⁹ , ¹⁰ ] Essentially, the stabilizing influence of these carbene ligands on gold(I) complexes is superior to that of phosphines and, in addition, it is relatively easy to modify the NHC substituents thus allowing a fine tuning of the electronic and steric properties of the complexes as well as of the biological target(s), eventually conferring a multimodal pharmacological activity.[ ¹¹ , ¹² ]

During the past decade, the synthesis and evaluation of several Au(I)‐ and Ag(I)‐NHC complexes were pursued with much interest due to their wide range of pharmacological properties. Nowadays, research on bioactive NHC complexes is a highly dynamic field within the medicinal bioinorganic chemistry, and great attention is devoted to the development of group 11 metal‐based NHCs.[ ⁹ , ¹³ , ¹⁴ ]

In this framework, some of us have recently developed and fully characterized a series of NHC‐based selenoureas and their Au(I) and Ag(I) related metal complexes (Scheme 1). ^[25] The NHC‐based selenourea ligands were obtained via a “weak base” ^[26] synthetic approach that allows good to excellent overall yields. Their Au(I) and Ag(I) derived metal complexes were readily accessed using our previously reported procedures (Scheme 1).[ ²⁵ , ²⁷ , ²⁸ ]

Scheme 1.

Synthesis and structure of the complexes used in this work.

In this report, the potential of these NHC‐based selenourea complexes as anticancer agents and the mechanism behind were extensively investigated. These complexes intrinsically possess an intriguing anticancer potential as they are holding biologically active ligands, i. e. selenoureas, which have been reported to exhibit a prominent anticancer effect.[ ²⁹ , ³⁰ , ³¹ ] Such bioactive moieties can exert a function complementary to that of the metal center, concurring to promote cancer cell death.

Since several oncogenic pathways are involved in cancer disease, a single‐molecular‐target approach may hold several restrictions in its efficacy.[ ³² , ³³ ] “Single‐acting” drug effectiveness may be compromised by robust cancer cell networks preventing major changes through multiple compensatory pathways; this can be more successfully tackled by means of a multifaceted pharmacological approach directed to multiple targets.

Previous studies highlighted that inhibition of TrxR by “single‐acting” small molecules yielded ineffective changes in cancer cell proliferation and spreading, suggesting that a “multi‐acting” system approach is needed to circumvent the inherent compensatory redundancy.[ ³⁴ , ³⁵ ] Consistently, multi‐acting gold(I) anticancer agents developed so far showed an outstanding anticancer profile, attesting that the conjugation of different pharmacophores within the same molecular entity allows to simultaneously deliver multiple bioactive molecules into cancer cells, thus boosting the neat anti‐proliferative potential of the resulting compound. ^[36] On this basis, the antitumor potential of the reported NHC‐based selenoureas and their Au(I) and Ag(I) related metal complexes was assayed in vitro, on 2D and 3D cancer cell systems. In addition, mechanistic studies aimed at detailing the mechanism accounting for their antiproliferative activity was explored, pointing the attention to their ability to hamper TrxR or other cellular redox sensors thus leading to oxidative stress induction.

Results and Discussion

Hydrolytic stability studies

Initially, stability tests were performed by dissolving each complex in a D₂O/DMSO‐d₆ (80 : 20) mixture and following up (at t=0, 24 h, 48 h and 9 days) via ¹H NMR analysis. However, only the Au(I) complexes were exhibiting high enough solubility to allow detection by our NMR equipment. Better follow up of all complexes was attained when we amended the D₂O/DMSO‐d₆ mixture ratio to 20 : 80. ^[37] The same protocol was repeated for all complexes using, this time, a 20 : 80 mixture of NaCl‐D₂O(100 mM)/DMSO‐d₆ (the NaCl solution is meant to approximate the standard [Cl⁻] in blood). Under these conditions, the most discernible trend observed was the good overall stability of all Ag(I) complexes in the D₂O/DMSO‐d₆ (20 : 80) solution, as evidenced by the unchangeable ¹H NMR signals even after 9 days. The same stability for all Ag(I) complexes was also observed in the NaCl‐D₂O(100 mM)/DMSO‐d₆ (20 : 80) solution.

In contrast, the Au(I) complexes showed a completely different behavior; except for [AuCl{Se(ICy)}], all gold complexes showed immediate signs of water coordination upon analysis right after dissolution (See Supporting Information). It seems that most Au(I) complexes were showing signals for 2 compounds in equilibrium, which we postulated to be the initial complex, [AuCl{Se(NHC)}], and the water‐coordinated one, [Au(H₂O){Se(NHC)}]Cl, which resulted from a water molecule displacing the chloride ligand into the metal's outer coordination sphere (Figure 1). This process is slow due to Cl⁻ being a strongly coordinating anion. To further support this hypothesis, [AuCl{Se(IPr)}] was dissolved in a 20 : 80 D₂O/DMSO‐d₆ mixture, and a proton NMR spectrum was recorded (Figure 1, middle spectrum); a mixture of α‐Cl/α‐H₂O in a 41 : 59 ratio was observed. This ratio slowly shifted over time in favor of the α‐H₂O compound, and complete chloride displacement was observed after 9 days (Figure S3, Supporting Information). The same displacement outcome can be obtained by adding stoichiometric amounts of AgNO₃ to [AuCl{Se(IPr)}], right after dissolving the latter in 20 : 80 D₂O/DMSO‐d₆ (Figure 1, top spectrum); this silver additive is known for chloride abstraction, via AgCl precipitation. Once the chloride is substituted by the nitrate, complete displacement towards [Au(H₂O){Se(NHC)}]NO₃ is quickly induced since NO₃ ⁻ is a weakly coordinating counterion and will thus prefer to remain in the metal's outer coordination sphere. Upon dissolving the same amount of [AuCl{Se(IPr)}] in a NaCl‐D₂O(100 mM)/DMSO‐d₆ (20 : 80) mixture, a 53 : 47 ratio of α‐Cl/α‐H₂O was observed (Figure 1, bottom spectrum); this ratio remained unchanged even after 9 days (Figure S4, Supporting Information). This indicated that the increase in [Cl⁻] (from the NaCl solution) has slightly shifted the equilibrium in favor of α‐Cl and has prevented any further chloride displacement over time. A similar displacement behavior was also observed with the Se(SIPr)‐ and Se(IAd)‐based Au(I) complexes in both D₂O/DMSO‐d₆ and NaCl‐D₂O(100 mM)/DMSO‐d₆ solutions (Figures S5, S6, S17 and S18).

