Unveiling the promising anticancer activity of palladium(II)-aryl complexes bearing diphosphine ligands: a structure-activity relationship analysis.

In continuation of our previous works on the cytotoxic properties of organopalladium compounds, in this contribution we describe the first systematic study of the anticancer activity of Pd(II)-aryl complexes. To this end, we have prepared and thoroughly characterized a wide range of palladium derivatives bearing different diphosphine, aryl and halide ligands, developing, when necessary, specific synthetic protocols. Most of the synthesized compounds showed remarkable cytotoxicity towards ovarian and breast cancer cell lines, with IC50 values often comparable to or lower than that of cisplatin. The most promising complexes ([PdI(Ph)(dppe)] and [PdI(p-CH3-Ph)(dppe)]), characterized by a diphosphine ligand with a low bite angle, exhibited, in addition to excellent cytotoxicity towards cancer cells, low activity on normal cells (MRC5 human lung fibroblasts). Specific immunofluorescence tests (cytochrome c and H2AX assays), performed to clarify the possible mechanism of action of this class of organopalladium derivatives, seemed to indicate DNA as the primary cellular target, whereas caspase 3/7 assays proved that the complex [PdI(Ph)(dppe)] was able to promote intrinsic apoptotic cell death. A detailed molecular docking analysis confirmed the importance of a diphosphine ligand with a reduced bite angle to ensure a strong DNA-complex interaction. Finally, one of the most promising complexes was tested towards patient-derived organoids, showing promising ex vivo cytotoxicity.

Platinum(0)-η2-1,2-(E)ditosylethene Complexes Bearing Phosphine, Isocyanide and N-Heterocyclic Carbene Ligands: Synthesis and Cytotoxicity towards Ovarian and Breast Cancer Cells.

A wide range of platinum(0)-η2-(E)-1,2-ditosylethene complexes bearing isocyanide, phosphine and N-heterocyclic carbene ancillary ligands have been prepared with high yields and selectivity. All the novel products underwent thorough characterization using spectroscopic techniques, including NMR and FT-IR analyses. Additionally, for some compounds, the solid-state structures were elucidated through X-ray diffractometry. The synthesized complexes were successively evaluated for their potential as anticancer agents against two ovarian cancer cell lines (A2780 and A2780cis) and one breast cancer cell line (MDA-MB-231). The majority of the compounds displayed promising cytotoxicity within the micromolar range against A2780 and MDA-MB-231 cells, with IC50 values comparable to or even surpassing those of cisplatin. However, only a subset of compounds was cytotoxic against cisplatin-resistant cancer cells (A2780cis). Furthermore, the assessment of antiproliferative activity on MRC-5 normal cells revealed certain compounds to exhibit in vitro selectivity. Notably, complexes 3d, 6a and 6b showed low cytotoxicity towards normal cells (IC50 > 100 µM) while concurrently displaying potent cytotoxicity against cancer cells.

Rational Design of Palladium(II) Indenyl and Allyl Complexes Bearing Phosphine and Isocyanide Ancillary Ligands with Promising Antitumor Activity.

A new class of palladium-indenyl complexes characterized by the presence of one bulky alkyl isocyanide and one aryl phosphine serving as ancillary ligands has been prepared, presenting high yields and selectivity. All the new products were completely characterized using spectroscopic and spectrometric techniques (NMR, FT-IR, and HRMS), and, for most of them, it was also possible to define their solid-state structures via X-ray diffractometry, revealing that the indenyl fragment always binds to the metal centre with a hapticity intermediate between ƞ3 and ƞ5. A reactivity study carried out using piperidine as a nucleophilic agent proved that the indenyl moiety is the eligible site of attack rather than the isocyanide ligand or the metal centre. All complexes were tested as potential anticancer agents against three ovarian cancer cell lines (A2780, A2780cis, and OVCAR-5) and one breast cancer cell line (MDA-MB-231), displaying comparable activity with respect to cisplatin, which was used as a positive control. Moreover, the similar cytotoxicity observed towards A2780 and A2780cis cells (cisplatin-sensitive and cisplatin-resistant, respectively) suggests that our palladium derivatives presumably act with a mechanism of action different than that of the clinically approved platinum drugs. For comparison, we also synthesized Pd-ƞ3-allyl derivatives, which generally showed a slightly higher activity towards ovarian cancer cells and lower activity towards breast cancer cells with respect to their Pd-indenyl congeners.

