# Expanding the Chemical Space of Allyl Palladates with Anticancer Properties

Alessia Schiavo, Giacomo Baio, Francis Bru, Irene Guerra, Victoria Mechrouk, Thierry Achard, Sara Baldissin, Stéphane Bellemin-laponnaz, Catherine S J Cazin, Cristina Tubaro, et al.

► To cite this version:

Alessia Schiavo, Giacomo Baio, Francis Bru, Irene Guerra, Victoria Mechrouk, et al. Expanding the Chemical Space of Allyl Palladates with Anticancer Properties. European Journal of Inorganic Chemistry, 2025, 28 (18), 10.1002/ejic.202500146. hal-05374236

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# Expanding the Chemical Space of Allyl Palladates with Anticancer Properties

Alessia Schiavo, Giacomo Baio, Francis Bru, Irene Guerra, Victoria Mechrouk, Thierry Achard, Sara Baldissin, Stéphane Bellemin-Laponnaz, Catherine S. J. Cazin, Cristina Tubaro, Fabiano Visentin, Isabella Caligiuri, Flavio Rizzolio,* Steven P. Nolan,* and Thomas Scattolin*

Allyl palladates are emerging as promising anticancer agents due to their unique mechanisms of action, including thioredoxin reductase inhibition and immunogenic cell death activation. However, their lack of selectivity toward cancer cells has hindered their therapeutic potential. Herein, a library of ten novel allyl palladates featuring diverse azolium ligands is synthesized via a solvent-free approach, including 1) ring-expanded azolium salts; 2) azolium salts functionalized with phosphonium, thioether, selenoether, or sulfoxide groups; 3) 12-membered cyclic

alkyl groups; or 4) bulky or methoxy-functionalized aryl groups. The newly synthesized compounds are tested against A2780, A2780cis, and U87 cancer cell lines, as well as MRC-5 non-cancerous cells. Results reveal a general excellent cytotoxicity (in the micromolar or submicromolar range), with allyl palladates 2c-d showing the greatest activity but limited selectivity. Compounds 2f and 2h, however, demonstrate improved selectivity toward cancer cells, highlighting the influence of thioether and selenoether functional groups.

## 1. Introduction

Metal-based compounds have played a significant role in the field of oncology since the introduction of cisplatin in the 1970s, which deeply revolutionized cancer treatment approach.[1] While platinum compounds like cisplatin, carboplatin, and oxaliplatin are still widely used, their clinical application is often marred by important issues such as severe side effects and acquired resistance in patients.[2-6] These limitations have prompted researchers to explore other metal-based agents as potential alternatives with improved therapeutic profiles. Metals such as ruthenium,[7-10] gold,[11-14] iron,[15-18] and palladium[19-23] are being investigated for their distinct properties and mechanisms of action, offering new hope for more effective cancer therapies.

In this vast scenario, palladium-based complexes have gained attention owing to their unique chemical properties, versatility in drug design, and potential for selective cancer cell targeting.[19-23] Unlike platinum compounds, which primarily rely on DNA cross-linking to induce cancer cell death, palladium complexes can be engineered to act through a broader range of mechanisms, enhancing their potential as anticancer agents. Moreover, palladium's ability to undergo facile ligand exchange reactions makes it a dynamic candidate for interaction with biological molecules, including proteins and DNA, offering novel avenues for therapeutic intervention.[24-26]

However, with the aim of controlling ligand dissociation processes and thus preventing complete hydrolysis of the complex before it reaches the biotarget of interest, a very common

A. Schiavo, G. Baio, I. Guerra, C. Tubaro, T. Scattolin
Dipartimento di Scienze Chimiche
Università degli Studi di Padova
via Marzolo 1, 35131 Padova, Italy
E-mail: thomas.scattolin@unipd.it

F. Bru, C. S. J. Cazin, S. P. Nolan
Department of Chemistry and Centre for Sustainable Chemistry
Ghent University
Krijgslaan 281, 9000 Ghent, Belgium
E-mail: steven.nolan@ugent.be

V. Mechrouk, S. Bellemin-Laponnaz
Institut de Physique et Chimie des Matériaux de Strasbourg
CNRS-Université de Strasbourg UMR7504
23 rue du Loess BP 43, 67034 Strasbourg, France

T. Achard
Aix Marseille Université
CNRS
Centrale Méditerranée
iSm2 UMR 7313
52 Av. Escadrille Normandie Niemen, 13013 Marseille, France

S. Baldissin, I. Caligiuri, F. Rizzolio
Pathology Unit
Department of Molecular Biology and Translational Research
Centro di Riferimento Oncologico di Aviano (CRO) IRCCS
via Franco Gallini 2, 33081 Aviano, Italy
E-mail: flavio.rizzolio@unive.it

F. Visentin, F. Rizzolio
Dipartimento di Scienze Molecolari e Nanosistemi
Università Ca' Foscari
Campus Scientifico Via Torino 155, 30174 Venezia-Mestre, Italy

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Supporting information for this article is available on the WWW under https://doi.org/10.1002/ejic.202500146

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© 2025 The Author(s). European Journal of Inorganic Chemistry published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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strategy is to incorporate at least one strong palladium-carbon bond into the structure of palladium-based anticancer agents. These organopalladium derivatives exhibit various modes of action, making them highly versatile compared to conventional platinum-based drugs.[19]

Some organopalladium compounds target DNA, similar to platinum compounds, thus interrupting processes like replication and transcription, which leads to cancer cell apoptosis.[27-30] In addition to DNA targeting, organopalladium compounds have been shown to generate reactive oxygen species (ROS) within cancer cells.[31,32] The accumulation of ROS leads to oxidative stress, causing cellular damage and triggering apoptosis. This dual mechanism—direct DNA interaction and oxidative stress induction—allows palladium-based compounds to kill cancer cells through multiple pathways, reducing the likelihood of developing drug resistance.