Figure 1.

Au(I) complexes behavior in aqueous solutions.

With the Se(IMes)‐ and Se(IPr^Me)‐based Au(I) complexes, complete displacement was observed after 24 h, and the new compounds, [Au(H₂O){Se(NHC)}]Cl, appeared to be stable even after 9 days (Figures S9 and S11). The same complexes, when dissolved in the NaCl‐D₂O(100 mM)/DMSO‐d₆ (20 : 80) solution, showed similar behavior to the Se(IPr)‐ and Se(SIPr)‐based Au(I) complexes (Figures S10 and S12); a persistent, stable equilibrium was observed between Cl‐ and H₂O‐based compounds, even after 9 days.

The Se(IPr^Cl)‐based Au(I) complex, when dissolved in 20 : 80 D₂O/DMSO‐d₆, showed an α‐Cl/α‐H₂O ratio of 59 : 41 which slowly shifted to a 50 : 50 ratio after 9 days (Figure S13). The same complex, when dissolved in 20 : 80 NaCl‐D₂O(100 mM)/DMSO‐d₆, showed a ratio of 67 : 33 in favor of the α‐Cl which remained stable even after 9 days (Figure S14). A similar behavior was also observed with the Se(SIMes)‐based Au(I) complex.

The Se(ICy)‐based Au(I) complex, when dissolved in 20 : 80 D₂O/DMSO‐d₆, showed signs of complete water displacement after 24 h, and the new signals remained stable even after 9 days (Figure S15). When dissolved in 20 : 80 NaCl‐D₂O(100 mM)/DMSO‐d₆, the same complex showed signs of complete water displacement only after 48 h (Figure S16). The new signals again remained unchanged up to 9 days.

The Au(I) complexes’ overall behavior does explain their better solubility compared to the Ag(I) analogues when dissolution was initially attempted in 80 : 20 D₂O/DMSO‐d₆ solution, since the hydrated form, [Au(H₂O){Se(NHC)}]Cl, is expected to be more water soluble. Apart from the aforementioned behavior of Au(I) complexes, both Au(I) and Ag(I) complexes did not show any signs of degradation/decomposition in all the measured timepoints.

Cytotoxicity studies

The ability of the tested complexes and of the corresponding uncoordinated ligands to promote cell death was evaluated in a panel of human cancer cell lines derived from solid tumors (2008 ovarian, HCT‐15 colon, PSN‐1 pancreatic, MCF‐7 breast, A431 cervical, H157 lung carcinoma cells and A375 melanoma cells). The cytotoxicity parameters, in terms of IC₅₀ obtained after 72 h of exposure to the MTT assay are reported in Tables 1 and S1. For comparison purposes, the cytotoxicity of cisplatin and auranofin were assessed under the same experimental conditions.

Table 1.

In vitro antitumor activity of Au(I) and Ag(I) selenourea complexes.

	IC50 (μM)±SD
	2008	HCT‐15	PSN‐1	MCF‐7	A375	A431	H157
[AuCl{Se(IPr)}]	6.2±0.9	8.3±0.4	4.1±0.7	7.4±1.3	0.6±0.1	9.2±0.9	3.0±0.7
[AuCl{Se(SIPr)}]	4.5±1.2	7.8±0.5	1.4±0.1	5.4±1.1	1.4±0.4	4.6±0.4	0.8±0.2
[AuCl{Se(SIMes)}]	2.3±0.7	3.2±0.8	2.8±0.3	0.7±0.2	1.3±0.4	1.8±0.3	0.6±0.1
[AuCl{Se(IMes)}]	1.8±0.3	2.7±0.4	1.1±0.1	0.6±0.1	0.7±0.1	2.8±1.5	0.5±0.05
[AuCl{Se(IPrMe)}]	1.0±0.1	1.6±0.3	0.3±0.05	3.5±0.6	1.0±0.1	0.3±0.1	0.4±0.1
[AuCl{Se(IPrCl)}]	1.4±0.2	1.8±0.8	0.9±0.3	8.2±0.8	1.3±0.3	0.4±0.1	1.3±0.4
[AuCl{Se(ICy)}]	2.5±0.6	4.4±1.6	2.7±0.6	3.2±0.3	0.4±0.1	3.5±0.3	0.6±0.05
[AuCl{Se(IAd)}]	2.3±0.4	2.9±0.5	3.1±0.7	2.7±0.1	0.6±0.1	3.3±0.9	1.7±0.2
[AgCl{Se(IPr)}]	1.1±0.3	5.4±0.1	7.0±1.8	3.8±0.2	0.5±1.1	0.4±0.3	1.1±0.4
[AgCl{Se(SIPr)}]	3.5±0.8	3.9±0.2	5.1±0.9	6.7±0.4	0.4±0.1	0.6±0.1	1.4±0.4
[AgCl{Se(SIMes)}]	1.6±0.4	4.0±0.4	1.2±0.2	2.6±0.6	0.6±0.2	0.5±0.3	1.0±0.2
[AgCl{Se(IMes)}]	3.1±1.1	4.0±0.3	0.3±0.3	2.8±0.8	0.7±0.2	0.4±0.2	2.0±0.7
[AgCl{Se(IPrMe)}]	3.0±0.9	2.2±0.3	0.3±0.1	1.6±0.6	0.4±0.1	0.9±0.3	1.0±0.4
[AgCl{Se(IPrCl)}]	4.4±1.2	2.3±0.9	0.6±0.6	2.9±0.9	0.8±0.1	1.8±0.9	1.7±0.1
[AgCl{Se(ICy)}]	3.3±0.9	8.3±1.1	1.4±0.5	1.3±0.4	1.1±0.1	4.1±0.5	4.8±0.9
Cisplatin	2.2±1.3	11.3±1.5	12.1±2.9	8.8±1.0	3.1±0.4	1.4±0.3	5.9±1.1
Auranofin	1.0±0.2	0.1±0.02	1.0±0.4	0.9±0.3	2.3±1.0	2.0±0.8	1.6±0.3

SD=standard deviation. Cells (3–8×10³ mL⁻¹) were treated for 72 h with increasing concentrations of the tested compounds. The cytotoxicity was assessed by the MTT test. IC50 values were calculated by a four‐parameter logistic model 4‐PL (P<0.05).

