Rational design of gold(I)-NHC complexes as anticancer agents: induction of necroptosis and paraptosis in lung adenocarcinoma†

Sayari Dewan, Himanshu Sonker, Kajal Chaudhary and Ritika Gautam Singh*
Department of Chemistry, Indian Institute of Technology, Kanpur, India – 208016. E-mail: rgautam@iitk.ac.in

First published on 19th June 2025

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The serendipitous discovery of cisplatin as an anticancer agent catalyzed an unprecedented surge in the exploration of metal-based complexes as potential chemotherapeutic agents. Among these, gold compounds have garnered significant attention due to their dual functionality as both anticancer and immunomodulatory agents. This remarkable bioactivity is inherently linked to gold's unique position in the periodic table, endowing it with the highest electronegativity, electron affinity, and redox potential among transition metals, thereby conferring distinctive reactivity and biochemical interactions.¹ Auranofin, a well-established gold(I)-based drug, has been extensively utilized in the treatment of rheumatoid arthritis, owing to its potent anti-inflammatory and immunomodulatory properties.^2,3 Thioredoxin reductase (TrXR) has been identified as a primary molecular target for gold-based compounds,^4–6a wherein their interaction disrupts redox homeostasis, leading to aberrant reactive oxygen species (ROS) accumulation and subsequent induction of apoptotic pathways. While TrxR is a well-recognized target for Au(I) and Au(III) based compounds, several alternative non-TrxR-mediated targets are also worth considering. Notably, components of the ubiquitin–proteasome system (UPS) have been identified as key targets for gold and ruthenium complexes, disrupting protein degradation pathways.^6b–d Aquaglyceroporin-3 (AQP3), which facilitates glycerol and H₂O₂ transport, has been linked to gold(I) compound activity, potentially modulating ROS homeostasis.^6e Additionally, poly(ADP-ribose) polymerases (PARPs), via interactions with zinc finger domains, may offer another mechanistic route relevant to both cytotoxic and antimicrobial effects.^6f Additionally, several gold complexes exhibit the capacity to interact with DNA and inhibit proteasomal activity, further contributing to their multifaceted cytotoxic mechanisms.^7–9

The pharmacological efficacy of gold-based therapeutics is profoundly influenced by their ligand architecture, which governs their interaction with human serum albumin (HSA), often modulating their bioavailability and therapeutic activity. Furthermore, Au(I) exhibits a pronounced tendency for disproportionation into Au(III) and Au(0), particularly under physiological conditions. This inherent instability necessitates coordination with strong σ-donor and π-acceptor ligands, such as N-heterocyclic carbenes (NHCs), which play a pivotal role in stabilizing gold in its +1 oxidation state, thereby enhancing its biochemical persistence and reactivity.¹⁰ However, sterically encumbered N-heterocyclic carbene (NHC) ligands have been reported to attenuate the cytotoxicity of gold complexes, primarily by imposing spatial constraints that hinder the direct interaction of the metal center with cellular targets. This steric shielding effect may reduce cellular uptake and limit engagement with key biomolecular pathways, thereby modulating the overall therapeutic potential of these complexes.^11,12 Consequently, significant efforts have been directed toward enhancing the antiproliferative efficacy of gold complexes through strategic incorporation of nitrogen (N), sulfur (S), and selenium (Se) donor ligands. These heteroatoms not only modulate the electronic environment of the metal center but also facilitate targeted biomolecular interactions, thereby augmenting the therapeutic potential of gold-based chemotherapeutics.^13–15a (Fig. 1).

The resulting [Au(NHC)(Ln)] complexes consistently exhibited enhanced cytotoxicity compared to their precursor [Au(NHC)X] counterparts, highlighting the pivotal role of auxiliary ligand coordination in modulating biological activity. However, gold complexes of the general framework [(NHC)AuX], incorporating sterically demanding NHC ligands while retaining potent cytotoxicity against cancer cells, remain relatively unexplored, underscoring a critical gap in the development of structurally optimized gold-based chemotherapeutics. Thus, there exists substantial scope for the rational design and development of novel gold complexes of the [(NHC)AuCl] framework, aimed at enhancing cytotoxic potency against cancerous cells. Strategic modifications in ligand architecture could further optimize their stability, bioavailability, and mechanistic engagement with oncogenic pathways, paving the way for next-generation gold-based chemotherapeutics. In our previous study,^15b we reported the synthesis of lipophilic and sterically demanding 1,10-phenanthroline-based N-heterocyclic carbene (NHC) precursors, which exhibited a distinctive mode of cytotoxicity in A549 lung cancer cells, characterized by dynamin-dependent endocytosis and vacuolization-mediated cell death. Coinage metal-based N-heterocyclic carbene (NHC) complexes have been extensively explored for their therapeutic potential as both anticancer and antibacterial agents. Motivated by these advancements, we synthesized a series of Au(I)-NHC complexes derived from imidazolium chlorides and systematically evaluated their cytotoxic profiles against cancerous cells.