Combined Treatment of Cancer Cells Using Allyl Palladium Complexes Bearing Purine-Based NHC Ligands and Molecules Targeting MicroRNAs miR-221-3p and miR-222-3p: Synergistic Effects on Apoptosis.

Combined treatments employing lower concentrations of different drugs are used and studied to develop new and more effective anticancer therapeutic approaches. The combination therapy could be of great interest in the controlling of cancer. Regarding this, our research group has recently shown that peptide nucleic acids (PNAs) that target miR-221 are very effective and functional in inducing apoptosis of many tumor cells, including glioblastoma and colon cancer cells. Moreover, in a recent paper, we described a series of new palladium allyl complexes showing a strong antiproliferative activity on different tumor cell lines. The present study was aimed to analyze and validate the biological effects of the most active compounds tested, in combination with antagomiRNA molecules targeting two miRNAs, miR-221-3p and miR-222-3p. The obtained results show that a "combination therapy", produced by combining the antagomiRNAs targeting miR-221-3p, miR-222-3p and the palladium allyl complex 4d, is very effective in inducing apoptosis, supporting the concept that the combination treatment of cancer cells with antagomiRNAs targeting a specific upregulated oncomiRNAs (in this study miR-221-3p and miR-222-3p) and metal-based compounds represents a promising therapeutic strategy to increase the efficacy of the antitumor protocol, reducing side effects at the same time.

Reactions of proteins with a few organopalladium compounds of medicinal interest.

Pd compounds form a promising class of experimental anticancer drug candidates whose mechanism of action is still largely unknown; in particular, a few organopalladium compounds seem very attractive. To gain mechanistic insight into medicinal palladium compounds, we have explored here - through ESI MS analysis - the interactions of four organopalladium agents (1-4) - showing remarkable in vitro antiproliferative properties - with a few representative model proteins, i.e., lysozyme (HEWL), ribonuclease A (RNase), and carbonic anhydrase (hCAI). The tested panel included three Pd allyl compounds with one or two carbene ligands and a palladacyclopentadienyl complex. Notably, the Pd allyl compounds turned out to manifest, on the whole, a modest tendency to react with the above proteins. Only complex 3 produced small amounts of characteristic adducts with hCAI bearing either one or two Pd allyl groups. In contrast, the palladacyclopentadienyl complex 4 manifested a greater and peculiar reactivity with all the above proteins generating invariably protein adducts with a mass increase of +256 Da where a butadienyl group - with no associated Pd - is attached to the proteins. Afterwards, we extended our investigations to the C-terminal dodecapeptide of thioredoxin reductase bearing the -Cys-Sec- reactive motif. In this latter case adducts were formed with all tested Pd compounds; however, complex 4 manifested towards this dodecapeptide a type of reactivity deeply different from that observed with HEWL, RNase A and hCAI. The mechanistic implications of these findings are discussed.

Cationic palladium(II)-indenyl complexes bearing phosphines as ancillary ligands: synthesis, and study of indenyl amination and anticancer activity.

The reactivity of palladium(II) indenyl derivatives and their applications are topics relatively less studied, though in recent times these compounds have been used as pre-catalysts able to promote challenging cross-coupling processes. Herein, we propose the first systematic study concerning the nucleophilic attack on the palladium(II) coordinated indenyl fragment and, for this purpose, we have prepared a library of new Pd-indenyl complexes bearing mono- or bidentate phosphines as spectator ligands, developing specific synthetic strategies. All novel compounds are thoroughly characterized, highlighting that the indenyl ligand presents always a hapticity intermediate between η3 and η5. Secondary amines have been chosen as nucleophiles for the present study and indenyl amination has been monitored by UV-Vis and NMR spectroscopies, deriving a second order rate law, with dependence on both complex and amine concentrations. The rate-determining step of the process is the initial attack of the amine to the coordinated indenyl fragment, and this conclusion has been supported also by DFT calculations. The determination of second order rate constants has allowed us to assess the impact of the phosphine ligands on the kinetics of the process and identify the steric and electronic descriptors most suitable for predicting the reactivity of these systems. Finally, in vitro tests have proven that these organometallic compounds promote antiproliferative activity towards ovarian cancer cells better than cisplatin and possibly by adopting a different mechanism of action.

