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The substitution inert platinum agent [Pt(1 S,2 S-diaminocyclohexane)(5,6-dimethyl-1,10-phenanthroline)]2+ (56MeSS, 5) is a potent cytotoxic metallodrug. In contrast to conventional cisplatin or oxaliplatin, the mechanism of action (MoA) of 5 is fundamentally different. However, details of the mechanism by which the 5,6-dimethyl-1,10-phenanthroline ligand contributes to the cytotoxicity of 5 and its derivatives have not been sufficiently clarified so far. Here, we show that 5 and its Pt(IV) derivatives exhibit an intriguing potency in the triple-negative breast cancer cells MDA-MB-231. Moreover, we show that the Pt(IV) derivatives of 5 act by multimodal MoA resulting in the global biological effects, that is, they damage nuclear DNA, reduce the mitochondrial membrane potential, induce the epigenetic processes, and last but not least, the data provide evidence that changes in the organization of cytoskeleton networks are functionally important for 5 and its derivatives, in contrast to clinically used platinum cytostatics, to kill cancer cells.

Dual- or multi-action PtIV prodrugs represent a new generation of platinum anticancer drugs. The important property of these PtIV prodrugs is that their antitumor action combines several different mechanisms owing to the presence of biologically active axial ligands. This work describes the synthesis and some biological properties of a "triple-action" prodrug that releases in cancer cells cisplatin and two different epigenetically acting moieties, octanoate and phenylbutyrate. It is demonstrated, with the aid of modern methods of molecular and cellular biology and pharmacology, that the presence of three different functionalities in a single molecule of the PtIV prodrug results in a selective and high potency in tumor cells including those resistant to cisplatin [the IC50 values in the screened malignant cell lines ranged from as low as 9 nm (HCT-116) to 74 nm (MDA-MB-231)]. It is also demonstrated that cellular activation of the PtIV prodrug results in covalent modification of DNA through the release of the platinum moiety accompanied by inhibition of the activity of histone deacetylases caused by phenylbutyrate and by global hypermethylation of DNA by octanoate. Thus, the PtIV prodrug introduced in this study acts as a true "multi-action" prodrug, which is over two orders of magnitude more active than clinically used cisplatin, in both 2D monolayer culture and 3D spheroid cancer cells.

We developed a novel PtIV prodrug that simultaneously releases four different bioactive moieties inside the cancer cell. Its cytotoxicity against monolayer cultures (2D) and spheroid (3D) cancer cells is significantly better than cisplatin. It is 200-450-fold more potent than cisplatin against KRAS mutated pancreatic and colon cancers and is 40-fold more selective towards KRAS mutated cells compared to non-cancerous. This is important since RAS proteins play a role in regulating cell differentiation, proliferation, and survival and KRAS is mutated in 90 % of pancreatic adenocarcinomas, 45 % of colorectal cancers, and 35 % of lung adenocarcinomas. The selectivity index, determined by dividing the IC50 value in non-cancerous cells by that of a cancerous cell line, is two-fold better than cisplatin, attesting to preferential cytotoxicity towards neoplastic cells.

The discrepancy between the in vitro cytotoxic results and the in vivo performance of Pt56MeSS prompted us to look into its interactions and those of its PtIV derivatives with human serum (HS), human serum albumin (HSA), lipoproteins, and serum-supplemented cell culture media. The PtII complex, Pt56MeSS, binds noncovalently and reversibly to slow-tumbling proteins in HS and in cell culture media and interacts through the phenanthroline group with HSA, with a Kd value of ∼1.5×10-6 m. All PtIV complexes were found to be stable toward reduction in HS, but those with axial carboxylate ligands, cct-[Pt(1S,2S-DACH)(5,6-dimethyl-1,10-phenantroline)(acetato)2 ](TFA)2 (Pt56MeSS(OAc)2 ) and cct-[Pt(1S,2S-DACH)(5,6-dimehtyl-1,10-phenantroline)(phenylbutyrato)2 ](TFA)2 (Pt56MeSS(PhB)2 ), were spontaneously reduced at pH 7 or higher in phosphate buffer, but not in Tris buffer (pH 8). HS also decreased the rate of reduction by ascorbate of the PtIV complexes relative to the reduction rates in phosphate buffer, suggesting that for this compound class, phosphate buffer is not a good model for HS.