Herein, we report the development of sterically demanding N-heterocyclic carbene (NHC)-based Au(I) complexes exhibiting highly potent cytotoxicity against human lung cancer cells (A549). This was achieved through strategic incorporation of electron-withdrawing substituents within the NHC framework, based on the hypothesis that the synergistic interplay between the intrinsic cytotoxic properties of the parent imidazolium salts and the Au(I) center would yield novel molecular scaffolds with enhanced antineoplastic efficacy. Notably, the resulting complexes demonstrated a promising selectivity index compared to their parent imidazolium ligands, highlighting a new class of gold-based chemotherapeutics with robust antiproliferative activity.

Results and discussion

Synthesis and characterization of Au-L1–Au-L7

In our previous studies, we reported the synthesis of N-heterocyclic carbene (NHC) ligand precursors. Typically, Au(I)-NHC complexes are synthesized via two primary routes: (i) an indirect approach involving the formation of Ag(I)-NHC intermediates using silver salts such as Ag₂O, followed by transmetalation to the Au(I) center, or (ii) a direct method wherein imidazolium salts react with Au salts in the presence of a base such as K₂CO₃. Given its efficiency and streamlined single-step protocol, we adopted the latter strategy for the synthesis of our target Au(I)-NHC complexes. Subsequently, seven gold target complexes were synthesized by the reaction of imidazolium salt with Au(THT)Cl (THT = tetrahydrothiophene) and K₂CO₃ in dichloromethane following a reported procedure¹⁶ (Fig. 2). The successful formation of the Au(I)-NHC complexes was substantiated by the disappearance of the characteristic imidazolium proton (NCHN) signal at approximately 10.5 ppm in the ¹H NMR spectra of the corresponding ligand precursors, indicating the deprotonation and subsequent coordination of the NHC ligand to the gold center (Fig. S10–S23†).

The ¹³C(1H) NMR spectra of complexes AuL6 and AuL7 exhibited a distinct peak around 200 ppm, indicative of successful Au–C bond formation. This pronounced downfield shift of the C2 carbon, relative to the corresponding ligand precursors, strongly suggests the formation of bis(carbene) species of the type [Au(NHC)₂]⁺, further corroborating the coordination environment of the gold center.¹⁷ In certain cases, the characteristic peak corresponding to the carbenic carbon in the ¹³C(1H) NMR spectra of the gold complexes were not observed, a phenomenon that has also been documented in previous literature reports. This absence is often attributed to rapid relaxation effects, low signal intensity, or dynamic ligand behavior in solution, which can render the carbenic carbon signal elusive.¹⁸ Interestingly, in the case of AuL1, the ¹³C(¹H) NMR spectrum exhibited a distinct resonance for the carbenic carbon at approximately 172 ppm, strongly suggesting the predominant existence of a monocarbenic species in solution. This observation contrasts with the bis(carbene) formation noted in other complexes and underscores the influence of ligand architecture on the coordination environment of gold.

The ESI-MS analysis of all the complexes further corroborated the formation of bis(carbene) species, as evidenced by the characteristic mass signals corresponding to the [Au(NHC)₂]⁺ fragment. This reinforces the structural assignment of these gold complexes and aligns with the observations from ¹³C(¹H) NMR spectroscopy, highlighting the tendency of Au(I) to adopt a bis(carbene) coordination mode under the given synthetic conditions (Fig. S17–S23†). However, in solid state study via single-crystal X-ray diffraction (SC-XRD) studies of AuL2, AuL3, AuL5, and AuL7 unambiguously confirmed that these gold complexes adopt a neutral monocarbenic form, [(NHC)AuCl], (Fig. 3). All crystallographically characterized complexes exhibited a linear coordination geometry around the Au(I) center, with the NHC ligand and halide positioned in a trans configuration. This structural arrangement is consistent with the expected d¹⁰ electronic configuration of Au(I), reinforcing the stability and preferential geometry of these [(NHC)AuCl] complexes in the solid state (Fig. 3). The Au–C1 bond distances were determined to be 1.978(2) Å, 1.983(3) Å, 1.987(3) Å, and 1.967(4) Å for AuL2, AuL3, AuL5, and AuL7, respectively. These values are in excellent agreement with previously reported Au–C bond lengths for structurally analogous [(NHC)AuCl] complexes, further validating the integrity of the monocarbenic coordination mode in the solid state.¹⁹ The Au–Cl bond lengths were measured as 2.2671(6) Å, 2.2625(7) Å, 2.2689(9) Å, and 2.289(15) Å for AuL2, AuL3, AuL5, and AuL7, respectively, aligning well with literature-reported values for [(NHC)AuCl] complexes. Notably, AuL2 and AuL3 crystallized in the orthorhombic crystal system, whereas AuL5 and AuL7 adopted a monoclinic crystallographic arrangement. A comprehensive structural description, including detailed bond lengths and bond angles, is provided in Tables S1 and S2.†