Other classes of organopalladium derivatives present mito-
chondria as their primary target. Particularly noteworthy are some
cationic palladium-allyl[33,34] and palladium-indenyl[35,36] com-
plexes, as well as a palladium(I) dimer bearing two diNHC ligands
(NHC = N-heterocyclic carbene) synthesized by some of us.[37]

Very recently, we have discovered the first example of organopalladium compounds, namely, the allyl palladates, that exert their antitumor activity through a particularly interesting mechanism, known as immunogenic cell death (ICD).[38] Allyl palladates are a class of compounds that not only exhibit an unusual mechanism of action in the landscape of organopalladium compounds but also possess an exceptionally unique molecular structure. This is evidenced by an unusual and strong interaction between a proton from an azolium salt and two chlorides of a palladium-allyl fragment.[39] These compounds, initially isolated and studied as key intermediates in the preparation of [Pd(NHC)Cl(allyl)]

**Previous works (2022-2024)**

<figure><img src="image_6.png" /><figcaption>R₁ = Me, ¹Pr, CH(Ph)₂
R₂ = H, Me</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Immunogenic
Cell Death (ICD)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Ovarian Cancer
Therapy (OCT)</figcaption></figure>

This work

<figure><img src="image_10.png" /><figcaption>Allyl palladates with azolium salts bearing bulky or methoxy-functionalized aryl groups</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Allyl palladates with saturated and unsaturated azolium salts
bearing 12-membered cyclic alkyl groups</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>Allyl palladates with azolium salts bearing phosphonium, thioether, sulfoxide and selenoether groups</figcaption></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>Allyl palladates bearing ring-expanded
azolium salts</figcaption></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

**Scheme 1.** Chemical space of allyl palladate complexes.
<figure><img src="image_20.png" /></figure>

precatalysts via the weak base route,[40] can intriguingly be syn-
thesized in quantitative yields by simply grinding the [PdCl(allyl)]₂
dimer and the desired azolium salt with a mortar and pestle.[38–40]
If the consistency of the azolium salt does not allow for efficient
grinding, the synthesis can be alternatively carried out using an
organic solvent, such as acetone or methanol, under aerobic and
mild conditions.

In our two previous works, we discussed the preparation of allyl palladates derived from conventional imidazol(in)ium and bis(imino)acenaphthene-NHC (BIAN-NHC) salts (see **Scheme 1**) and their high cytotoxicity toward a wide range of 2D and 3D ovarian cancer models.[38,39] The proposed mechanism of action for this class of compounds appears to be independent of the nature of the azolium salt used and involves the activation of typical ICD pathways, along with effective inhibition of thioredoxin reductase (TrxR). These two phenomena, as observed in some organogold complexes,[41,42] seem to be closely connected.

It should be remembered that ICD is a recently discovered
form of programmed cell death that triggers an immune
response against dying cancer cells.[43-47] Unlike regular cell
death, ICD releases damage-associated molecular patterns that
activate the immune system, promoting recognition and elimina-
tion of cancer cells.[48] Key hallmarks of ICD include the exposure
of calreticulin on the cell surface, release of ATP, and secretion of
high-mobility group box 1 protein, which stimulate dendritic cells
and T-cells.[49-52]

By promoting both cytotoxicity and long-lasting immune
memory, ICD-inducing metallodrugs may prevent tumor recur-
rence and metastasis, potentially enhancing the overall efficacy
of cancer therapy compared to non-immunogenic treatments.[53]

In this work, we aim to significantly expand the scope of
allyl palladates in the hope of improving their selectivity toward
cancer cells, which is currently the main limitation of this
intriguing class of compounds. In previous studies, a comparable,
albeit slightly lower, cytotoxicity of allyl palladates toward
non-cancerous cells compared to cancer cells was observed.[38]

The azolium salts chosen for this study complement and expand
the library of compounds examined in our previous works by
varying the steric hindrance, the nature of the imidazole ring,
as well as introducing interesting functional groups or extended
cyclic alkyl substituents (Scheme 1).

**2. Results and Discussion**

**2.1. Synthesis of Allyl Palladates with Azolium Salts Bearing Bulky or Methoxy-Functionalized Aryl Groups**

The first class of allyl palladates examined in this work was
synthesized from azolium salts **1a** and **1b**, which are character-
ized by a high steric hindrance and the presence of heteroatoms
(methoxy groups) directly attached to the aryl substituents,
respectively. In one of our previous studies, it was observed that
the cytotoxicity of allyl palladates increases with the steric
demand of the aryl substituents or by the introduction of a het-
eroatom (e.g., Cl) into the imidazole backbone.[39] Following this
trend, we first sought to investigate whether further increasing
the steric hindrance of the azolium salt compared to the complex
containing the IPr\*.HCl precursor (IPr\*.HCl = 1,3-bis(2,6-bis
(diphenylmethyl)-4-methylphenyl)imidazolium chloride), which
was reported in our previous work, would lead to an additional
increase in cytotoxicity.

To this end, we selected the imidazolium salt **1a** (IPr\*Tol·HCl), which contains tolyl substituents instead of the phenyl groups present in IPr\*.HCl. Gratifyingly, by grinding the highly hindered azolium salt **1a** with 0.5 equivalents of the [PdCl(allyl)]₂ dimer using mortar and pestle, the corresponding allyl palladate complex **2a** was obtained in quantitative yield (**Scheme 2**). As observed with all allyl palladates studied prior to this work,[38–40] the final product exhibits a ¹H NMR spectrum that shows the same number of signals as the sum of the two reagents used, but with different chemical shifts compared to the starting

<figure><img src="image_21.png" /><figcaption>**Scheme 2.** Synthetic route to allyl palladates with azolium salts bearing bulky or methoxy-functionalized aryl groups.</figcaption></figure>
<figure><img src="image_22.png" /></figure>

materials. As usual, a particularly diagnostic feature is the shift to lower chemical shift values for the imidazolium NCHN proton signal (12.7 vs 12.0 ppm), resulting from the formation of a strong interaction with the two chlorides of the Pd-allyl fragment. Additionally, significant shifts are observed in the three sets of allyl signals (central, syn, and anti protons).