Uncoordinated selenoureas possess a moderate cytotoxic potency that was, on average, from 2 to 10 times lower than that of the corresponding metal complexes (Table S1). All tested complexes showed a very promising cytotoxic potential, eliciting IC₅₀ values in the low‐/sub‐micromolar range towards all the human cancer cell lines belonging to the in‐house panel.

In particular, all derivatives were more effective than cisplatin in reducing cancer cell viability, with IC₅₀ average values up to 11.6 times lower than that of the reference Pt‐based drug, and some of them also possess a cytotoxic potency comparable or even better than that of auranofin. Overall, all selenourea metal complexes were more effective than auranofin in inducing cancer cell killing effects towards A375 melanoma cancer cells. In addition, Au(I) and Ag(I) complexes bearing the [Se(IPr^Me)] ligand were, on average, slightly more effective than auranofin against tested cancer cell lines whereas complexes with the [Se(IMes)] ligand showed an in vitro antitumor potency comparable to that of the reference gold(I) complex. Metal‐complexes with the [Se(IPr)] and [Se(SIPr)] ligands were the weakest among the series whereas those characterized by [Se(IPr^Me)] ligands were the most cytotoxic derivatives. Though no clear correlation could be established between the antitumor activity of these complexes and either of their electronic, steric or hydrolytic properties, we have noticed that [Se(IPr^Me)]‐based complexes were the least soluble in the aqueous conditions used during the hydrolytic stability tests.

In general, Ag(I) complexes were much more effective than the corresponding Au(I) complexes, except in the case of complexes bearing the [Se(ICy)] ligand; in this case, complex [AgCl{Se(ICy)}] was on average slightly less effective than the equivalent Au(I) complex, [AuCl{Se(ICy)}]. This trend again correlates to the lower solubility of all Ag(I) complexes, compared to their Au(I) analogues, in the aqueous conditions used when conducting the hydrolytic stability tests. This in turn can be attributed to the Ag(I) complexes’ resistance to water coordination.

The causes of the clinical failure of chemotherapy are numerous but drug resistance represents a key determinant for the variable efficacy of platinum‐based anticancer therapy. To assess the ability of metal‐[Se(NHC)] complexes to bypass acquired drug resistance, Au(I) and Ag(I) complexes were also evaluated for their antiproliferative activity against specific cell line pairs selected for sensitivity/resistance to oxaliplatin, LoVo and LoVo‐OXP human colon cancer cell, and Multi‐Drug Resistant (MDR) cancer cells, LoVo MDR and human ovarian adenocarcinoma A2780 and A2780 ADR cells. The degree of resistance was evaluated by means of a resistant factor (RF), which is defined as the ratio between IC₅₀ (obtained with MTT assay after 72 h of drug exposure) calculated for the resistant cells and those arising from the sensitive ones (Table 2).

Table 2.

Cross‐resistance profiles of Au(I) and Ag(I) selenourea complexes.

	IC50 (μM)±SD
	LoVo	LoVo‐OXP	RF	LoVo MDR	RF	A2780	A2780 ADR	RF
[AuCl{Se(IPr)}]	2.0±0.3	2.1±0.4	1.1	3.9±1.1	2.0	0.6±0.1	0.3±0.05	0.5
[AuCl{Se(SIPr)}]	4.2±1.3	3.4±0.5	0.8	4.2±0.8	1.0	0.6±0.1	0.3±0.04	0.5
[AuCl{Se(SIMes)}]	1.6±0.4	2.5±0.5	1.6	3.0±0.4	1.9	0.7±0.2	0.7±0.1	1
[AuCl{Se(IMes)}]	1.7±0.1	2.3±0.6	3.3	3.2±0.5	1.9	0.4±0.1	0.3±0.03	0.8
[AuCl{Se(IPrMe)}]	1.0±0.04	0.9±0.3	0.9	1.9±0.1	1.9	0.5±0.1	0.3±0.02	0.6
[AuCl{Se(IPrCl)}]	3.9±0.4	4.5±0.9	1.2	4.1	1.1	0.4±0.05	0.2±0.02	0.5
[AuCl{Se(ICy)}]	2.6±0.3	2.1±0.3	0.8	3.7±0.3	1.4	0.3±0.05	0.2±0.02	0.7
[AuCl{Se(IAd)}]	3.5±0.1	4.9±1.1	1.4	4.4±0.9	1.3	0.6±0.2	0.3±0.1	0.5
[AgCl{Se(IPr)}]	3.1±1.1	3.6±0.6	1.2	5.4±1.2	1.7	0.6±0.1	0.3±0.05	0.5
[AgCl{Se(SIPr)}]	2.7±1.3	4.5±0.1	1.7	1.2±0.4	0.4	0.6±0.1	0.3±0.04	0.5
[AgCl{Se(SIMes)}]	3.2±1.2	2.4±0.8	0.8	1.4±0.6	0.4	0.7±0.2	0.7±0.1	1.0
[AgCl{Se(IMes)}]	2.7±0.5	0.9±0.1	0.3	2.3±1.0	0.9	0.4±0.1	0.3±0.03	0.8
[AgCl{Se(IPrMe)}]	3.6±0.6	0.6±0.2	0.2	1.3±0.2	0.4	0.5±0.1	0.3±0.02	0.6
[AgCl{Se(IPrCl)}]	1.8±0.6	0.7±0.1	0.4	1.5±0.3	0.8	0.4±0.05	0.2±0.02	0.5
[AgCl{Se(ICy)}]	4.7±0.8	4.4±0.3	0.9	6.9±1.1	1.5	3.1±1.0	3.3±0.8	1.1
Doxorubicin (oxaliplatin)	1.1±0.2 (1.1±0.6)	ND (14.3±0.3)	ND 13.0	19.2±2.3 ND	17.5 –	0.01±0.001 ND	0.16±0.03 ND	16.0 –

ND=not detected. SD=standard deviation. Cells (3–8×10³ mL⁻¹) were treated for 72 h with increasing concentrations of the tested compounds. The cytotoxicity was assessed by the MTT test. IC₅₀ values were calculated by a four‐parameter logistic model 4‐PL (P<0.05). RF=IC₅₀ resistant cells/IC₅₀ sensitive cells.