Glutathione (GSH) plays an important role in the development of resistance to platinum-based chemotherapy, since it can prevent drug binding to DNA and resulting apoptosis of tumor cells. The recently re-discovered drug candidate kiteplatin was found active toward cisplatin- and oxaliplatin-resistant tumor cells, and this could be related to a different interplay of drug-inactivation/DNA-interaction processes. In this study GSH and GMP have been chosen as simple models of platinophiles and DNA, respectively, and the reactivity of kiteplatin has been tested toward GMP, after previous interaction with GSH; toward GSH, after previous interaction with GMP; and toward GMP and GSH simultaneously.

Dual- or multi-action Pt

A series of triple action Pt(iv) prodrugs was designed to test the hypothesis that multi-action compounds, where each bioactive moiety intervenes in several cellular processes, might be more effective than a single agent at killing cancer cells., A series of triple action Pt(iv) prodrugs was designed to test the hypothesis that multi-action compounds, where each bioactive moiety intervenes in several cellular processes, might be more effective than a single agent at killing cancer cells. In particular, “triple action” Pt(iv) derivatives of cisplatin, where the axial ligands are inhibitors of cyclooxygenase (COXi), histone deacetylase (HDACi) or pyruvate dehydrogenase kinase (PDKi) were developed. All compounds, ctc-[Pt(NH3)2(COXi)(PDKi)Cl2], ctc-[Pt(NH3)2(COXi)(HDACi)Cl2] and ctc-[Pt(NH3)2(HDACi)(PDKi)Cl2], where COXi = aspirin or ibuprofen, PDKi = dichloroacetate and HDACi = valproate or phenylbutyrate, were significantly more cytotoxic than cisplatin against all cell lines of an in-house panel of human cancer cells. They were particularly effective against thyroid and pancreatic cancer cells in monolayer cytotoxicity tests. Remarkably, in 3D spheroid cancer cell cultures, some triple action compounds showed an antitumor potency up to 50-fold higher than cisplatin against a KRAS mutated pancreatic cancer cell line (PSN-1 cells). Standard biochemical assays classically employed to explore structure activity relationships of platinum drugs, such as cellular uptake and binding to potential biological targets (DNA, HDAC, mitochondria, and COX), do not provide linear correlations with the overall cytotoxicity data. We observed a preferential induction of ROS production and of an anti-mitochondrial effect in cancer cells compared to rapidly dividing non-cancerous cells. Thus, we propose that these new triple action Pt(iv) derivatives of cisplatin are a novel and interesting class of potent and selective cytotoxic agents.

A microwave-assisted synthesis of cisplatin, cis-[Pt(NH3)2Cl2], has been developed and optimized on both a 0.2 and 0.05 millimolar scale. The optimized synthetic procedure was modeled after the Lebedinskii-Golovnya method and is suitable for incorporating the radionuclide, (195m)Pt, into cisplatin for biological studies. Highest yields (47%) and purity are obtained using a K2PtCl4 : NH4OAc : KCl molar ratio of 1 : 4 : 2 at a temperature of 100 °C. The entire synthesis and purification procedure requires approximately 80 min. At a reaction temperature of 150 °C, the trans isomer is the exclusive product, suggesting that complexes of the general form, trans-[Pt(RNH2)2Cl2], can be synthesized directly from K2PtCl4 using [RNH3]OAc (R = alkyl or aryl moieties) via a microwave process. Two novel separation procedures have been developed which efficiently remove the major impurity (1 : 1 Magnus-type salt) from the crude reaction product, yielding a product of purity comparable to that obtained by the Dhara method and suitable for biological studies. These procedures are applicable to both the micro- and macro-scale of synthesis. The question of whether this microwave-assisted synthesis of cisplatin will be a preferred method for incorporating (195m)Pt into cisplatin is yet to be determined.