Regarding the enhanced antitumor activity observed when a heteroatom is introduced into the imidazole backbone, we questioned whether a similar effect might occur by introducing heteroatoms into the *N*-substituents. Specifically, we selected azolium salt **1b** as a model, which features methoxy groups at the *ortho* positions of the aryl substituents bonded to the nitrogen atoms. Using a procedure analogous to the previous case, the precursor [PdCl(allyl)]₂ and azolium salt **1b** were ground together for 5 min, yielding allyl palladate complex **2b** (Scheme 2).

The ¹H NMR and ¹³C NMR spectra display all the characteristic signals of the desired palladate species. In particular, the ¹H NMR spectrum shows the presence of two doublets at ≈2.8 and 3.9 ppm (with *J* = 12.0 and 6.7 Hz, respectively), corresponding to the *anti* and *syn* allyl protons, respectively. The central allyl proton resonates as a multiplet at 5.3 ppm, while the methyl protons of the methoxy groups appear as a single singlet at 3.9 ppm, indicating free rotation around the aryl–N bond at room temperature. Moreover, an upfield shift of the imidazolium proton NCHN (Δδ = 0.2 ppm) compared to the starting material further indicates interaction between the imidazolium moiety and the palladium-allyl fragment.

## 2.2. Synthesis of Allyl Palladates with Azolium Salts Bearing 12-Membered Cyclic Alkyl Groups

Since previous studies have never considered cyclic alkyl substituents, focusing solely on *N*-aryl groups, we opted to prepare two allyl palladates (**2c-d**) containing saturated or unsaturated azolium salts with 12-membered cyclic alkyl substituents.

The choice of these substituents was made with the intent of maintaining significant steric bulkiness, which, as mentioned previously, might be an important factor for the cytotoxicity of this class of compounds. The azolium salts **1c-d**, which are white solids, possess a suitable consistency for direct reaction with the [PdCl(allyl)]₂ dimer using the standard solvent-free method (Scheme 3).

In the ¹H NMR spectra of the target complexes, signals for the methylene protons of the 12-membered rings are observed as multiplets between 1.2 and 2.2 ppm, as well as a multiplet at

4.9 ppm corresponding to the CH group of the cyclic alkyl moiety directly attached to the imidazole nitrogen.

A notable difference between complexes **2c** and **2d** is in the position of the azolium proton NCHN signal, which appears at 9.4 and 10.6 ppm, respectively. In both cases, there was the typical shift of about 0.6–0.9 ppm toward lower chemical shifts compared to the starting azolium salts **1c-d**.

## 2.3. Synthesis of Allyl Palladates with Azolium Salts Bearing Phosphonium, Thioether, Selenoether, and Sulfoxide Groups

The third modification we considered for the palladate complexes involves the introduction of biologically relevant functional groups into one arm of the imidazole ring, while keeping constant the substituent on the other nitrogen atom (a benzyl group).

In particular, the first complex described in this section features an azolium salt equipped with a phosphonium (—PPh₃⁺) group. *N*-heterocyclic carbene (NHC) ligands derived from this class of azolium salts have been recently investigated by Canac group and some of us, with a special attention on their coordination chemistry toward rhodium, palladium, and gold.[54–60] Interestingly, the authors proved that NHC-phosphonium ligands can sometimes act as bidentate chelating ligands, known as NHC-phosphonium ylides.

However, since this work concerns the coordination chemistry of azolium salts rather than their carbene derivatives, we believe the primary advantage of having a phosphonium group lies in its ability to act as a delocalized lipophilic cation.[61] This property should enable the corresponding palladate complex to efficiently penetrate and accumulate in mitochondria.[61,62] This feature, combined with the fact that the membrane potential is higher in cancer cells than in non-cancerous ones, is expected to enhance selectivity toward cancer cells, thereby reducing side effects.

Armed with all these valuable information, we prepared azolium salt **1e** following the procedure reported in the literature, with the only modification being the exchange of the triflate anion for chloride using an ion-exchange resin.

Subsequently, azolium salt **1e** was ground with the [PdCl(allyl)]₂ dimer, yielding the corresponding palladate complex **2e** as a yellow and sticky solid (Scheme 4). The change in consistency, as well as the change in color, were observed during the grinding process, indicating the progress of the reaction.

The presence of a singlet at 24.4 ppm in the ³¹P NMR spectra of both the starting azolium salt (**1e**) and the product (**2e**) demonstrates the inability of the phosphonium cation to compete

<figure><img src="image_23.png" /><figcaption>**Scheme 3.** Synthetic route to allyl palladates with azolium salts bearing 12-membered cyclic alkyl groups.</figcaption></figure>
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>**Scheme 4.** Synthetic route to allyl palladates with azolium salts bearing phosphonium, thioether, selenoether and sulfoxide groups.</figcaption></figure>

with the imidazolium cation in interacting with the chlorides of the palladium-allyl fragment. The reaction thus selectively produces a complex with the standard allyl palladate structure, as evidenced by the shift of the imidazolium proton NCHN signal compared to the starting material (from 10.7 to 10.4 ppm), as well as the presence of the three sets of allyl signals (central allyl-H at 5.3 ppm, syn allyl-H at 3.9 ppm, and anti allyl-H at 2.8 ppm) and the aromatic signals in the range of 7.3–8.4 ppm.

The library of allyl palladates shown in Scheme 4 includes three additional types of compounds, which feature thioether (2f), sulfoxide (2g), and selenoether (2h) functional groups.

The idea of using azolium salts bearing these functional groups stems from recent studies that have described not only the coordination chemistry of the corresponding NHC ligands with various metals such as ruthenium and gold but also the benefits of employing such complexes as promising anticancer agents.[63-68] In fact, thioethers, sulfoxides, or selenoethers can act as efficient chemoprotectors in metal complexes where they are incorporated, as they partially protect the metal center from interactions with sulfur/selenium-containing biomolecules (e.g., glutathione).[63-68] This parasitic reaction often reduces the amount of the complex that reaches the desired biological target and increases metal speciation, which frequently causes increased side effects.