The main molecular mechanisms involved in oxaliplatin resistance appears to depend upon: (i) decreased cellular accumulation, which is thought to be related to a greater activity of the ATP7B exporter rather than to the activity of P‐glycoprotein (P‐gp) and multidrug resistance protein 1 (MRP1), and (ii) more efficient repair of oxaliplatin‐induced DNA‐damage by NER (Nucleotide Excision Repair). ^[38] As previously reported, LoVo OXP cells (derived from LoVo cells grown in the presence of increased concentration of oxaliplatin) were about 13‐fold more resistant to oxaliplatin than parental cells. ^[39] Interestingly, all complexes were equally effective against sensitive (LoVo) and resistant (LoVo‐OXP) colon cancer cells, thus attesting their ability to overcome the oxaliplatin resistance.

Table 2 illustrates the data obtained by testing selenourea metal complexes towards two MDR sublines, human colon LoVo MDR and ovarian A2780 ADR cancer cells, in which the resistance to doxorubicin, a drug belonging to the MDR spectrum, is associated with an overexpression of drug transporters, such as the 170 kDa P‐gp. ^[40] All compounds possess resistant factors much lower than that of doxorubicin, thus confirming their ability to overcome the MDR phenomena and to not act as P‐gp substrates.

To further evaluate the anticancer potential of the new Au(I) and Ag(I) selenourea compounds, they were also screened against 3D spheroids of lung (H157) and ovarian (A2780) cancer cells. Conventional 2D cell cultures scarcely represent the complexity of in vivo tumors, thus being barely reliable for predicting the in vivo anticancer activity. In contrast, 3D cell cultures possess several features that more closely mimic the heterogeneity and complexity of in vivo tumors, being consequently more predictive for in vivo effectiveness. ^[41] The cancer spheroids were treated with tested complexes or cisplatin for 72 h, and cell viability was assessed by means of the acid phosphatase (APH) assay (Table 3).

Table 3.

Cytotoxicity towards human cancer cell spheroids.

	IC50 (μM)±SD
	[AuCl{Se(IPr)}]	[AuCl{Se(SIPr)}]	[AuCl{Se(SIMes)}]	[AuCl{Se(IMes)}]	[AuCl{Se(IPrMe)}]	[AuCl{Se(IPrCl)}]	[AuCl{Se(ICy)}]	[AuCl{Se(IAd)}]	Cisplatin
H157	28.2±5.2	8.3±0.4	8.6±0.4	7.6±0.8	6.4±0.1	8.7±1.1	5.3±0.1	5.8±0.7	52.5±1.3
A2780	8.7±1.5	5.7±1.8	8.2±1.0	7.2±1.5	9.0±2.5	9.2±1.0	2.2±0.4	4.4±1.1	74.2±8.9
	IC50 (μM)±SD
	[AgCl{Se(IPr)}]	[AgCl{Se(SIPr)}]	[AgCl{Se(SIMes)}]	[AgCl{Se(IMes)}]	[AgCl{Se(IPrMe)}]	[AgCl{Se(IPrCl)}]	[AgCl{Se(ICy)}]	Cisplatin
H157	34.1±2.2	24.4±1.5	23.4±4.2	8.7±1.5	11.4±4.6	12.2±5	15.6±2.0	52.5±1.3
A2780	21.6±5.0	23.5±0.5	21.3±4.1	17.4±2.7	18.1±3.5	29.1±4.4	23.1±2.2	74.2±8.9

SD=standard deviation. Spheroids were treated for 72 h with increasing concentrations of tested compounds. The growth inhibitory effect was evaluated by APH. IC₅₀ values were calculated by a four‐parameter logistic model 4‐PL (p<0.05).

Notably, all complexes were much more effective than cisplatin against both three‐dimensional models. However, in contrast to the cytotoxicity studies performed on monolayer cell cultures, Au(I) complexes proved to be much more effective than the corresponding Ag(I) derivatives in these cancer spheroid models. In addition, the cytotoxicity pattern was similar to that observed in 2D studies for Ag(I) derivatives, with [AgCl{Se(IMes)}] and [AgCl{Se(IPr^Me)}] being the most effective complexes, whereas [AuCl{Se(ICy)}] and [AuCl{Se(IAd)}] were the most cytotoxic Au(I) selenourea compounds.

Interestingly, by comparing the cytotoxicity results in 3D and the octanol‐water partition coefficient (logP), predicted for [Se(NHC)] ligands by means of the Molinspiration calculator (www.molinspiration.com), no strong linear and/or direct correlations were found, thus suggesting that the hydro/lipophilic balance of the tested complexes does not completely impact their ability to penetrate cancer 3D cell systems and to kill inner and outer core cancer cell spheroids (Figure S1). It should be mentioned that Se(ICy) and Se(IAd) are the only ligands with alkyl substitution on the nitrogen atom, with the former being the smallest ligand of the two and of the whole ligand series.

Cellular uptake

Although to a different extent, all NHC‐complexes, accumulated into cancer cells (Figure 2). Notably, the intracellular levels of Ag(I) were significantly higher compared to Au(I) ones, thus suggesting that Ag(I) complexes are more effective in crossing cancer cell membranes; the fact that all Ag(I) complexes showed better resistance to hydration, compared to the Au(I) complexes during the hydrolytic tests, could be responsible for this difference in behavior. In addition, by comparing metal levels with the calculated LogP values, no direct or linear correlation were found for both Au(I) and Ag(I) complexes (R²=0.08 and 0.23, respectively) (Figure S2, panels A and B) and no correlation was established comparing cellular uptake with IC₅₀ values obtained with the tested complexes in H157 cancer cells (Figure S2, panels C and D). The intracellular selenium content was also monitored after treatment with our selenourea complexes. By analyzing the results obtained in Figure 2, we clearly observe that the cellular Se content is not in a 1 : 1 molar ratio with either metal content for all tested complexes; more particularly, the Se/Au ratio seem to be slightly more coherent than the Se/Ag one.

