FIELD OF THE INVENTION

The present invention relates to the sector of chemotherapy compounds and in particular of the metal complexes used for the purpose. The invention also relates to a process for the synthesis of these complexes and the therapeutic use thereof.

STATE OF THE ART

The discovery of the antitumour properties of cisplatin (cis-diamine- di chi oropl atinum(II)), and its approval by the Food and Drug Administration in 1978, paved the way for the use of inorganic compounds as chemotherapy agents. However, cisplatin has some important limitations, connected on one hand with its nephro- and neuro-toxicity and, on the other hand, with its inefficacy towards some types of tumours or induced resistance phenomena on others.

Platinum compounds have appeared on the market that have fewer side effects (carboplatin and oxaliplatin); however, these compounds, having the same action mechanism proposed for cisplatin (platination of DNA) turned out to be ineffective towards cisplatin- resistant tumours.

Many research groups have therefore focused in recent decades on the synthesis and study of the antitumour properties of compounds containing different metals from platinum. Among the most investigated compounds in this area, gold and ruthenium can be listed, whereas palladium compounds have only recently been taken into consideration.

Although it belongs to the same group as platinum, this element enables complexes to be obtained that are more soluble in water and moreover in some cases have a different primary target from the DNA. This latter characteristic explains the marked anti -proliferative activity of some of these compounds towards cisplatin-resistant cell lines.

A critical feature of palladium compounds relates to their higher hydrolysis speed with respect to platinum ones, with a consequent dissociation of the ligands and serious speciation problems (i.e. the formation of different species containing palladium derived from the starting one); this can cause greater collateral toxicity, due to the lack of control over the species effectively introduced into the organism.

However, it has recently been demonstrated that the use of ancillary ligands strongly anchored to a metal centre of Pd(II) significantly reduces or eliminates the problem connected with the dissociation of ligands in solution.

A category of ligands that have been widely studied in this sense in recent times is N- heterocyclic carbenes (NHCs). Pd(II) complexes containing these ligands have provoked a certain amount of interest, thanks to their high anti-proliferative activity on different, both cisplatin-sensitive and cisplatin-resistant, tumour lines. Pd(II) complexes of this type are for example the subject matter of patent applications US 2018/194789 A1 and CN 108558952 A.

There are not however, in the state of the art, any studies relating to the antitumour properties of palladium in oxidation state +1 (Pd(I)). These complexes are definitely less common than Pd(II) ones, since they are generally not very stable both in the solid state and in solution, being sensitive to oxygen and water. Among the few examples present in the literature, there are dimeric species of Pd(I), in which there is a Pd-Pd bond and in which every Pd atom is complexed by a phosphine and is bonded to a halogen atom. Examples of complexes of this type are reported in patent applications US 2012/190873 Al, US 2014/187803 A1 and WO 2018/073559 Al. These compounds have been studied so far only in view of their application as catalysts for chemical reactions, e.g. in cross-coupling processes.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a new family of palladium (I) complexes provided with antitumour activity, and to provide the process for the production thereof and the methods for the use of these complexes for therapeutic purposes.

These objects are achieved with the present invention which in a first aspect thereof relates to a compound of general formula (A) or of general formula (B):

(A) (B) wherein:

- R1 is an alkyl radical or a phenyl radical optionally substituted with one or more Ci- C3 alkyl groups;

- R2 is a linear or branched C1-C6 alkyl radical; - R3 is a linear or branched C1-C6 alkyl radical;

- m = 0, 1 or 2;

- n = 0, 1, 2, 3 or 4;

- the notation 2C ^Q means two pharmaceutically acceptable monovalent anions or a pharmaceutically acceptable bivalent anion. In a second aspect thereof, the invention relates to a process for producing compounds of general formula (A) or (B), which consists in reacting a compound of general formula (C) or of general formula (D) with a palladium (II) complex, according to the following diagrams: wherein Rl, R2, R3, m, n and X have the meanings reported above, Cpx(Pd(II)) indicates a complex of palladium in oxidation state +2, and MX generally indicates a salt containing the monovalent or bivalent anion X defined above. The stoichiometric coefficient p is 2 if Cpx(Pd(II)) contains a single Pd atom, and is 1 if Cpx(Pd(II)) contains two Pd atoms, so as to keep a 1 : 1 molar ratio between compound (C) or (D) and Pd. The stoichiometric coefficient q is 1 in the event in which the anion X is bivalent and is 2 in the event in which the anion X is monovalent, regardless of the oxidation number of the metal M. The salt will have the formula MiXi in the event in which the metal M and the anion X are both monovalent (respectively, cation with oxidation number +1 and anion with charge -1) or both bivalent (cation with oxidation number +2 and anion with charge -2); formula M1X2 (cation with oxidation number +2 and anion with charge -1); or formula M2X1 (cation with oxidation number +1 and anion with charge -2).