Due to the sticky consistency of azolium salts 1f–h, the corresponding palladates 2f–h were obtained using acetone or an acetone/methanol mixture as solvents, under aerobic conditions at 60 °C for 10 min. This synthetic approach had already been explored and validated in a previous study.[40]

The ¹H NMR spectra confirm the successful formation of the products, showing the characteristic shift of the imidazolium proton NCHN compared to the starting material, as well as the presence of allyl signals and all peaks expected from the methylene

and aromatic protons of the thioether, sulfoxide, and selenoether substituents.

## 2.4. Synthesis of Allyl Palladates with Ring-Expanded Azolium Salts

The final class of allyl palladates investigated in this work is characterized by ring-expanded azolium salts as starting materials (six- and seven-membered heterocyclic rings). These cationic species have been studied over the past decade as precursors to ring-expanded NHCs. The latter are a versatile class of ligands that have gained significant attention in coordination chemistry and catalysis due to their unique electronic and steric properties.[69-71] Compared to traditional NHCs, which typically feature five-membered heterocyclic rings, ring-expanded NHCs (often with six- to eight-membered rings) exhibit distinct structural characteristics that enhance their donor abilities and flexibility. These modifications can lead to improved stability and tunability in metal complexes, making them highly desirable for a wide range of applications. In particular, ring-expanded NHC ligands have shown promise in organometallic catalysis, small-molecule activation, and the stabilization of reactive intermediates, offering potential advancements in both industrial processes and academic research.[69-71]

As demonstrated in a very recent contribution by Bertrand and colleagues, the ability of ring-expanded NHCs to activate small molecules such as CO, terminal alkynes, ammonia, and isocyanides is attributed to the amphiphilic nature of this unique class of carbenes.[72]

In fact, the nitrogen atoms in larger ring systems exhibit less π-donation to the carbene center, making it less electron-rich than in traditional five-membered NHCs.
<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /><figcaption>**Scheme 5.** Synthetic route to allyl palladates with ring-expanded azolium salts.</figcaption></figure>

However, ring-expanded NHCs and their azolium precursors have rarely been investigated for the development of potential metallodrugs. To partially address this gap in the literature, we selected azolium salts **1i** and **1j**, which are derived from biologically relevant molecules such as 1,3-diazepines and 1,3,5-diazocines, and reacted them with the [PdCl(allyl)]₂ dimer using a 5:1 acetone/methanol mixture as the solvent at 60 °C for 30 min (**Scheme 5**). After simple solvent removal, the target allyl palladates **2i–j** were obtained in quantitative yields and characterized by ¹H and ¹³C NMR spectroscopies.

Notably, the ¹H NMR spectra show several signals common to both complexes, specifically those corresponding to the allyl protons (central, syn, and anti) and the mesityl substituents.

Regarding the methylene protons of the expanded heterocyclic ring, complex **2i** exhibits a quintet at 2.7 ppm and a triplet at 4.2 ppm, while complex **2j** shows two multiplets at 2.6 and 4.5 ppm. As in all previously examined cases, the characteristic upfield shift of the azolium NCHN proton signal compared to the starting materials serves as a diagnostic marker for the formation of the palladate species of interest.

## 2.5. Antiproliferative Activity toward Ovarian and Brain Cancer Cell Lines

The anticancer activity of the newly synthesized allyl palladates was tested in vitro against a selection of human cancer cell lines, which included A2780 (cisplatin-sensitive ovarian cancer), A2780cis (cisplatin-resistant ovarian cancer) and U87 (glioblastoma) cells, as well as on MRC-5 noncancerous cells. To facilitate comparison, the performance of cisplatin, a benchmark metal-based drug, was also evaluated under identical experimental conditions. Cytotoxicity was quantified using IC₅₀ values, which are summarized in **Table 1**.

Preliminarily, the stability of the allyl palladates was assessed in a 1:10 DMSO/H₂O solution containing 100 mM NaCl and

0.05 mM of the complex using UV-vis spectroscopy. Over a 24 h period, no significant alterations were observed in the spectra, suggesting that the complexes retained their structural integrity.

Analyzing the data presented in Table 1, it can be stated that the majority of the synthesized compounds exhibit good cytotoxicity (in the micromolar or submicromolar range) against the three tumor cell lines investigated (A2780, A2780cis, and U87).

In the A2780 cell line, the least active compounds (with IC₅₀ values at least 10 times higher than that of cisplatin) were those bearing extremely bulky N-substituents (**2a**) or phosphonium/sulfoxide groups (**2e** and **2g**). Conversely, the allyl palladates **2c–d**, containing 12-membered cyclic alkyl groups, were the most active compounds, displaying IC₅₀ values an order of magnitude lower than that of cisplatin. All other compounds (**2b**, **2f**, **2h–j**) demonstrated cytotoxicity comparable to or slightly lower than that of cisplatin.

For the cisplatin-resistant ovarian cancer cell line (A2780cis), the compound with a sulfoxide group (**2g**) confirmed the low cytotoxicity observed in the A2780 line, whereas all other compounds exhibited cytotoxicity one to two orders of magnitude higher than cisplatin. Once again, the leading compounds were the allyl palladates **2c–d**. In general, all compounds either maintained or improved their antiproliferative activity when transitioning from the cisplatin-sensitive (A2780) to the cisplatin-resistant (A2780cis) cell line. This observation supports the hypothesis that allyl palladates act through a mechanism distinct from that of cisplatin, as recently studied by our group.[³⁸]