Figure 2.

Cellular uptake. H157 cells were incubated for 24 h with 1 μM of the tested complexes. The intracellular amount of Au, Ag and Se were estimated using graphite furnace atomic absorption spectrometry (GF‐AAS). Error bars indicate SD.

For almost all the silver(I) selenourea complexes, the metal content was rather different than that of the chalcogen and, in many cases, the intracellular amount of Se was significantly higher than that of Ag. For example, cells treated with [AgCl{Se(IPr^Cl)}] showed an intracellular selenium content about 8 times higher compared with that of silver. A similar trend is observed when cells are treated with [AuCl{Se(IPr)}], [AuCl{Se(SIPr)}], [AuCl{Se(IPr^Me)}] and [AuCl{Se(IPr^Cl)}] complexes, which coincidentally are all IPr‐core‐based complexes with comparable structural size.

Mechanistic Studies

Au(I) and Ag(I) complexes have been widely described to be effective inhibitors of redox‐active Sec‐containing TrxR. ^[3] Selenium containing molecules are also known to act as cell redox sensors through the modulation of cellular redox enzymes, such as TrxR.[ ³¹ , ⁴² ] Hence, all compounds were evaluated for their inhibitory potential toward human TrxR1 both in cell‐free systems and in intact human lung H157 cancer cells and their activity was compared to that of auranofin, a well‐known inhibitor of TrxR. Enzyme activity was measured according to standard procedures described in the Experimental Section, and results are shown in Figure 3, panels A and B. In in vitro experiments, all selenourea complexes proved to be strongly effective in inhibiting cytosolic mammalian TrxR1 in a concentration‐dependent manner. In general, the Au(I) selenourea complexes were shown to be more effective than their Ag(I) counterparts in hampering the selenoenzyme; with [AuCl{Se(SIMes)}] being the most effective derivative, and able to almost completely abolish TrxR1 activity even at 0.5 nM (Figure 3, panel A). Remarkably, with the exception of [AuCl{Se(IAd)}], all Au(I) complexes showed an enzyme inhibition profile better than that of auranofin. In contrast, among the Ag(I) selenourea complexes, only [AgCl{Se(ICy)}] was as effective as the reference gold‐based drug (Figure 3, panel B). These results, highlighting the superior activity of gold with respect to silver complexes, are in line with previously reported studies on other Au(I)‐ and Ag(I)‐NHC systems.[ ⁴³ , ⁴⁴ ] It is important to underline that all the [Se(NHC)]‐based complexes were ineffective in reducing GR activity in vitro up to 1 μM concentration (data not shown), thus indicating a selectivity against the selenoenzyme compared to GR.

Figure 3.

TrxR and GR inhibition. Effects of tested compounds on redox enzymes. (A and B) TrxR1 activity was assayed by measuring NADPH‐dependent reduction of DTNB at 412 nm as described in the Experimental Section. Error bars indicate S.D. (C and D) H157 cells were incubated for 24 h with the tested compounds. Subsequently, cells were washed twice with PBS and lysed. TrxR activity was tested by measuring NADPH‐dependent reduction of DTNB at 412 nm (C) and GR activity was followed spectrophotometrically at 340 nm (D). Error bars indicate SD.

TrxR and GR inhibition were also evaluated in H157 cells treated for 24 h with equimolar concentrations (1 μM) of all derivatives. TrxR activity was assayed by measuring at 412 nm NADPH‐dependent reduction of DTNB (Figure 3, panel C) and GR activity was assayed at 340 nm (Figure 3, panel D). These studies confirmed the ability of all [Se(NHC)]‐based complexes to target TrxR whereas GR activity was only very minimally affected. All Au(I) complexes were almost equally or more effective than auranofin in decreasing the selenoenzyme activity, except for [AuCl{Se(IPr^Cl)}] which was shown to be ineffective. Similarly, all Ag(I) complexes were almost equally or more effective than auranofin in decreasing TrxR activity in intact cells, with [AgCl{Se(SIPr)}] being even 2.4 times more effective than the reference gold‐based drug in targeting the selenoenzyme (Figure 3, panel C). Concerning GR activity, only complex [AgCl{Se(IPr)}] was able to significantly reduce enzyme activity, concluding that all other derivatives were endowed with a preferential selectivity towards TrxR.

The Trx system plays a fundamental role in cellular redox regulation, and inhibition of this redox regulatory system has been shown to determine loss of cellular redox homeostasis in terms of sulfhydryl redox status and promote an increase in cellular basal production of ROS. ^[2]

On these bases, the effect induced by Au(I) and Ag(I) complexes on total cellular sulfhydryl content and on ROS production was assayed in H157‐treated human lung cancer cells.

Although to a differ extent, all tested complexes were effective in modulating total thiol content in H157‐treated cells (Figure 4, panel A). In particular, the reduction of cellular sulfhydryl content was similar to that induced by auranofin.

Figure 4.

Effects on sulfhydryl content and ROS production. (A) Sulfhydryl content in H157‐treated cancer cells incubated for 48 h with tested compounds. The sulfhydryl group amount was determined by the DTNB assay. Error bars indicate S.D. (B) effect of gold(I) and silver(I) compounds on hydrogen peroxide formation in H157 cells. H157 cells were pre‐incubated in PBS/10 mM glucose medium for 20 min at 37 °C in presence of 10 μM CM–DCFDA and then treated with IC₅₀ of the tested compounds.