In addition, HMBC spectra of all the complexes (Fig. S32–S38†) in CDCl₃ demonstrated the carbenic carbon to be resonating around 165 ppm, hinting at formation of monocarbene gold complexes. The combined findings from ¹³C(¹H) NMR spectroscopy, ESI-MS analysis, and SC-XRD, HMBC spectral studies strongly suggest the existence of a dynamic equilibrium between the bis(carbene) [(NHC)₂Au]⁺Cl⁻ and the monocarbenic [(NHC)AuCl] species in solution. This phenomenon, widely reported for both Ag(I)-NHC and Au(I)-NHC complexes, underscores the inherent fluxionality of these coordination environments, potentially influenced by ligand electronic properties, steric factors, and solvent effects.^20–23

Hence, conductometric analysis was further performed to elucidate the solution-phase structure of these gold complexes. Silver nitrate (AgNO₃) was used as a reference, and the molar conductance (Λ_m) of 1 mM solution of the selected gold complexes, AuL2 and AuL6, was measured in DMSO to assess their ionic nature and potential speciation in solution.

In the literature, 1:1 electrolytes are reported to exhibit molar conductivity values in the range of 35–90 S cm² mol⁻¹ in DMSO, serving as a reference for assessing the ionic nature of the gold complexes under investigation.^24,25 In our study, the complex AuL2 exhibited a molar conductance of 0.53 S cm² mol⁻¹, while AuL6 displayed a slightly higher value of 1.08 S cm² mol⁻¹ in DMSO. These remarkably low conductance values, well below the expected range for 1:1 electrolytes (35–90 S cm² mol⁻¹), strongly indicate that both complexes predominantly exist as neutral molecular species in solution. Furthermore, the molar conductance (Λm) values remained unchanged over 24 hours, suggesting high solution-phase stability of these complexes, with minimal dissociation or ionization under the experimental conditions (Table 1). These findings further corroborate that the neutral monocarbenic form remains the dominant species in DMSO, with little to no presence of the ionic bis(carbene) form, even after 24 hours of dissolution. This observation aligns well with SC-XRD results, reinforcing the stability and preference of the [(NHC)AuCl] framework in solution.

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UV-vis spectral analysis of AuL1–AuL7

The UV-vis spectra of the synthesized ligands (L1–L7 ) and their corresponding gold(I) complexes (AuL1–AuL7), recorded in DMSO, are presented in Fig. S25.† The spectra of the ligands exhibit characteristic absorption bands at 306 nm and 319 nm, attributed to π–π* transitions localized on the aromatic rings, along with an additional band at 335 nm, corresponding to the n–π* excitation involving the lone pair of electrons on the nitrogen atoms to the π* orbitals of the aromatic rings. Upon complexation with Au(I), a bathochromic shift in these absorption bands was observed, with the peaks shifting to 321 nm, 334 nm, and 349 nm. This redshift in absorption confirms the electronic perturbation induced by metal coordination, further supporting the successful formation of gold(I) complexes.

Stability of gold complexes in physiological buffers

Gold complexes, particularly those featuring N-heterocyclic carbene (NHC) ligands, are well-documented for their exceptional stability due to the strong Au–C bond. To evaluate their stability under physiological conditions and predict their potential biological activity, the synthesized gold complexes were subjected to UV-vis spectroscopy over a 48-hour period in phosphate-buffered saline (PBS, pH 7.4) containing 1% DMSO. As depicted in Fig. S26,† no bathochromic or hypsochromic shifts were observed in the absorption spectra, and no formation or disappearance of new peaks was detected, even after 48 hours. These findings confirm that the gold complexes remain structurally intact under simulated physiological conditions at room temperature. Furthermore, this high stability is consistent with previous reports on (NHC)Au(I)X complexes, where chloride-containing complexes demonstrated superior stability compared to their iodide analogues.²⁶

Anti-proliferative screening of L1–L8 against cancer cells

The cytotoxic efficacy of the synthesized gold NHC complexes (AuL1–AuL8) was systematically evaluated against human lung adenocarcinoma (A549) cells and non-cancerous human embryonic kidney (HEK-293) cells using an MTT assay over a 48-hour incubation period (Table 2). Among the tested complexes, only AuL4 exhibited a significant IC₅₀ value, whereas all other complexes, including AuL7, displayed markedly lower cytotoxicity in comparison to cisplatin. This trend aligns with previous studies on sterically demanding Au-NHC complexes, which often exhibit reduced anticancer activity due to hindered cellular interactions.