Regarding the glioblastoma cell line (U87), complexes **2b**, **2e**, and **2h** exhibited slightly lower cytotoxicity than cisplatin, whereas complexes **2a** and **2g** showed poor cytotoxicity. Encouraging results were observed with complexes **2c–d**, **2f**, and **2i–j**, which displayed IC₅₀ values comparable to or lower than those of cisplatin.
<table><caption>**Table 1.** Effects of the allyl palladate complexes on the proliferation of A2780, A2780cis, and U87 cancer cells and MRC-5 noncancerous cells. S.I. = selectivity index. The inhibition of cell growth is represented as IC₅₀.</caption><thead><tr><th>Complex<sup>a)</sup></th><th>A2780</th><th>A2780cis</th><th>U87</th><th>MRC-5</th><th>S.I.</th></tr></thead><tbody><tr><td>CisPt</td><td>3.4 ± 0.2</td><td>60 ± 10</td><td>6.0 ± 0.9</td><td>9 ± 1</td><td>0.39</td></tr><tr><td>2a</td><td>50 ± 10</td><td>6.5 ± 0.2</td><td>60 ± 10</td><td>80 ± 10</td><td>2.0</td></tr><tr><td>2b</td><td>7.2 ± 0.6</td><td>2.8 ± 0.9</td><td>16 ± 3</td><td>5.0 ± 0.2</td><td>0.6</td></tr><tr><td>2c</td><td>0.28 ± 0.04</td><td>0.23 ± 0.04</td><td>0.5 ± 0.1</td><td>0.30 ± 0.05</td><td>0.9</td></tr><tr><td>2d</td><td>0.25 ± 0.03</td><td>0.18 ± 0.04</td><td>0.47 ± 0.03</td><td>0.21 ± 0.06</td><td>0.7</td></tr><tr><td>2e</td><td>50 ± 20</td><td>2.2 ± 0.1</td><td>17 ± 2</td><td>80 ± 10</td><td>3.5</td></tr><tr><td>2f</td><td>14 ± 4</td><td>4.5 ± 0.2</td><td>3.9 ± 0.1</td><td>90 ± 10</td><td>12.0</td></tr><tr><td>2g</td><td>70 ± 20</td><td>40 ± 10</td><td>60 ± 10</td><td>70 ± 10</td><td>1.2</td></tr><tr><td>2h</td><td>5.2 ± 0.5</td><td>8.5 ± 0.2</td><td>27 ± 4</td><td>80 ± 20</td><td>5.9</td></tr><tr><td>2i</td><td>2.2 ± 0.2</td><td>2.2 ± 0.4</td><td>5.2 ± 0.2</td><td>4.5 ± 0.3</td><td>1.4</td></tr><tr><td>2j</td><td>3.2 ± 0.1</td><td>5 ± 1</td><td>6.9 ± 0.5</td><td>10 ± 1</td><td>2.0</td></tr></tbody></table>

Examining the data obtained for MRC-5 noncancerous cells, it is evident that although compounds **2c-d** are the most promising due to their high cytotoxicity in the three tumor lines studied, they also showed high cytotoxicity toward normal cells (selectivity index, SI = 0.7-0.9; SI is calculated as the quotient of the average IC₅₀ toward normal cells divided by the average IC₅₀ for malignant cells). Given that selectivity toward cancer cells is a critical factor in the in vitro screening of potential metallo-drugs, we believe that complexes **2f** and **2h** can be considered as the most interesting among those developed in this study, having SI = 12 and 5.9, respectively.

### 3. Conclusion

Recent studies on allyl palladates bearing classical NHC or BIAN-NHC ligands have pointed out that these compounds exhibit remarkable cytotoxicity against various tumor cell lines, strong inhibition of TrxR, and activation of ICD. This singular behavior that has not precedents among organopalladium compounds with anticancer properties has motivated us to significantly expand the diversity of azolium ligands in this study. Our aim was to select compounds having a good selectivity for cancer cells over non-cancerous ones, an issue that has thus far been a critical limitation for allyl palladates bearing classical NHCs.

In this work, we successfully synthesized a library of ten novel allyl palladates using a solvent-free approach. These compounds are characterized by: 1) ring-expanded azolium salts; 2) azolium salts functionalized with phosphonium, thioether, selenoether, or sulfoxide groups; 3) 12-membered cyclic alkyl groups; or 4) bulky or methoxy-functionalized aryl groups. The synthesized compounds were characterized using ¹H and ¹³C NMR spectroscopy and elemental analysis. They were subsequently evaluated for their cytotoxicity against a panel of human cancer cell lines, including A2780 (cisplatin-sensitive ovarian cancer), A2780cis

(cisplatin-resistant ovarian cancer), and U87 (glioblastoma), as well as on MRC-5 noncancerous cells.

Most compounds demonstrated good cytotoxicity in the micromolar or submicromolar range. The allyl palladates **2c-d**, featuring 12-membered cyclic alkyl groups emerged as the most active compounds, with IC₅₀ values significantly lower than cisplatin in all tested tumor cell lines. Conversely, compounds with bulky N-substituents (**2a**) or a sulfoxide group (**2g**) exhibited reduced activity in most of the examined cancer cells. Notably, all active compounds either maintained or improved their cytotoxicity when transitioning from cisplatin-sensitive (A2780) to cisplatin-resistant (A2780cis) ovarian cancer cells, suggesting a mechanism distinct from that of cisplatin. Despite the high cytotoxicity of **2c-d** against tumor cells, their lack of selectivity (SI = 0.7-0.9) toward non-cancerous MRC-5 cells limits their therapeutic potential. Compounds **2f** and **2h**, with selectivity indices of 12 and 5.9, respectively, emerged as the most promising candidates for further development. It appears that introducing a thioether or selenoether as a protective group not only ensures good cytotoxicity against cancer cells but also minimizes activity toward noncancerous cells.

In summary, within the broad landscape of allyl palladates, those containing thioglucosidic,[73] thioether, and selenoether groups emerge as the most promising candidates for further investigation in more complex biological systems, such as patient-derived organoids and animal models. These experiments are currently ongoing in our laboratories, alongside the development of allyl palladates with similar (or better) selective anticancer properties.

### 4. Experimental Section

#### Materials and Methods

The azolium salts **1a-d** were synthesized according to previously published procedures.[74-78] The azolium salts **1e-g** and **1i** were

<footnote><sup>a)</sup>Data after 96 h of incubation. Stock solutions in DMSO for all complexes; stock solutions in H₂O for cisplatin. A2780 and A2780cis (cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines), MDA-MB231 (triple-negative breast cancer cell line), DLD1 (colon cancer cell line), and MRC-5 (normal lung fibroblasts).</footnote>
<figure><img src="image_28.png" /></figure>

synthesized according to previously published procedures[55,63,67] and transformed in their chloride version using a DOWEX-21KCI resin and methanol as the solvent. All ¹H NMR spectra of azolium salts are reported in Supporting Information. Other reagents (e.g., [PdCl(allyl)]₂) and solvents were purchased and used as received without further purification, unless otherwise stated. All cell lines were purchased from Sigma-Aldrich (Merck).