Consistently, treatment of H157 cells with either Au(I) or Ag(I) complexes determined a substantial time‐dependent increase in cellular basal ROS production (Figure 4, panel B). Notably, treatment with all complexes determined a substantial increase in basal hydrogen peroxide formation which was slightly lower to that obtained with antimycin, a classic inhibitor of the mitochondrial respiratory chain at the level of complex III. Overall, these results demonstrate that the newly developed [Se(NHC)]‐based complexes induce an oxidative shift in the redox status of H157 cells. These results are coherent with those highlighting their ability to target TrxR and support the hypothesis that TrxR represents a major cellular target for this class of complexes.

A persistent increase in the rate of ROS production and the induction of thiol redox stress can in turn prompt the collapse of mitochondrial membrane potential (MMP) as well as loss of mitochondrial shape and integrity (swelling), possibly leading to the induction of cell apoptosis. ^[2] We hence evaluated the effect determined by treatment with selenourea complexes in terms of modification of mitochondrial pathophysiological characteristics, such as mitochondrial membrane potential and morphological changes, and induction of cell death through apoptosis.

For MMP detection, H157 cells were treated with IC₅₀ concentrations of the tested complexes, and the percentage of cells with hypopolarized mitochondrial membrane potential was determined by means of the Mito‐ID® Membrane Potential Kit. As evident by results depicted in Figure 5, panel A, the percentage of hypopolarized cells induced by treatment with the tested complexes was of about 20 % and was rather similar to that induced by the reference compound, carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP). It should be mentioned that no substantial differences between Au(I) and Ag(I) selenourea complexes were found in their ability to induce the loss of the mitochondrial membrane potential.

Figure 5.

Antimitochondrial effect and induction of apoptosis. A. H157 cells were treated for 24 h with IC₅₀ concentrations of the tested complexes or CCCP (3 μM). The percentage of cells with hypopolarized mitochondrial membrane potential was determined by Mito‐ID® Membrane Potential Kit. Data are the means of three independent experiments. Error bars indicate S.D. The percentage of cells with a hypopolarized mitochondrial membrane potential was determined fluorimetrically. B. TEM analysis. Transmission electron micrographs of H157 cells after 24 h treatment with selenourea complexes [AuCl{Se(SIMes)}] and [AgCl{Se(SIMes)}]. (a) and (b): controls; (c) and (d): auranofin; (e) and (f): [AuCl{Se(SIMes)}]; (g) and (h): [AgCl{Se(SIMes)}]. C. Hoechst staining of H157 cells incubated for 48 h with IC₅₀ doses of [AuCl{Se(SIMes)}] (b and c) or [AgCl{Se(SIMes)}] (d and e). Panel a represents untreated H157 cells as control.

In addition, TEM analyses were performed on H157 cells after 24 h treatment with IC₅₀ concentrations of the representative complexes [AuCl{Se(SIMes)}] and [AgCl{Se(SIMes)}] as well as of auranofin.

Images c and d from Panel B of Figure 5 demonstrate that the gold‐based drug auranofin induced a dramatic swelling of the mitochondria, associated with decreased electron density of the inner membrane and matrix regions, as clear signs of its antimitochondrial effect. Similarly, cells treated with the gold(I) [Se(NHC)]‐based complex [AuCl{Se(SIMes)}] showed mitochondria with disrupted cristae and a significant increase in mitochondrial volume (swelling) with respect to control cells (Figure 5, panel B, e and f), confirming that AuCl{Se(SIMes)}] elicited a substantial modification of mitochondria physiology. Concerning the Ag(I) [Se(NHC)]‐based complex, [AgCl{Se(SIMes)}], in addition to a clear evidence of mitochondrial loss of classical morphological characteristics and induction of massive swelling, classical mitophagic signs were detected, thus suggesting the induction of mitophagy as a tentative way of cancer cells to recover from a direct mitochondrial damage induced by the Ag(I) complex (Figure 5, panel B, g and h). Finally, we assessed the ability of selected complexes to induce cancer cell death by means of apoptosis. Figure 5 (Panel C) shows the results obtained upon monitoring cellular morphological changes in H157 cells treated for 48 h with IC₅₀ doses of [AuCl{Se(SIMes)}], [AgCl{Se(SIMes)}] and auranofin, and stained with Hoechst 33258 fluorescent probe. Compared with control cells, cells treated with the tested complexes presented brightly stained nuclei and morphological features typical of cells undergoing apoptosis, such as chromatin condensation, thus confirming the ability of [Se(NHC)]‐based complexes to induce cancer cell death by means of apoptosis.

Conclusions

A series of NHC‐based selenourea Au(I) and Ag(I) complexes were developed and fully characterized by using our previously reported procedures. All derivatives were evaluated in vitro for their cytotoxic potential against a wide panel of human cancer cells derived from solid tumors, and showed to possess an outstanding activity which was, both in 2D and in 3D cancer cell systems, comparable or even better than that of the reference metallodrug auranofin. In addition, all tested complexes were able to overcome both platinum‐based and multi‐drug resistances.

By mechanistic studies, both Ag(I) and Au(I) selenourea complexes were found to selectively and strongly inhibit mammalian TrxR, being even much more effective than the reference metallodrug auranofin. On the contrary, GR activity was only very minimally affected by tested complexes.

Inhibition of TrxR in human lung H157 cancer cells was accompanied by a significant decrease of the total sulfhydryl groups and overproduction of ROS, thus resulting in the loss of mitochondrial shape and functioning, ultimately leading to cancer cell death via apoptosis. These results render the new selenourea metal complexes especially useful towards cancer cells that have acquired drug resistance by activating mechanisms of DNA damage repair.

To the best of our knowledge, this is the first example of studies reporting the in vitro anticancer potential of selenourea‐based Au(I) and Ag(I) complexes. Despite the considerable number of tested complexes, the investigation of their biological and chemico‐physical properties did not allow the elucidation of structure‐activity relationships. Both Au(I) and Ag(I) complexes were markedly effective, without significant differences, in terms of antiproliferative activity and cellular redox state alterations, thus suggesting that the selenourea moiety plays a key role in cancer cell response to metal complexes.

Besides confirming the potency of Ag(I) and Au(I) complexes as anticancer agents, the very promising results obtained in our studies open an intriguing new scenario in the development of group 11 metal complexes, bearing selenium‐based moieties as anticancer metallodrugs, to selectively target cancer cell redox regulation.