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Notably, AuL4, featuring an electron-withdrawing fluorine (F) substituent in the NHC backbone, demonstrated the highest cytotoxic potency against A549 cells, with an IC₅₀ of 5.73 ± 0.42 μM (Fig. 4). This is comparable to cisplatin (IC₅₀ = 5.09 ± 1.10 μM), emphasizing the therapeutic relevance of fluorinated gold(I) complexes in anticancer applications. The superior activity of AuL4 can be attributed to the small atomic radius and strong electronegativity of fluorine, which fine-tunes charge distribution, acidity/basicity, and lipophilicity, thereby influencing molecular interactions within cancer cells.²⁷

To investigate whether reducing steric hindrance around the Au(I) center could enhance cytotoxicity, AuL7 was synthesized. However, contrary to expectations, all synthesized complexes, including AuL7, exhibited poor cytotoxicity, highlighting that electronic effects play a more crucial role than steric factors in governing the anticancer activity of these gold(I) NHC complexes. Notably, the introduction of electron-donating substituents (–OMe) or lipophilic groups (phenyl, n-butyl, tert-butyl) failed to enhance cytotoxic potency, further underscoring the dominance of electronic contributions over steric influences in dictating biological activity.

Moreover, while cisplatin, used as a positive control, exhibited poor selectivity toward cancerous cells (selectivity index (SI) = 0.26), AuL4 demonstrated a significantly higher selectivity index of ∼4 (Fig. 4). This suggests that AuL4 possesses a markedly improved selectivity profile, potentially enabling targeted anticancer action with reduced off-target toxicity, thereby positioning fluorinated Au(I)-NHC complexes as promising candidates for further therapeutic development.

According to the Table S6,† lipophilic substituents like –n-butyl, –tert-butyl group in L2 and L3 resulted in higher clogP values. We hypothesized that these may facilitate the higher cytotoxicity of the corresponding metal complexes in A549 cells in comparison to L4, L5, L6 with lower clogP. But, surprisingly, AuL4, the metal complex of L4 (lowest clogP of 5.2) showed promising toxicity towards A549 cells, unlike other complexes which had higher clogP values. Hence, the differences in the cytotoxic profiles of the metal complexes can be attributed to variations in the lipophilicity of their corresponding ligand frameworks. It is important to note that Lipinski's rule of five suggests that, for drugs to exhibit high bioavailability, their lipophilicity (logP) should not exceed a value of 5.

In our case, complexes bearing ligands with very high clogP values—thereby violating Lipinski's rule—exhibited minimal toxicity toward cancer cells. In contrast, AuL4, which possesses a borderline clogP value, demonstrated significant cytotoxicity. These observations highlight the critical role of lipophilicity, and by extension hydrophobicity, in influencing the bioavailability and cytotoxic potential of this class of metal complexes.

Density functional theory (DFT) calculations

As previously discussed, the IC₅₀ values of the gold complexes suggest that electronic effects play a crucial role in determining their cytotoxicity. To gain deeper insights into this correlation, DFT calculations were carried out for selected complexes: AuL3 (bearing an electron-donating tert-butyl group), AuL4 (featuring an electron-withdrawing fluorine substituent in the NHC backbone), and AuL7 (which replaces the –CH₂Ph group at the ortho-position of the aniline moiety with an isopropyl substituent to assess steric influences near the Au(I) center).

The calculations were performed in the gas phase using the BP86 functional, providing insights into the electronic structures and stability of these gold complexes. This computational approach helps elucidate the impact of substituent effects on the electronic distribution and potential anticancer activity of these molecular scaffolds.^28,29

To further elucidate the electronic properties of the synthesized gold complexes, DFT calculations were performed utilizing the def2-TZVP basis set for all atoms,³⁰ with a relativistic effective core potential (ECP) for gold.³¹ The starting geometries for optimization were derived from single-crystal X-ray diffraction (SC-XRD) data, with solvent molecules omitted for computational efficiency. Molecular orbital analysis revealed that the LUMO localization remained largely unaltered regardless of the electronic nature of the substituents. This invariance can be attributed to the quinoline-benzimidazolium fragment, which consistently served as the primary electronic reservoir in all three gold complexes. Additionally, the aniline moiety was found to exert minimal influence on the overall electron density distribution of the complexes. The computed molecular orbital distributions are depicted in Fig. 5. The cartesian coordinates of the complexes have been depicted in Tables S3–S5.† The optimised structures of the complexes as obtained from DFT calculations are demonstrated in Fig. S27.† Interestingly, despite the enhanced cytotoxic activity observed for AuL4, DFT calculations did not provide a direct electronic rationale for its superior bioactivity. This suggests that alternative factors such as lipophilicity, cellular uptake, or interaction with biological targets may play a more significant role in determining the anticancer potency of these gold complexes.