¹H and ¹³C[¹H] NMR spectra were recorded on a Bruker Advance 400 spectrometer at room temperature (298 K). Elemental analysis was carried out using an Elemental CHN "CUBO Micro Vario" analyzer.

## Synthesis of Azolium Salt 1h

In a glove box, a solution of (PhSe)₂ (0.40 g, 1.3 mmol) in THF/ethanol 1:1 (2 mL) was added dropwise to a flask with a suspension of NaBH₄ (0.14 g, 3.8 mmol) in CH₂Cl₂ (10 mL). The mixture was stirred overnight at room temperature evolving from bright yellow to white. Out of the glove box, the reaction mixture was washed with HCl 1 M (5 mL), water (2 × 5 mL), and brine (5 mL). The organic phase was dried over MgSO₄, filtered, and collected in a flask. The solvent was evaporated and a yellow liquid was obtained. N-benzyl imidazole was directly added to the flask (0.41 g, 2.6 mmol, 1 eq.) and the reaction mixture was heated at 70 °C under reflux for 26 h. To the resulting oil, diethyl ether was added and the precipitation of a solid was observed. The solid was isolated and dissolved in chloroform. A second precipitation in hexane affords the product as a white solid which was dried under vacuum (0.162 g, 0.45 mmol, yield: 17%). The solid is hygroscopic and must be stored under nitrogen.

¹H NMR (300 MHz, CDCl₃): δ = 10.22 (s, 1 H, NCHN), 7.18–7.45 (12 H, aryl-H), 5.83 (s, J₅ₐ⁻H = 17.4 Hz, 2 H, NCH₂Se), 5.42 (s, 2 H, NCH₂Ph).

¹³C NMR (100 MHz, CDCl₃): δ = 137.1 (NCHN), 134.9 (d, J₅ₐ⁻C = 9.2 Hz, Aryl-CH), 133.1 (Aryl-C), 129.9 (Aryl-C), 129.4 (Aryl-C), 129.3 (Aryl-C), 128.8 (Aryl-C), 126.1 (Aryl-C), 122.2 (CH = CH¹¹m), 122.0 (CH = CH¹¹m), 53.4 (NCH₂Se), 44.7 (NCH₂Ph).

## Solvent-Free Synthesis of Allyl Palladates 2a–e (General Procedure)

In air, the azolium salt (NHC·HCl) and the dimeric precursor [PdCl(allyl)]₂ were added to a mortar. The two solids were mixed and grinded using a pestle for 5 min. A crystalline solid was obtained.

### Synthesis of Complex 2a

Following the general procedure from [PdCl(allyl)]₂ (8.6 mg, 0.024 mmol) and 1a (49.7 mg, 0.048 mmol), the product was obtained as a pale-yellow powder in a quantitative yield.

¹H-NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 12.03 (s, 1 H, NCHN), 7.17 (d, *J* = 7.2 Hz, 8 H, aryl-H), 7.06 (d, *J* = 7.3 Hz, 8 H, aryl-H), 6.83 (d, *J* = 7.7 Hz, 8 H, aryl-H), 6.72 (s, 4 H, aryl-H), 6.62 (d, *J* = 7.8 Hz, 8 H, aryl-H), 5.48 (s, 2 H, CH·CH¹¹m), 5.35 (s, 4 H, CH(Tol)₂), 5.18 (m, 1 H, central allyl-H), 3.85 (bd, 2 H, syn allyl-H), 2.71 (bd, 2 H, anti allyl-H), 2.28 (s, 12 H, CH₃Tol), 2.25 (s, 12 H, CH₃Tol), 2.17 (s, 6 H, *p*-CH₃).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 140.8, 140.5, 140.0, 139.8, 136.1, 135.9, 130.9, 130.6, 130.2, 129.2, 129.1, 129.0, 123.1, 109.5 (allyl-CH), 60.9 (allyl-CH₂), 50.2, 21.9, 21.1, 21.0.

Elemental analysis calcd (%) for C₈₀H₇₈Cl₂N₂Pd: C, 77.13, H, 6.39, N, 2.25; found: C, 77.50, H, 6.18, N, 2.09.

## Synthesis of Complex 2b

Following the general procedure from [PdCl(allyl)]₂ (22.5 mg, 0.062 mmol) and 1b (50.0 mg, 0.124 mmol), the product was obtained as a pale-yellow powder in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 8.08 (s, 1 H, NCHN), 7.33 (t, *J* = 8.5 Hz, 2 H, aryl-H), 6.65 (d, *J* = 8.5 Hz, 4 H, aryl-H), 5.33–5.21 (m, 1 H, central allyl-H), 4.61 (s, 4 H, CH₂-CH₂), 3.92 (s, 12 H, OCH₃), 3.90 (d, *J* = 6.7 Hz, 2 H, syn allyl-H), 2.81 (d, *J* = 12.0 Hz, 2 H, anti allyl-H).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 160.1, 155.2, 131.0, 112.9, 109.5 (allyl-CH), 104.6, 61.0 (allyl-CH₂), 56.7, 51.7.

Elemental analysis calcd (%) for C₂₂H₂₈Cl₂N₂O₄Pd: C, 47.04, H, 5.02, N, 4.99; found: C, 47.36, H, 4.79, N, 4.81.

### Synthesis of Complex 2c

Following the general procedure from [PdCl(allyl)]₂ (20.9 mg, 0.057 mmol) and 1c (50.1 mg, 0.114 mmol), the product was obtained as a pale-yellow powder in a quantitative yield.