Experimental Section

Materials and general methods

The 15 compounds used in this study are depicted in Scheme 1. These were synthesized in accordance with our previous reports (see Supporting Information).[ ²⁵ , ²⁷ , ²⁸ ] Selenium, AgCl, triethylamine and potassium carbonate were obtained from commercial sources (Sigma Chemical Co. or CarlRoth) and used as supplied. [AuCl(SMe₂)] is prepared according to a known procedure. ^[45] Reagent grade solvents (VWR, Sigma Chemical Co.) were always used as received unless otherwise stated.

Experiments with cultured human cancer cells

Au(I) and Ag(I) complexes, the corresponding uncoordinated ligands and auranofin (Sigma Chemical Co.) were dissolved in DMSO just before the experiment, and a calculated amount of drug solution was added to the cell growth medium to a final solvent concentration of 0.5 %, which had no detectable effects on cell viability. Cisplatin (Sigma Chemical Co, St. Louis, USA) and oxaliplatin (Sigma Chemical Co.) were dissolved in 0.9 % sodium chloride solution.

Cell cultures

Human lung (H157), breast (MCF‐7), colon (HCT‐15 and LoVo), and pancreatic (PSN‐1) carcinoma cell lines along with melanoma (A375) cells were obtained by American Type Culture Collection (ATCC, Rockville, MD, USA). Human cervical carcinoma A431 cells were kindly provided by Prof. F. Zunino (Division of Experimental Oncology B, Istituto Nazionale dei Tumori, Milan). The 2008 cells are human ovarian adenocarcinoma cell lines that were kindly provided by Professor G. Marverti (Dipartimento di Scienze Biomediche, Università di Modena University, Italy). Human ovarian carcinoma A2780 cells and their ADR‐resistant counterpart (A2780 ADR) were kindly provided by Prof. M.P. Rigobello (Dipartimento di Scienze Biomediche, Università di Padova, Italy) The LoVo‐OXP cells were derived, using a standard protocol, by growing LoVo cells in increasing concentrations of OXP and following 17 months of selection of resistant clones, as previously described. ^[39]

Cell lines were maintained in the logarithmic phase at 37 °C in a 5 % carbon dioxide atmosphere using the following culture media containing 10 % fetal calf serum (EuroClone, Milan, Italy), antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin) and 2 mM l‐glutamine: (i) RPMI‐1640 medium (EuroClone) for A431, A431/Pt, PSN‐1, H157, HCT‐15, A2780, A2780 ADR and 2008 cells; (ii) F‐12 HAM'S (Sigma Chemical Co.) for LoVo, LoVo MDR and LoVo‐OXP cells; (iii) DMEM for A375 and MCF‐7cells.

MTT assay

The growth inhibitory effect towards tumor cells was evaluated by means of MTT assay. ^[39] Briefly, 3–8×10³ cells/well, dependent upon the growth characteristics of the cell line, were seeded in 96‐well microplates in growth medium (100 μL). After 24 h, the medium was removed and replaced with a fresh one containing the compound to be studied at the appropriate concentration. Triplicate cultures were established for each treatment. After 72 h, each well was treated with 10 μL of a 5 mg/mL MTT saline solution, and following 5 h of incubation, 100 μL of a sodium dodecyl sulfate (SDS) solution in HCl 0.01 M were added. After an overnight incubation, cell growth inhibition was detected by measuring the absorbance of each well at 570 nm using a Bio‐Rad 680 microplate reader. Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted vs. drug concentration. IC₅₀ values, the drug concentrations that reduce the mean absorbance at 570 nm to 50 % of those in the untreated control wells, were calculated by the four‐parameter logistic (4‐PL) model. Evaluation was based on means from at least four independent experiments.

Spheroid cultures

Spheroid cultures were obtained by seeding 2.5×10³ H157 and A2780 cancer cells/well in a round bottom non‐treated tissue culture 96 well‐plate (Greiner Bio‐one, Kremsmünster, Austria) in phenol red free RPMI‐1640 medium (Sigma Chemical Co.) containing 10 % fetal calf serum and supplemented with 20 % methyl cellulose stock solution.

Acid phosphatase (APH) assay

An APH modified assay was used for determining cell viability in 3D spheroids, as previously described. ^[46] IC₅₀ values were calculated with a four‐parameter logistic (4‐PL) model.

Au, Ag and Se cellular accumulation

H157 cells (2.5×10⁶) were seeded in 75 cm² flasks in growth medium (20 mL). After 24 h, the medium was replaced, and the cells incubated for 24 h with tested complexes. Monolayers were then washed twice with ice cold phosphate‐buffered saline (PBS), harvested, and counted. Cell samples were subjected to five freeze‐thaw cycles at −80 °C, and then vigorously vortexed. The samples were treated with highly pure nitric acid (1 mL; Ag≤0.01 μg kg⁻¹, Au≤0.02 μg kg⁻¹, Au≤0.005 μg kg⁻¹, Trace‐SELECT Ultra, Sigma Chemical Co.) and transferred into a microwave Teflon vessel. Samples were then submitted to standard mineralization procedures and analyzed for Au, Ag and Se amount by using a Varian AA Duo graphite furnace atomic absorption spectrometer (Varian, Palo Alto, CA; USA) at 324, 242.795 and 196 nm for Ag, Au and Se respectively. The calibration curve was obtained using known concentrations of standard solutions purchased from Sigma Chemical Co.

In vitro TrxR1 inhibition

The assay was performed in 0.2 M Na‐K‐phosphate buffer pH 7.4, containing 5 mM EDTA, 0.250 mM nicotinamide adenine dinucleotide phosphate (NADPH) and 75 nmol of TrxR1 (IMCO, Sweden). Tested complexes as well as auranofin were pre‐incubated for 5 min at room temperature; the reaction started with 1 mM DTNB (5,5′‐dithiobis(2‐nitrobenzoic acid)), and the increase of absorbance was monitored at 412 nm over 5 min at 25 °C. Enzyme activity was calculated taking into account that 1 mol of NADPH yields 2 mol of CNTP anion (carboxy‐nitro‐thiophenol, reduced DTNB).