Role of solution-phase speciation on biological activity

Previous studies have elegantly demonstrated that Au(I)-NHC complexes exhibit a solvent-dependent equilibrium in aqueous media, wherein monocarbenic species [(NHC)AuCl] exist in equilibrium with their biscarbenic counterparts [(NHC)₂Au]⁺Cl⁻. This ligand scrambling phenomenon was found to be particularly pronounced in aqueous and protic environments, whereas in dry, aprotic solvents like DMF, the mono-NHC-Au(I)-X complexes remained stable over at least 72 hours. In our case, ¹³C NMR measurements were carried out in deuterated DMSO, a hygroscopic solvent that is highly susceptible to residual water contamination. Indeed, all spectra exhibited a discernible peak at 3.3 ppm, consistent with the presence of water. We propose that this trace moisture facilitates partial ligand scrambling in solution, shifting the equilibrium toward the bis-NHC-Au(I) species detectable by NMR. This hypothesis is strongly supported by conductometric studies performed using molecular biology grade, anhydrous DMSO. Under these rigorously dry conditions, AuL6 consistently exhibited the behavior of a neutral, monocarbenic species—suggesting that in the absence of water, the complex remains predominantly in the mono-NHC form.

Notably, prior literature reports have indicated that [(NHC)₂Au]⁺ species often exhibit significantly enhanced cytotoxicity compared to their neutral [(NHC)AuX] counterparts, in some cases by as much as fivefold. However, it has also been observed that for [(NHC)AuCl], the ligand scrambling in aqueous solutions is relatively slow compared to analogous complexes with heavier halides (e.g., Br⁻, I⁻), implying a nuanced and halide-dependent behavior of these equilibria. Taken together, our findings align well with existing literature and underscore the importance of solvent purity and experimental conditions in accurately interpreting the speciation and behavior of Au(I)-NHC complexes in solution.

Mitochondrial dysfunction induced by AuL4 in A549 cells

Encouraged by the promising cytotoxic activity of the gold complex AuL4, we sought to explore its potential impact on mitochondrial function, given the central role of mitochondria in cellular bioenergetics and apoptosis regulation. To assess whether AuL4 induces mitochondrial membrane potential (ΔΨm) disruption, we employed tetramethylrhodamine methyl ester perchlorate (TMRM), a well-established fluorescent probe for detecting mitochondrial depolarization. This approach allowed us to systematically investigate the extent of mitochondrial dysfunction following AuL4 treatment, shedding light on its mechanism of action at the cellular level.³² A significant reduction in mean fluorescence intensity (MFI) was observed in AuL4-treated A549 cells (MFI = 4.17) compared to the control group (MFI = 14.21), indicating a marked disruption in the mitochondrial energy cascade. This substantial decline in fluorescence intensity strongly suggests AuL4-induced mitochondrial depolarization, which could serve as a crucial precursor to mitochondria-mediated apoptotic cell death (Fig. 6a and S28†). To further investigate the impact of mitochondrial dysfunction on the cytotoxicity of AuL4, we evaluated the relative cell viability of A549 cells in the presence of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) over a 24-hour period. CCCP is a well-known mitochondrial uncoupler that disrupts mitochondrial membrane potential (ΔΨm) by facilitating proton transport across the inner mitochondrial membrane, thereby dissipating the electrochemical gradient essential for ATP synthesis. This experiment aimed to determine whether mitochondrial impairment contributes to the cytotoxic effects of AuL4. A significant reduction in cell viability in CCCP-treated cells, when co-incubated with AuL4, would reinforce the hypothesis that mitochondrial perturbation plays a key role in AuL4-mediated cell death (Fig. 6b).³³ The cell viability in the presence of CCCP was observed to be ∼108%, whereas cells treated with AuL4 at 2× concentration exhibited significantly reduced viability (∼54%). These findings strongly suggest that AuL4 disrupts oxidative phosphorylation, leading to impaired electron transport chain (ETC) function, thereby affecting cellular respiration and ATP production. Such mitochondrial dysfunction is a well-established trigger for cancer cell apoptosis (Fig. 6b).