The anticancer activity of an air-stable Pd(I)-NHC (NHC = N-heterocyclic carbene) dimer.

A new dinuclear Pd(i) complex coordinating two bis(NHC) ligands revealed an unsuspected stability despite the unsaturation of the two metal centres. Even more surprisingly, the compound showed high and selective antiproliferative activity against different cancer cell lines and ovarian cancer tumoroids, and the mechanism of action was different from that of cisplatin.

Palladium(II)-η3 -Allyl Complexes Bearing N-Trifluoromethyl N-Heterocyclic Carbenes: A New Generation of Anticancer Agents that Restrain the Growth of High-Grade Serous Ovarian Cancer Tumoroids.

The first palladium organometallic compounds bearing N-trifluoromethyl N-heterocyclic carbenes have been synthesized. These η3 -allyl complexes are potent antiproliferative agents against different cancer lines (for the most part, IC50 values fall in the range 0.02-0.5 µm). By choosing 1,3,5-triaza-7-phosphaadamantane (PTA) as co-ligand, we can improve the selectivity toward tumor cells, whereas the introduction of 2-methyl substituents generally reduces the antitumor activity slightly. A series of biochemical assays, aimed at defining the cellular targets of these palladium complexes, has shown that mitochondria are damaged before DNA, thus revealing a behavior substantially different from that of cisplatin and its derivatives. We assume that the specific mechanism of action of these organometallic compounds involves nucleophilic attack on the η3 -allyl fragment. The effectiveness of a representative complex, 4 c, was verified on ovarian cancer tumoroids derived from patients. The results are promising: unlike carboplatin, our compound turned out to be very active and showed a low toxicity toward normal liver organoids.

Chemoselective oxidative addition of vinyl sulfones mediated by palladium complexes bearing picolyl-N-heterocyclic carbene ligands.

The manifold interactions of (E)- or (Z)-1,2-ditosylethene with a palladium(0) centre bearing picolyl-NHC carbene ligands have been studied thoroughly. (E)-1,2-Ditosylethene produces the expected and stable η2-olefin palladium complexes, whereas the coordination of the Z derivative alternatively promotes the isomerization of the olefin itself or an oxidative addition process depending on the steric bulkiness of carbene substituents and/or the adopted synthetic procedure. Remarkably, the oxidative addition pathway involves a selective S-vinyl (not S-aryl) breaking and produces selectively the S- rather than O-coordinated sulfinate. A mechanistic study has clarified the reasons of the chemoselectivity of the process, which was proved to be kinetically controlled. All the involved species have been isolated and exhaustively characterized. In particular, we report the first example of the X-ray crystal structure of a complex bearing one vinyl and one S-sulfinate fragment coordinated to palladium.

Synthesis and in-depth studies on the anticancer activity of novel palladacyclopentadienyl complexes stabilized by N-Heterocyclic carbene ligands.

New palladacyclopentadienyl complexes with bis-N-heterocyclic carbenes as spectator ligands have been synthesized and exhaustively characterized. The crystal structure of complex 1a has been also determined by X-ray diffraction analysis. Their in vitro cytotoxicity and that of other palladacyclopentadienyl derivatives coordinating different ancillary ligands has been determined against different cancer cell lines. Many complexes have shown an antiproliferative activity toward tumor cells often definitely better than cisplatin, whereas they have resulted practically inactive against the non-cancer MRC-5 cell line. The mechanism of action of bis-NHC derivative 1a, particularly active against ovarian cancer cell lines was studied in depth. Through a longitudinally analysis, it is shown that compound 1a induces apoptosis via DNA damage and release of cytochrome C. We propose compound 1a as a powerful and specific drug for the therapy of a deadly disease such as high grade serous ovarian cancer.

Synthesis of new allyl palladium complexes bearing purine-based NHC ligands with antiproliferative and proapoptotic activities on human ovarian cancer cell lines.