In vitro glutathione reductase (GR) inhibition

Glutathione reductase activity was estimated at 25 °C in 0.1 M Tris‐HCl (pH 8.1) containing 0.2 mM NADPH. Tested complexes as well as auranofin were pre‐incubated for 5 min at room temperature; reactions were started by the addition of 1 mM GSSG and followed spectrophotometrically at 340 nm.

Inhibition of TrxR and GR in cancer cells

H157 cells (1×10⁶) were grown in 75 cm² flasks at confluence and treated for 18 h with the metal complexes at increasing concentrations. Cell monolayers were harvested, washed with PBS and centrifuged. Each sample was lysed with RIPA buffer added immediately before the use of an antiprotease cocktail (Roche, Basel, Switzerland). Aliquots were employed for the determination of protein content by means of the BioRad assay (BioRad). The samples were tested for TrxR activity as described above for the in vitro assay. Briefly, the assay was performed in 0.2 M Na‐phosphate buffer (pH 7.4) containing 5 mM EDTA, 0.25 mM NADPH and 50 μg protein per ml. The reaction was initiated by the addition of 3 mM DTNB to both the sample and the reference and the increase in absorbance was monitored at 412 nm for about 15 min at 25 °C. The GR activity was estimated on 50 μg of the protein in 0.2 M Tris‐HCl buffer (pH 8.1), 1 mM EDTA, and 0.25 mM NADPH. The measurement was started after adding 1 mM GSSG and followed spectrophotometrically at 340 nm.

Reactive oxygen species (ROS) production

The production of ROS was measured in H157 cells (10⁴ per well) grown for 24 h in a 96‐well plate in RPMI medium without phenol red (Sigma Chemical Co.). Cells were then washed with PBS and loaded with 10 μM 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate acetyl ester (CM–H₂DCFDA) (Molecular Probes‐Invitrogen, Eugene, OR) for 25 min, in the dark. Afterwards, cells were washed with PBS and incubated with increasing concentrations of tested compounds. Fluorescence increase was estimated utilizing a plate reader (Tecan Infinite M200 PRO, Männedorf, Switzerland) at 485 nm (excitation) and 527 nm (emission). Antimycin (3 μM, Sigma Chemical Co), a potent inhibitor of Complex III in the electron transport chain, and auranofin were used as positive controls.

Quantification of thiols

The H157 cells (2×10⁵) were seeded in a six‐well plate in growth medium (4 mL). After 24 h, cells were incubated for 24 h with increasing concentrations of tested compounds. Subsequently, the thiol content was measured as previously described. ^[47]

Mitochondrial membrane potential (ΔΨ)

The ΔΨ was assayed using the Mito‐ID® Membrane Potential Kit according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY). Briefly, H157 cells (8×10³ per well) were seeded in 96‐well plates; after 24 h, cells were washed with PBS and loaded with Mito‐ID Detection Reagent for 30 min at 37 °C in the dark. Afterwards, cells were incubated with increasing concentrations of tested complexes. Fluorescence intensity was estimated using a plate reader (Tecan Infinite M200 PRO, Männedorf, Switzerland) at 490 (excitation) and 590 nm (emission). Carbonyl cyanide m‐chlorophenyl hydrazone (CCCP, 4 μM), a chemical inhibitor of the oxidative phosphorylation, was used as positive control.

Transmission electron microscopy (TEM) analyses

About 10⁶ H157 cells were seeded in 24‐well plates and, after 24 h incubation, were treated with the tested compounds and incubated for additional 24 h. Cells were then washed with cold PBS, harvested, and directly fixed with 1.5 % glutaraldehyde buffer with 0.2 M sodium cacodylate, pH 7.4. After washing with buffer and post‐fixation with 1 % OsO4 in 0.2 M cacodylate buffer, specimens were dehydrated and embedded in epoxy resin (Epon Araldite). Sagittal serial sections (1 μm) were counterstained with toluidine blue; thin sections (90 nm) were given contrast by staining with uranyl acetate and lead citrate. Micrographs were taken with a Hitachi H‐600 electron microscope (Hitachi, Tokyo, Japan) operating at 75 kV. All photos were typeset in Corel Draw 11.

Apoptosis induction

H157 cells were seeded into 8‐well tissue‐culture slides (BD Falcon, Bedford, MA, USA) at 5×10⁴ cells/well (0.8 cm²). After 24 h, the cells were washed twice with PBS and, following 48 h of treatment with IC₅₀ doses of the tested compound, cells were stained for 5 min with 10 μg/mL of Hoechst 33258 (20‐(4‐hydroxyphenyl)‐5‐(4‐methyl‐1‐piperazinyl)‐2,50‐bi‐1H‐benzimidazole trihydrochloride hydrate, Sigma‐Aldrich, St.Louis, MI, USA) in PBS. Samples were examined at 5x and 40x magnification in a Zeiss LSM 800 confocal microscope using the Zeiss ZEN 2.3 software system.

Statistical analysis

All values are the means±SD of no less than three measurements starting from three different cell cultures. Multiple comparisons were made by ANOVA followed by the Tukey–Kramer multiple comparison test (*p<0.05, **p<0.01), using GraphPad software.

Hydrolytic stability studies

Stability was studied by dissolving a known quantity of the corresponding complex (ca. 4 mg) in DMSO‐d₆ (∼0.4 mL), followed by diluting the solution with D₂O to get a total volume of 0.5 mL. NMR spectra were acquired on a Bruker AV400 spectrometer with a BBFO‐z‐ATMA probe. ¹H NMR spectra were recorded at t=0, 24 h, 48 h and 9 days to investigate the stability of the complexes in aqueous solution. Stability of the corresponding complexes were additionally studied with a D₂O solution of 100 mM NaCl (approximating the standard [Cl⁻] in blood) instead of D₂O, using the same procedure.

Conflict of interest

The authors declare no conflict of interest.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

De Franco M., Saab M., Porchia M., Marzano C., Nolan S. P., Nahra F., Van Hecke K., Gandin V., Chem. Eur. J. 2022, 28, e202201898.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.