Moreover, mitochondrial impairment is often linked to overproduction of reactive oxygen species (ROS), which can exacerbate oxidative stress and promote cancer cell death. To further explore this, ROS generation in A549 cells post AuL4 treatment was assessed using H₂DCFDA staining, a fluorescent probe that detects intracellular ROS accumulation.³⁴ Surprisingly, fluorescence microscopy images (Fig. S29†) revealed no significant increase in ROS levels following AuL4 treatment. This suggests that although AuL4 disrupts mitochondrial function, leading to impaired oxidative phosphorylation and ATP depletion, it does not induce substantial ROS accumulation. These findings indicate that the cytotoxic mechanism of AuL4 is likely independent of ROS-mediated oxidative damage and may involve alternative apoptotic pathways.³⁵ Notably, no significant increase in cell viability was observed in AuL4-treated groups upon NAC co-treatment (Fig. S30†). These results reinforce the notion that AuL4-induced mitochondrial dysfunction and subsequent cytotoxicity are independent of ROS accumulation, suggesting an alternative mechanism of action, potentially involving direct mitochondrial targeting or disruption of bioenergetic homeostasis in A549 cells (Fig. S30†).³⁶

AuL4 triggers necroptosis and paraptosis in A549 cells

Given that AuL4-induced cytotoxicity was not mediated via ROS overproduction, we sought to elucidate its mechanism of action in A549 cells by assessing its impact on distinct cell death pathways. To this end, cells were pretreated with specific inhibitors targeting apoptosis, ferroptosis, autophagy, necroptosis, and paraptosis, followed by cell viability assessment after 24 hours at 2× IC₅₀ concentration of AuL4 (Fig. 7).

In the absence of inhibitors, the relative cell viability was 54.50% (Fig. 7). A marginal increase was observed upon preincubation with the ferroptosis inhibitor ferrostatin (56%) and the apoptosis inhibitor Q-VD-OPh (59%), indicating minimal involvement of these pathways. Similarly, pre-treatment with 3-methyl adenine (autophagy inhibitor) did not significantly alter cell viability. However, a notable increase in viability was observed upon preincubation with necrostatin (necroptosis inhibitor) and cycloheximide (paraptosis inhibitor), with cell survival rising to 82–84%, suggesting that AuL4 predominantly induces necroptosis and paraptosis.

The IC₅₀ of AuL4 was 5.12 ± 1.16 μM. But when inhibitor of necroptosis, necrostatin 1 was added, the IC₅₀ of AuL4 increased to 7.74 ± 2.15 μM. On the other hand, cycloheximide, inhibitor of paraptosis, increased the IC₅₀ of AuL4 to 8.71± 2.90 μM. When both necrostatin and cycloheximide was employed, the IC₅₀ of AuL4 was observed to be 13.22 ± 3.69 μM. This indicated both necroptosis and paraptosis played a significant role in AuL4 mediated cancer cell death (Fig. S31†).

Since gold complexes are traditionally reported to induce apoptosis in cancer cells,³⁷ their therapeutic efficacy may be compromised in apoptosis-resistant cancers. In contrast, AuL4′s ability to activate dual non-apoptotic cell death pathways distinguishes it as a promising chemotherapeutic candidate with potential efficacy against apoptosis-resistant malignancies.

AuL4 suppresses A549 cell metastasis and clonogenic growth

The ability of cancer cells to migrate and establish secondary tumors represents a significant challenge in cancer therapy. To assess whether AuL4 can inhibit the metastatic potential of A549 lung adenocarcinoma cells, a scratch wound healing assay was performed (Fig. 8).³⁸ The results revealed minimal cell migration in AuL4-treated groups compared to the control, indicating a significant reduction in the migratory capacity of A549 cells. This suggests that AuL4 effectively impairs tumor cell dissemination, thereby limiting the potential for secondary tumor formation.

Furthermore, the uncontrolled proliferation of malignant cells often hinders the effectiveness of therapeutic interventions. To evaluate the effect of AuL4 on long-term proliferative capacity, a clonogenic assay was conducted (Fig. 9).³⁹ AuL4-treated A549 cells exhibited a marked reduction in colony formation compared to the untreated control, suggesting that AuL4 successfully inhibits the clonal expansion of A549 cells. These findings underscore the therapeutic potential of AuL4 in restraining both metastatic progression and uncontrolled proliferation, making it a promising candidate for anti-cancer therapy.

Conclusion

In summary, this study presents a novel class of gold(I)-NHC complexes featuring sterically demanding ligands, among which AuL4 exhibited remarkable cytotoxic potency against A549 lung adenocarcinoma cells. Mechanistic investigations revealed that AuL4 induces mitochondrial dysfunction, disrupts oxidative phosphorylation, and uniquely triggers necroptosis and paraptosis—two non-apoptotic cell death pathways. This distinguishes AuL4 from conventional gold-based anticancer agents, which primarily rely on apoptosis, potentially limiting their efficacy in apoptosis-resistant cancers.