¹H-NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 9.41 (s, 1 H, NCHN), 5.32 (m, H central allyl-H), 4.12 (m, 2 H, NCH), 3.94 (d, *J* = 6.7 Hz, 2 H, syn allyl-H), 3.86 (s, 4 H, CH₂-CH₂), 2.86 (d, *J* = 12.0 Hz, 2 H, anti allyl-H), 1.79–1.32 (44 H, CH₂ C₁₂-rings).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 158.2, 109.5 (allyl-CH), 61.0 (allyl-CH₂), 55.6, 45.2, 27.8, 23.8, 23.5, 23.4, 21.7.

Elemental analysis calcd (%) for C₃₀H₅₆Cl₂N₂Pd: C, 57.92, H, 9.07, N, 4.50; found: C, 58.24, H, 8.95, N, 4.31.

## Synthesis of Complex 2d

Following the general procedure from [PdCl(allyl)]₂ (25.3 mg, 0.069 mmol) and 1d (59.8 mg, 0.138 mmol), the product was obtained as a yellow powder in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 10.59 (t, *J* = 1.6 Hz, 1 H, NCHN), 7.13 (d, *J* = 1.6 Hz, 2 H, CH·CH¹¹m), 5.34 (m, 1 H, central allyl-H), 4.90 (m, 2 H, NCH), 3.97 (d, *J* = 6.7 Hz, 2 H, syn allyl-H), 2.90 (d, *J* = 12.0 Hz, 2 H, anti allyl-H), 2.15-2.03 (4 H, CH₂ C₁₂-rings), 1.88–1.78 (4 H, CH₂ C₁₂-rings), 1.57–1.24 (36 H, CH₂ C₁₂-rings).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 138.3, 119.0, 118.9, 109.6 (allyl-CH), 61.09 (allyl-CH₂), 58.5, 30.3, 23.9, 23.8, 23.3, 23.2, 21.3.

Elemental analysis calcd (%) for C₃₀H₅₄Cl₂N₂Pd: C, 58.11, H, 8.78, N, 4.52; found: C, 58.37, H, 8.91, N, 4.40.

## Synthesis of Complex 2e

Following the general procedure from [PdCl(allyl)]₂ (24.1 mg, 0.062 mmol) and 1e (70.2 mg, 0.124 mmol), the product was obtained as a yellow powder in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 10.43 (s, 1 H, NCHN), 8.46 (s, 1 H, CH·CH¹¹m), 7.91–7.67 (15 H, PPh₃), 7.40–7.30 (5 H, Ph), 6.94 (s, 1 H, CH·CH¹¹m), 5.42 (s, 2 H, CH₂-Ph), 5.30 (m, 1 H, central allyl-H), 4.97 (t, *J* = 6.6 Hz, 2 H, CH₂-PPh₃), 3.92 (d, *J* = 6.6 Hz, 2 H, syn allyl-H), 3.83 (t, *J* = 14.7 Hz, 2 H, N-CH₂), 2.84 (d, *J* = 12.0 Hz, 2 H, anti allyl-H), 2.50 (m, 2 H, CH₂CH₂CH₂).

³¹P[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 24.4

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 135.0, 134.9, 134.0, 133.9, 132.9, 130.6, 130.5, 129.4, 129.4, 128.9, 125.0, 120.2, 118.5, 117.3, 109.6 (allyl-CH), 61.0 (allyl-CH₂), 53.4.
<figure><img src="image_29.png" /></figure>

Elemental analysis calcd (%) for C₃₄H₃₆Cl₃N₂PPd: C, 57.00, H, 5.07, N, 3.91; found: C, 56.73, H, 5.20, N, 4.07.

### Synthesis of Complex 2f

Azolium salt **1f** (22.5 mg, 0.068 mmol), [PdCl(allyl)]₂ (12.5 mg, 0.034 mmol), a magnetic stir bar, acetone (1 mL) and methanol (4 mL) were added into a round-bottom flask. The mixture was stirred at 60 °C for 30 min. The solvents were removed, and the product was dried under vacuum. The product was obtained as a yellow solid in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 10.75 (s, 1 H, NCHN), 7.21–7.43 (10 H, aryl-H), 7.14 (bs, 1 H, CH-CH¹²), 6.85 (bs, 1 H, CH-CH¹²), 5.52 (s, 2 H, NCH₂Ph), 5.39 (m, 1 H, central allyl-H), 4.64 (t, *J* = 6.1 Hz, 2 H, CH₂), 4.03 (d, *J* = 6.7 Hz, 2 H, syn allyl-H), 3.59 (t, *J* = 6.1 Hz, CH₂), 2.97 (d, *J* = 12.1 Hz, 2 H, *anti* allyl-H).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 138.9, 133.5, 132.9, 130.3, 129.6, 129.6, 129.6, 129.3, 127.4, 122.8, 120.3, 110.3 (allyl-CH), 61.8 (allyl-CH₂), 53.7, 49.3, 34.7.

### Synthesis of Complex 2g

Azolium salt **1g** (24.3 mg, 0.070 mmol), [PdCl(allyl)]₂ (13.0 mg, 0.035 mmol), a magnetic stir bar, acetone (1 mL), and methanol (4 mL) were added into a round-bottom flask. The mixture was stirred at 60 °C for 30 min. The solvents were removed, and the product was dried under vacuum. The product was obtained as a yellow solid in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 10.81 (t, *J* = 1.8 Hz, 1 H, NCHN), 7.40–7.70 (11 H, aryl-H, CH-CH¹²), 6.89 (t, *J* = 1.8 Hz, CH-CH¹²), 5.49 (d, *J* = 3.9 Hz, 2 H, NCH₂Ph), 5.35 (m, 1 H, central allyl-H), 4.70–5.01 (2 H, CH₂), 4.01 (d, *J* = 6.7 Hz, 2 H, syn allyl-H), 3.96 (m, 2 H, CH₂), 2.93 (d, *J* = 12.1 Hz, 2 H, *anti* allyl-H).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 141.9, 139.2, 132.6, 131.4, 129.8, 129.6, 129.3, 124.2, 123.4, 120.5, 110.0 (allyl-CH), 61.6 (allyl-CH₂), 55.4, 53.8, 43.5.