A series of new palladium allyl complexes bearing purine-based carbenes derived from caffeine, theophylline and theobromine have been prepared and characterized by NMR spectroscopy, and elemental analysis and in two cases by single crystal X-ray diffraction. The cytotoxic and proapoptotic activities of compounds have been determined in vitro on human ovarian cancer A2780 and SKOV-3 cell lines. These experiments have shown that the palladium-allyl fragment induces a general cytotoxicity, but the choice of the supporting ligands is of paramount importance for achieving the best results. In particular complexes 4c, 4d and 5d exhibit a higher antiproliferative effect (IC50: 0.09, 0.81 and 0.85 µM respectively) than cisplatin (IC50: 1.5 µM) on A2780 cells, and 4d (IC50: 1.7 µM vs. 5.94 µM) on SKOV-3 cell line. Moreover in many cases it has been proved that the cytotoxicity of our complexes is associated with the induction of apoptosis.

Reactivity of N-heterocyclic carbene-pyridine palladacyclopentadiene complexes toward halogen addition. The unpredictable course of the reaction.

As an extension of a previously published work we have reacted some palladacyclopentadiene complexes stabilized by bidentate N-heterocyclic carbene-pyridine or monodentate N-heterocyclic carbene-pyridine and isocyanide ligands with the halogens I2 and Br2. All the bidentate and monodentate complexes react with halogens to give at first the expected σ-coordinated butadienyl fragment. However, two of the less hindered NHC carbene-pyridine bidentate butadienyl iodo derivatives undergo a further rearrangement and novel Pd(ii) complexes characterized by a ten term coordinative ring were isolated and characterized. In the most favorable case we were able to carry out the kinetics of rearrangement and measure its reaction rate. Moreover, we have surmised a plausible mechanism on the basis of a dedicated computational approach and in one case the surprising structure characterized by the ten term coordinative ring was resolved by X-ray diffraction.

Isocyanide insertion across the Pd-C bond of allenyl and propargyl palladium complexes bearing phosphoquinoline as a spectator ligand. Synthesis of a palladium complex bearing a coordinated cyclobutenyl fragment.

We have studied the insertion of p-toluenesulfonylmethyl isocyanide (TosMIC) on selected allenyl and propargyl complexes of palladium bearing diphenylphosphine quinoline as a spectator ligand. The fast process gives different products depending on the tautomer involved in the reaction. Thus, the unsubstituted allenyl species yields an insertion complex with the isocyanide coordinated between the metal and the first allenyl carbon. On the other hand, a mixture of phenyl substituted allenyl and propargyl palladium complexes yields a novel species characterized by a cyclo-butenyl fragment directly coordinated to palladium. The solid state structure of such a complex together with an exhaustive kinetic study of the whole process is reported.

The unexpected case of reactions of halogens and interhalogens with halide substituted Pd(ii) σ-butadienyl complexes.

We have experimentally studied and theoretically interpreted the addition under stoichiometric conditions of halogens or interhalogens to σ-butadienyl palladium complexes bearing the heteroditopic thioquinolines as spectator ligands. The observed reactions do not involve the expected extrusion of the butadienyl fragment but rather the unpredictable substitution of the halide coordinated to palladium and in some cases also of that bound to the terminal butadienyl carbon. We have explained this peculiar reactivity with a mechanistic hypothesis based on a sequence of selective processes of oxidative addition and reductive elimination involving Pd(iv) intermediates.

The addition of bromine and iodine to palladacyclopentadienyl complexes bearing bidentate heteroditopic P-N spectator ligands derived from differently substituted quinolinic frames. The unexpected evolution of the reaction.