Additionally, AuL4 effectively suppresses both metastasis and clonal expansion of A549 cells, highlighting its potential to inhibit tumor progression. These findings open a new avenue for the design of gold(I)-NHC complexes as promising anticancer therapeutics with unique mechanisms of action, emphasizing their potential as next-generation chemotherapeutic agents against aggressive and resistant malignancies.

Since our in vitro results are promising, our future planned in vivo study will include:

a). In vivo tumor xenograft model: b). Evaluation of pharmacokinetic distribution of AuL4: we plan to elucidate the pharmacokinetic profile of AuL4 in BALB/c mice models, highlighting key data and parameters, such as T_max (h), C_max (μg mL⁻¹), AUC (0-infinite) (μg h mL⁻¹, Vd, CL, t_1/2 (h), F (%). For this purpose, we plan to use advanced techniques like ICP-MS, to evaluate the amount of gold present in different tissues at different time points as well as LC-MS-QTof to obtain the different pharmacokinetic parameters. c). Pulmonary metastasis model: given the inhibitory effects of AuL4 on cellular migration, we want to explore these effects in murine models also. Hence, we have planned to generate a pulmonary metastasis model by intravenous injection of cancerous cells. Post treatment with our gold complex AuL4 every alternate day, we would sacrifice the mice and harvest the lung tissue to count the number of metastatic nodules post H & E staining.

Experimental procedures

AuL1

White solid. Yield: 75%. ¹H NMR (δ ppm, DMSO-d₆, 500 MHz): δ 9.93 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.71–7.67 (m, 1H), 7.60 (dd, J = 11.0, 3.9 Hz, 1H), 7.34 (d, J = 9.6 Hz, 1H), 7.20–7.12 (m, 13H), 7.01–6.98 (m, 4H), 6.91 (d, J = 6.9 Hz, 4H), 6.77 (s,1H), 6.39 (s, 2H), 5.37 (s, 2H), 3.53 (s, 3H). ¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 172.81, 143.63, 142.17, 141.81, 129.49, 128.96, 127.28, 126.90, 55.46, 51.50. ESI-MS (m/z) calculated for {[Au(NHC)(Acn)] + H}⁺ 844.2602, observed 848.2616, calculated for {[Au(NHC)₂] + 2H}⁺ 1449.4723, observed 1449.6869.

AuL2

Off-white solid. Yield: 59%. ¹H NMR (δ ppm, DMSO-d₆, 500 MHz): δ 9.94 (d, J = 8.5 Hz, 1H), 7.84 (dd, J = 7.8, 1.5 Hz, 1H), 7.68 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.61 (td, J = 7.5, 1.1 Hz, 1H), 7.35 (d, J = 9.6 Hz, 1H), 7.21–7.12 (m, 13H), 6.97 (dd, J = 6.7, 2.8 Hz, 4H), 6.88 (dd, J = 7.6, 1.7 Hz, 4H), 6.86 (s, 1H), 6.76 (s, 2H), 5.34 (s, 2H), 1.39–1.32 (m, 2H), 1.21–1.13 (m, 2H), 0.76 (t, J = 7.3 Hz, 3H). ¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 142.41, 142.02, 141.70, 132.73, 129.47, 128.96, 128.87, 127.24, 127.06, 51.49, 35.04, 32.93, 21.68, 14.11. ESI-MS (m/z) calculated for {[Au(NHC)(Acn)]}⁺ 870.3122, observed 870.3090, calculated for {[Au(NHC)₂] + Me}⁺ 1476.6283, observed 1476.7350.

AuL3

Off-white solid. Yield: 74%. ¹H NMR (δ ppm, DMSO-d₆, 500 MHz): 9.92 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.70–7.65 (m, 1H), 7.61 (dd, J = 11.3, 4.6 Hz, 1H), 7.34 (d, J = 9.6 Hz, 1H), 7.22–7.12 (m, 15H), 6.98–6.92 (m, 6H), 6.89 (dd, J = 7.6, 1.4 Hz, 3H), 5.43 (s, 2H), 1.04 (s, 9H). ¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 142.46, 141.35, 129.45, 128.99, 127.18, 127.10, 126.18, 117.37, 116.38, 51.85, 35.15, 31.20. ESI-MS (m/z) calculated for {[Au(NHC)(Acn)]}⁺870.3122, observed 870.3105, calculated for {[Au(NHC)₂] + Me}⁺ 1476.6283, observed 1476.7384.