Finally, the third aspect of the invention relates to the use of the compounds described above as antitumour therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be illustrated below with reference to the Figures, in which:

- Figure 1 shows the ¹ H-NMR spectrum of a compound of the invention;

- Figure 2 shows the ¹³ C{ ¹ H}-NMR spectrum of the same compound of Figure 1;

- Figure 3 shows a perspective image of the three-dimensional structure of the compound of Figure 1, reconstructed with the single-crystal X-ray diffraction method.

PET ATT /ED DESCRIPTION OF THE INVENTION

In the first aspect thereof the invention relates to new palladium (I) complexes, provided with antitumour activity, having general formula (A) or (B).

The compounds of the invention are formed by heterocyclic units, in particular imidazolidines (general formula (A)) and benzimidazolidines (general formula (B)) with a carbon of the carbene type between two nitrogen atoms in the heterocyclic ring; the carbene carbon atoms complex two palladium atoms, which have a direct bond with each other, producing a dimeric species in which palladium is in oxidation state +1 (Pd(I)).

In general formulae (A) and (B), R1 is an alkyl radical or a phenyl radical optionally substituted with one or more C1-C3 alkyl groups. The preferred alkyl radicals for the purposes of the invention are tert-butyl and 1-adamantyl, whereas preferred aromatic radicals are 2,4,6- trimethylphenyl (commonly referred to as mesityl) and 2,6-diisopropylphenyl.

The substituents R2 and R3 may be a linear or branched C1-C6 alkyl radical. The subscript m, related to the substituents R2 in general formula (A), may be equal to 0, 1 or 2; the preferred case is m = 0, wherein the carbons of the imidazolidine ring are saturated with hydrogen atoms. Likewise, the subscript n, related to the substituents R3 in general formula (B), may be equal to 0, 1, 2, 3 or 4, and the preferred case is n = 0.

Finally, X is a pharmaceutically acceptable monovalent or bivalent anion, chosen among those known by the skilled in the art; preferred monovalent anions for the purposes of the invention are chloride, perchlorate (CIO-T), tetraflurob orate (BF-f) and hexafluorophosphate (PF6-); examples of possible bivalent anions, typically used in the pharmaceutical sector, are sulphate, oxalate, maleate and fumarate.

The preferred compounds of the invention have the formulae Al and Bl reported below:

(Al) (Bl) wherein Mes indicates the mesityl radical.

In its second aspect the invention relates to a process for the synthesis of the compounds of general formula (A) and (B), consisting in reacting a compound of general formula (C) or of general formula (D) with a palladium (II) complex, as reported above. The compound of general formula (C) reported above, which is used as a reactant in the process of the invention for the preparation of a compound of general formula (A), can be obtained by reaction between the corresponding imidazolium salt and silver oxide according to the conditions reported in literature (“An unusually static, sterically hindered silver bis(n- heterocyclic carbene) complex and its use in transmetalation”, Y. A. Wanniarachchi el al ., Organometallics, 2004, 23, 5881-5884).

The compound of general formula (D) reported above, which is used as a reactant in the process of the invention for preparing a compound of general formula (B), can be synthesized, starting from the related bis-imidazolium salt, in quite similar conditions as those described for compound (C). For the preparation of the specific compounds (Al) and (Bl), the compounds (Cl) and (Dl), the formula of which is shown below, are used as reactants:

(Cl) (Dl)

The second reactant of the process of the invention is a Pd(II) complex, indicated generally as Cpx(Pd(II)). The palladium complex must have one or more sufficiently labile coordination sites for the introduction of the biscarbene ligand and a sufficiently hindered allyl fragment.

Preferred palladium complexes are [Pd(p-Cl)(p ³ -l,l-dimethylallyl)]2 and [(MePyCH2SPh)Pd(p ³ -l,l-dimethylallyl)]C104, having the following structural formulae:

[Pd(y-Cl)(rf-l,l-dimethylallyl)] ₂ [b/lePyCH ₂ SPh)Pd(rf-l,l-dimethylallyl) ]C10 ₄

For simplicity, in the rest of the description, these two complexes shall be simply indicated as Pd(l,l-dimethylallyl)Cl2 and [Pd(NS)(l,l-dimethylallyl)]X.