### Synthesis of Complex 2h

Azolium salt **1h** (40.0 mg, 0.11 mmol), [PdCl(allyl)]₂ (20.0 mg, 0.055 mmol), a magnetic stir bar, acetone (10 mL), and methanol (1 mL) were added into a round-bottom flask. The mixture was stirred at 60 °C for 30 min. The solvents were removed, and the product was dried under vacuum. The product was obtained as a yellow solid in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 10.26 (s, 1 H, NCHN), 7.27–7.56 (10 H, aryl-H), 7.15 (t, *J* = 1.8 Hz, 1 H, CH-CH¹²), 7.03 (t, *J* = 1.8 Hz, 1 H, CH-CH¹²), 5.88 (s, 2 H, NCH₂Se), 5.46 (s, 2 H, NCH₂Ph), 5.37 (m, 1 H, central allyl-H), 4.00 (d, *J* = 6.7 Hz, 2 H, syn allyl-H), 2.93 (d, *J* = 12.1 Hz, 2 H, *anti* allyl-H).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 138.1, 134.9 (d, *J*<sub>se-c</sub> = 10.3 Hz), 133.1, 130.0, 129.5, 129.4, 129.4, 126.3, 121.9, 121.4, 110.1 (allyl-CH), 61.6 (allyl-CH₂), 53.5, 44.9 (d, *J*<sub>se-c</sub> = 92.3 Hz).

### Synthesis of Complex 2i

Azolium salt **1i** (69.7 mg, 0.196 mmol), [PdCl(allyl)]₂ (35.7 mg, 0.098 mmol), a magnetic stir bar, acetone (10 mL), and methanol (2 mL) were added into a round-bottom flask. The mixture was stirred at 60 °C for 30 min. The solvents were removed, and the product was dried under vacuum. The product was obtained as a yellow solid in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 7.54 (s, 1 H, NCHN), 6.94 (s, 4 H, aryl-H), 5.33 (m, 1 H, central allyl-H), 4.18 (t, *J* = 5.3 Hz, 4 H, NCH₂), 3.97 (d, *J* = 6.6 Hz, 2 H, syn allyl-H), 2.89 (d, *J* = 12.1 Hz, 2 H, *anti* allyl-H), 2.73 (m, 2 H, CH₂-CH₂), 2.34 (s, 12 H, *o*-CH₃ mesityl), 2.27 (s, 6 H, *p*-CH₃ mesityl).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 153.6, 140.3, 136.7, 134.5, 130.0, 109.5 (allyl-CH), 61.0 (allyl-CH₂), 47.3, 21.0, 19.6, 18.0.

Elemental analysis calcd (%) for C₂₅H₃₄Cl₂N₂Pd: C, 55.62, H, 6.35, N, 5.19; found: C, 55.97, H, 6.02, N, 5.08.

### Synthesis of Complex 2j

Azolium salt **1j** (100.0 mg, 0.270 mmol), [PdCl(allyl)]₂ (49.2 mg, 0.135 mmol), a magnetic stir bar, acetone (10 mL), and methanol (2 mL) were added into a round-bottom flask. The mixture was stirred at 60 °C for 30 min. The solvents were removed, and the product was dried under vacuum. The product was obtained as a yellow solid in a quantitative yield.

¹H NMR (400 MHz, CDCl₃, *T* = 298 K, ppm) δ: 7.21 (s, 1 H, NCHN), 6.88 (s, 4 H, aryl-H), 5.26 (m, 1 H, central allyl-H), 4.46 (bt, 4 H, NCH₂), 3.89 (d, *J* = 6.7 Hz, 2 H, syn allyl-H), 2.80 (d, *J* = 12.0 Hz, 2 H, *anti* allyl-H), 2.55 (mt, 4 H, CH₂-CH₂), 2.35 (s, 12 H, *o*-CH₃ mesityl), 2.21 (s, 6 H, *p*-CH₃ mesityl).

¹³C[¹H]-NMR (CDCl₃, *T* = 298 K, ppm) δ: 157.8, 140.1, 139.6, 133.9, 130.1, 109.7 (allyl-CH), 61.2 (allyl-CH₂), 55.0, 25.4, 21.0, 18.4.

Elemental analysis calcd (%) for C₂₅H₃₄Cl₂N₂Pd: C, 56.38, H, 6.55, N, 5.06; found: C, 56.70, H, 6.13, N, 4.94.

### Cell Viability Assay

Cells were grown in agreement with the supplier and incubated at 37 °C (5% of CO₂). 500 cells were plated in 96 wells and treated with six different concentrations of allyl palladate complexes (0.001, 0.01, 0.1, 1, 10, and 100 µM). After 96 h, cell viability was evaluated with CellTiter glow assay (Promega, Madison, WI, USA) with a Tecan M1000 instrument. IC₅₀ values were obtained from triplicates, and error bars are standard deviations.

With a similar procedure, organoids were plated as single cells as possible (around 1000 cells) in five replicates and treated with six serial dilutions of the compounds (0.001, 0.01, 0.1, 1, 10, and 100 µM) and analyzed after 96 h.

### Acknowledgements

The authors are grateful to the SBO (D2M to SPN) and the BOF (starting and senior grants to SPN) as well as the iBOF C3 project for financial support. F.R. was financially supported by Fondazione AIRC per la Ricerca sul Cancro (Grant AIRC IG23566) and VC from Ministero della Salute – Ricerca Corrente.

Open access publishing facilitated by Universita degli Studi di Padova, as part of the Wiley - CRUI-CARE agreement.

### Conflict of Interest

The authors declare no conflict of interest.
<figure><img src="image_30.png" /></figure>

Data Availability Statement

The data that support the findings of this study are available in
the supplementary material of this article.

Keywords: allyl palladate complexes • anticancer activity • mechanochemistry • N-heterocyclic carbenes

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Manuscript received: March 20, 2025

Revised manuscript received: May 20, 2025

Version of record online: June 4, 2025