AuL4

Off-white solid. Yield: 72%. ¹H NMR (δ ppm, DMSO-d₆, 500 MHz): δ 9.89 (d, J = 8.5 Hz, 1H), 7.86–7.84 (m, 1H), 7.71–7.67 (m, 1H), 7.63–7.59 (m, 1H), 7.38–7.33 (m, 2H), 7.22–7.13 (m, 13H), 7.04–6.97 (m, 5H), 6.94–6.91 (m, 2H), 6.68 (d, J = 9.3 Hz, 2H), 5.40 (s, 2H). ¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 141.59, 141.30, 129.52, 129.44, 129.08, 127.48, 117.51, 117.33, 116.35, 116.24, 116.00, 51.47. ESI-MS (m/z) calculated for {[Au(NHC)(Acn)]}⁺ 832.2402, observed 832.2401, calculated for {[Au(NHC)₂]}⁺ 1385.4608, observed 1385.4601.

AuL5

White solid. Yield 84%. ¹H NMR (500 MHz): ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (d, J = 8.4 Hz, 1H), 7.84 (dd, J = 7.7, 1.5 Hz, 1H), 7.72–7.66 (m, 1H), 7.63–7.61 (m, 1H), 7.36 (d, J = 9.6 Hz, 1H), 7.21–7.18 (m, 6H), 7.17–7.14 (m, 5H), 6.98–6.96 (m, 4H), 6.93–6.91 (m, 4H), 6.88 (s, 2H), 6.81 (s, 1H), 6.69–6.65 (m, 1H), 6.57 (d, J = 7.1 Hz, 1H), 5.42 (s, 2H). ¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 144.74, 141.53, 141.17, 129.44, 129.12, 127.50, 127.40, 51.56. ESI-MS (m/z) calculated for {[Au(NHC)(Acn)]}⁺ 848.2106, observed 848.2108, calculated for {[Au(NHC)₂] + 2H}⁺ 1419.4173, observed 1419.4045.

AuL6

White solid. Yield 89%. ¹H NMR (500 MHz) δ 9.91 (d, J = 8.5 Hz), 7.84 (d, J = 7.7 Hz), 7.70–7.66 (m), 7.60 (td, J = 7.5, 1.1 Hz), 7.35 (d, J = 9.6 Hz), 7.22–7.18 (m), 7.14 (t, J = 5.1 Hz), 7.05 (t, J = 6.0 Hz), 6.99 (s), 6.93 (d, J = 7.1 Hz), 6.86 (dd, J = 7.6, 1.8 Hz), 6.78 (dd, J = 6.6, 3.0 Hz), 6.70 (s), 5.55 (s), 5.31 (s). ¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 201.45, 151.56, 143.74, 142.05, 141.63, 130.23, 129.38, 128.81, 126.88, 126.83, 117.50, 55.43, 51.43. ESI-MS (m/z) calculated for {[Au(NHC)Cl] + H₂O}⁺ 992.2807, observed 992.3008, calculated for {[Au(NHC)₂] + H}⁺ 1682.6439, observed 1682.6320.

AuL7

Off-white solid. Yield: 56%. ¹H NMR (δ ppm, DMSO-d₆, 500 MHz): δ 10.11 (d, J = 8.5 Hz, 1H), 8.28 (s, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.74 (t, J = 7.2 Hz, 1H), 7.65 (t, J = 7.3 Hz, 1H), 7.51 (q, J = 9.6 Hz, 2H), 7.32 (t, J = 7.6 Hz,), 7.22 (t, J = 7.3 Hz, 4H), 7.14 (d, J = 7.4 Hz, 2H), 7.09 (s, 2H), 5.74 (s, 1H), 2.19 (dt, J = 13.6, 6.8 Hz, 2H), 1.10 (d, J = 6.8 Hz, 6H), 0.97 (d, J = 6.8 Hz, 6H). ¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 206.56, 146.26, 145.37, 144.19, 130.30, 129.60, 128.98, 126.97, 125.13, 118.18, 98.81, 56.18, 28.47, 24.58, 24.30. ESI-MS (m/z) calculated for {[Au(NHC)(Acn)]}⁺ 732.2653, observed 732.2626, calculated for {[Au(NHC)₂]}⁺ 1185.5109, observed 1185.5067.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

S. D. executed the synthesis, characterization, and mammalian cell assays, DFT calculation. H. S. analysed the SC-XRD structures. K. C. performed the UV-vis, IR characterization. All authors contributed to the manuscript writing and approved the final manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This work was supported by the Indian Council of Medical Research, DST SERB Start-up Research Grant (Grant 2021435) and INSPIRE Faculty Research Grant (Grant 2019295), CSIR-ASPIRE Grant - 01WS/028/2023-24/EMR-II/ASPIRE, DST Antimicrobial resistance (AMR) (TPN 94103) vide file no. DST/TDT/TC/AMR/2023/01.

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Footnote

† Electronic supplementary information (ESI) available: Other materials; NMR, HRMS, UV-vis spectra; single crystal X-ray diffraction data. CCDC 2432303–2432305 and 2433312. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5md00290g

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