These Pd(II) complexes can be prepared with methods known to those skilled in the art of coordination chemistry.

For example, the complex Pd(l,l-dimethylallyl)Cl2 can be prepared by suspending PdCh powders in a solution of 1,1 -dimethyl-propyl- 1-ene in methanol; both these reactants are commonly available on the market.

Likewise, the complex [Pd(NS)(l,l-dimethylallyl)]X can be prepared by dissolving the complex Pd(l,l-dimethylallyl)Cl2 in dichloromethane in the presence of the ligand 2-(phenyl- thiomethyl)-6-methyl-pyridine, according to the methods described in the article “Mechanism of the reaction of allyl amination of Pd(II) allyl complexes containing chelating pyridine- chalcogen ligands. A surprisingly low influence of the chalcogen atom”, L. Canovese, Polyhedron 20 (2001) 3171-3181. In particular, the synthesis of the ligand 2-(phenyl- thiomethyl)-6-methyl-pyridine consists of reacting thiophenol and 2-chloromethyl-6- methylpyridine hydrochloride in a solution of potassium hydroxide in dimethyl sulfoxide, followed by usual work-up operations (addition of water, separation of the phases, concentration of the organic phase and purification of the product by chromatography).

Finally, the reactant MX has the function of introducing the anion X into the reaction, to compensate for the charge of the Pd(I) complex that is produced in the process. As mentioned, the anion is selected from the pharmaceutically acceptable ones, and preferably from chloride, perchlorate, tetrafluob orate and hexafluorophosphate, whereas the cation is preferably selected from silver and alkali metals (Li, Na and K).

The reaction is carried out in an organic solvent, preferably dichloromethane, at a temperature between 0 °C and 35 °C, for times generally between 5 minutes and 1 hour. The stoichiometry of the reaction requires the use of the compound of formula (C) or (D) in a molar ratio 1 : 1 with respect to the Pd(II) complex when this contains a single Pd atom, and in a molar ratio 2: 1 with respect to the Pd(II) complex when this contains two Pd atoms.

Finally, the invention relates to a method for treating tumours that uses the Pd(I) complexes described above, and which consists of administering said complexes to a patient affected by a tumour, possibly in combination with at least one cytostatic or cytotoxic agent and, possibly, in the form of a combined preparation for simultaneous, separate or sequential use in the antitumour therapy.

The complexes of the invention are particularly effective in the treatment of ovarian, cervical, colon and lung cancers.

Said complexes may be administered by the typical routes of chemotherapy, for example in the form of intravenous solutions (the most frequent), intramuscular or subcutaneous, or orally through capsules or tablets.

The invention will be further illustrated by the following examples.

INSTRUMENTS, METHODS AND EXPERIMENTAL CONDITIONS

Ή-NMR and ¹³ C-NMR:

Bruker 400 Avance spectrometer.

XRD:

XRD1 beamline at Elettra Sincrotrone, Trieste (Italy).

ANTIPROLIFERATIVE ACTIVITY:

Tecan analyser, mod. Infinite ^® M1000.

EXAMPLE 1

This example relates to the preparation of the compound (Al) of the invention.

In a 50 mL round-bottom flask, kept in an argon atmosphere, 0.0690 g (0.164 mmol) of the precursor Pd( 1,1 -dimethylallyl)Cl2 were dissolved in 15 mL of anhydrous dichloromethane; then 0.2485 g (0.3270 mmol) of compound (Cl), previously dissolved in 5 mL of dichloromethane, and 0.0678 g (0.3270 mmol) of AgCICL were added thereto. The reaction mixture was kept under agitation for 15 minutes; during the reaction, the temperature fluctuated in the range 25 ± 2 °C. At the end of this period, the reaction mixture was filtered to remove the silver bromide that had formed. The volume of the solution obtained was reduced using a rotary evaporator. The addition of diethylether (Et20) induced the precipitation of a mixture of the compound (Al) (dimer complex of Pd(I)) and, as a by-product, of a palladium complex with a single metal centre.

The solid fraction was dissolved in a mixture of CH2Cl2/Et20 (30 mL:70 mL) and the system was allowed to rest at 5 °C overnight. The selective precipitation of the dimeric species of Pd(I) (Compound (Al)) in the form of a pink solid was obtained, which was filtered with a Gooch crucible (yield: 0.0354 g). The compound is stable both in the solid state and in solution and was characterised using NMR spectroscopy.