We have synthesized two palladacyclopentadienyl derivatives bearing bidentate ligands heteroditopic 8-(diphenylphosphino)quinoline or 8-(diphenylphosphino)-2-methylquinoline. We have reacted the palladacyclopentadienyl complexes with Br2 and I2 to gain clues on the formation mechanism of the corresponding σ-butadienyl derivatives. We were able to obtain the pure σ-butadienyl derivative only in the case of Br2 reacting with the palladacyclopentadienyl complex bearing the unsubstituted quinoline. However, an equilibrium mixture of the σ-butadienyl and a novel zwitterionic species was obtained when the same complex reacts with I2. Furthermore, we have obtained exclusively an unprecedented zwitterionic complex when I2 reacts with the palladacyclopentadienyl complex bearing the substituted quinoline and a different ratio of an equilibrium mixture of σ-butadienyl and the zwitterionic species when the latter derivative reacts with Br2. The solid state structures of one σ-butadienyl complex and of the two novel zwitterionic derivatives were determined and an interpretation of the observed reactivity based on kinetic data and a computational study has been suggested.

Synthesis, characterization, dynamics and reactivity toward amination of η3-allyl palladium complexes bearing mixed ancillary ligands. Evaluation of the electronic characteristics of the ligands from kinetic data.

On the basis of an original protocol, we have synthesized several complexes of the type [Pd(η(3)-C(3)H(3)R(2))(LL')]ClO(4) (R = H, Me; L, L' = PPh(3), P(OEt)(3), 2,6-dimethylphenylisocyanide, t-butylisocyanide, 1,3-dimesitylimidazolidine, 1,3-dimesitylimidazol-2-ylidene). The complexes, some of which are completely new species, were fully characterized and their behaviour in solution was studied by means of (1)H NMR. The reactions of the complexes bearing the symmetric allyl moiety [Pd(η(3)-C(3)H(5))(LL')]ClO(4) with piperidine in the presence of the olefin dimethylfumarate were followed under kinetically controlled conditions. Formation of allyl-amine and of the palladium(0) derivatives [Pd(η(2)-dmfu)(LL'] was observed. The reaction rates k(2) proved to be strongly dependent on the ancillary ligand nature and allowed a direct comparison among the electronic characteristics of the ligands. The reactivity trend determined appears to be mainly influenced by the capability of the ancillary ligands in transferring electron density to the metal centre and consequently on the allyl fragment.

Substitution reactions between bis-chelate ligands in palladium(II) alkenyl complexes: an unusual way to form unstable trans-P complexes. A study on the isomerization mechanism.

The substitution reactions between asymmetric bis-chelate ligands and alkenyl chloro derivatives of palladium(II) of the type [Pd(L-L')(Rx)Cl] (L-L' = 2-phenylsulfanylmethyl-pyridine (HN-SPh), 2-methyl-6-phenylsulfanylmethyl-pyridine (MeN-SPh), 2,2'-bipyridinyl (BiPy), Rx = -CCOOMe=CMeCOOMe (Ra), -CCOOEt=CMeCOOEt (Rb), -CCOOt-Bu=CMeCOOt-Bu (Rc), -(CCOOMe=CCOOMe)(2)Me (Rd)) with phosphoquinoline moieties (8-diphenylphosphanyl-quinoline (DPPQ), 8-diphenylphosphanyl-2-methyl-quinoline (DPPQ-Me)) usually leads to the formation of the stable geometrical isomer bearing these groups in the cis position thanks to the mutual trans influence of the alkenyl and phosphine groups. However, when the leaving group MeN-SPh and the entering ligand DPPQ are involved, the fast and quantitative substitution reaction leads to the formation of a couple of geometrical isomers [Pd(DPPQ)(Rx)Cl]-trans P and [Pd(DPPQ)(Rx)Cl]-cis P (Rx = Ra, Rb, Rc, Rd) in which the alkenyl and the phosphine groups are in mutual trans or cis position. The substrate [Pd(DPPQ)(Rx)Cl]-trans P (Rx = Ra, Rb, Rc) slowly interconverts into its thermodynamically stable -cis P counterpart while the bulky [Pd(DPPQ)(Rd)Cl]-trans P displays no tendency to isomerize, thereby allowing separation of the two geometrical forms. Also, the ligand DPPQ-Me induces the formation of the -trans P geometrical isomer which is only detectable at low temperature since it rapidly interconverts into the -cis P derivative at RT. The kinetics of the interconversion process, a reasonable explanation of the observed phenomenon based on theoretical calculations, and eventually an unequivocal structure determination of the stable [Pd(DPPQ)(Rx)Cl]-cis P substrate are reported in the present paper.