The ^I-NMR and ¹³ C{ ¹ H}-NMR spectra of the compound are reported respectively in Figures 1 and 2. The listing and the attribution of the peaks in the two spectra are provided below. ¾-NMR (300 MHz, d6-DMSO, T = 298 K, ppm) d: 1.26 (s, 12H, 4 o-aryl-Cft), 1.59 (s, 12H, 4 o-aryl-Cft), 2.43 (s, 12H, 4 p-aryl-Cft), 6.51 and 7.23 (AB system, J = 13.6 Hz, 4H, 2NCH2N), 6.95-7.07 (8H, Ar-H), 7.45-8.03 (8H, CH ^Im ).

¹³ C{ ¹ H}-NMR (d6-DMSO, T = 298 K, ppm) d: 16.8 (CH ₃ , o-Mesityl-CH ₃ ), 17.2 (CH ₃ , o-Mesityl-CH ₃ ), 21.2 (CH ₃ , p-Mesityl-CH ₃ ), 64.0 (CH ₂ , NCH2N), 122.6-125.1 (CH, CH=CH ^Im ), 129.6-139.3 (Ar-C), 182.8 (C, carbene).

EXAMPLE 2

This example relates to an alternative preparation of the compound (Al) of the invention.

In a 50 mL round-bottom flask, kept in an argon atmosphere, 0.1603 g (0.3270 mmol) of the precursor [Pd(NS)(l,l-dimethylallyl)]Cl4 were dissolved in 15 mL of dichloromethane anhydrous; then 0.2485 g (0.3270 mmol) of compound (Cl), previously dissolved in 5 mL of dichloromethane, were added thereto. The reaction mixture was kept under agitation for 15 minutes; during the reaction, the temperature fluctuated in the range 25 ± 2 °C. At the end of this period, the reaction mixture was filtered to remove the formed silver bromide. The volume of the solution obtained was reduced using a rotary evaporator. The addition of diethylether induced the precipitation of a mixture of the compound (Al) (dimer complex of Pd(I)) and, as a by-product, of a palladium complex with a single metal centre.

The solid fraction was dissolved in a mixture of CH2Cl2/Et20 (30 mL:70 mL) and the system was allowed to rest at 5 °C overnight. The selective precipitation of the dimeric species of Pd(I) (Compound (Al)) in the form of a pink solid was obtained, which was filtered with a Gooch crucible (yield: 0.0354 g). The compound is stable both in the solid state and in solution and was characterised using NMR spectroscopy. The ¹ H-NMR and ¹³ C{ ¹ H}-NMR spectra of the compound obtained coincide with those of Example 1.

EXAMPLE 3

This example relates to the structural characterization of the compound (Al) produced in Example 1.

The crystallographic data relating to compound (Al) were collected at the temperature of 100 K at the XRDl beamline of Elettra Sincrotrone of Trieste, using a monochromatic light with wavelength of 0.620 A. Figure 3 shows a perspective view of the spatial structure of the compound obtained by X-ray analysis.

EXAMPLE 4

This example relates to the evaluation of the therapeutic efficacy of the compounds of the invention.

Tests were performed for the purpose of determining the inhibitory concentration (IC50) of a compound of the invention towards tumour cell lines and on a control cell line; for comparative purposes, the same tests were performed with the cisplatin compound. The tested cell lines were: A2780, A2780cw, OVCAR5, OVCAR3 e KURAMOCHI

(ovarian cancer), A549 (lung cancer), HeLa (cervical cancer), DLD1 (colon cancer) and MRC- 5 (control; lung fibroblasts). The mother solutions were obtained in dimethyl sulfoxide for the Pd(I) compound and in H2O for cisplatin.

The cells were kept, according to the protocols indicated by the suppliers, at 37 °C in an atmosphere containing 5% carbon dioxide. About 500 cells were plated on 96 different well- plates and kept under such conditions for 96 hours.

The viability of the cells was determined, after treatment of the cells with a 1 : 1 CellTiter- Glo ^® solution (registered trademark of Promega Corp., Madison, Wisconsin, USA) and DPBS, with the Tecan Infinite ^® M1000 instrument. The IC50 values were calculated by the response curves to the dose. The means and standard deviations were obtained from triplicate measurements. The test results are shown in the following table.

Table 1 The values reported in table 1 show that the compound of the invention is very active towards all the examined tumour lines. Furthermore, it was substantially inactive (ICso >100 mM) towards normal cells (MRC-5 lung fibroblasts), and therefore does not have the toxicity side effects encountered with cisplatin.