This report describes the synthesis, characterization and biological activity of a series of platinum(iv) derivatives of [Pt(1S,2S-DACH)(5,6-dimethyl-1,10-phenanthroline)] (Pt56MeSS) with non-bioactive, lipophilic and bioactive axial ligands. In an attempt to explore the anticancer activity potential of the Pt(iv) derivatives, 2D and 3D cytotoxic screening and a preliminary in vivo study were performed. The average IC50 values of the platinum(iv) derivatives ranged from 1.26 to 5.39 μM, compared with 1.24 μM for Pt56MeSS, suggesting that the axial ligands have a relatively minor effect on the potency of the compounds. Preliminary in vivo studies indicate that the platinum(iv) derivatives of Pt56MeSS are active in vivo and can reduce the tumor to a similar extent to cisplatin.

Kiteplatin, (cis-1,4-DACH)dichloridoplatinum(ii), contains an isomeric form of the carrier ligand present in the successful antitumor drug oxaliplatin and has been recently found to be very active against oxaliplatin-resistant colon cancers, confirming that, by changing the nature of the amine ligand, it is possible to obtain platinum drugs that are not cross-resistant to those already in clinical use. Apart from interaction with DNA, another factor that can affect the activity of platinum drugs is their metabolic fate in the cellular environment. Therefore, kiteplatin has been reacted with S-donor biomolecules, such as glutathione, cysteine, and methionine. The investigation has further confirmed the different reactivity of methionine as compared to cysteine-containing peptides and has unraveled the possibility of cis-1,4-DACH to become mono-coordinated with one free end (a situation never seen for isomeric 1,2-DACH ligands) and to labilize cis ligands as a consequence of its large steric hindrance. The reaction of kiteplatin-GSH adducts with 5'-GMP has also shown how the reaction products can be different depending upon the aerobic or anaerobic reaction conditions used.

The discrepancy between the in vitro cytotoxic results and the in vivo performance of Pt56MeSS prompted us to look into its interactions and those of its Pt

Although the encouraging antitumor activity of [PtCl2(cis-1,4-DACH)] (1; DACH = diaminocyclohexane) was shown in early studies almost 20 years ago, the compound has remained nearly neglected. In contrast, oxaliplatin, containing isomeric 1R,2R-DACH carrier ligand, enjoys worldwide clinic application as a most important therapeutic agent in the treatment of colorectal cancer. By extending the investigation to human chemo-resistant cancer cells, we have demonstrated the real effectiveness of 1 in circumventing cisplatin and oxaliplatin resistance in LoVo colon cancer cells. The uptake of compound 1 by the latter cells was similar to that of sensitive LoVo cells. This is not the case for all other compounds considered in this investigation. Interaction with ds-DNA, investigated by a biosensor assay and by QM/MM geometry optimization of the 1,2-GG intrastrand cross-link, does not show significant differences between 1 and oxaliplatin. However, the DNA adducts of 1 are removed from repair systems with lower efficiency and are more effective in inhibiting DNA and RNA polymerase.

The anticancer activity of cisplatin is triggered by its formation of intrastrand adducts involving adjacent G residues of DNA. To obtain information on the different conformers that can be formed{,} carrier ligands such as 2{,}2[prime or minute]-bipiperidine{,} which provide large steric bulk near the platinum coordination plane and decrease the dynamic motion about the Pt-N7 bonds{,} were introduced ({"}retro-modelling{"} approach). In the present study we investigate the effect of cis-1{,}4-diaminocyclohexane (cis-1{,}4-DACH) on the formation{,} stability{,} and stereochemistry of (cis-1{,}4-DACH)Pt(ss-oligo) adducts (ss-oligo = d(GpG) with 3[prime or minute]- and/or 5[prime or minute]-substituents). Interesting features of this ligand{,} absent in previous retro-modelling studies{,} include the large bite angle (expected to impede the ease of interconversion between possible conformers){,} the presence of two protons on each nitrogen (a characteristic associated with antitumor activity){,} and the absence of chiral centres. The use of cis-1{,}4-DACH has made it possible to detect different conformers in a system containing a primary diamine carrier ligand associated with anticancer activity and to confirm the previous hypothesis that the coexistence of different conformers established in studies of retro models having relatively bulky ligands is not an artefact resulting from carrier-ligand bulk. Moreover{,} the data for the (cis-1{,}4-DACH)Pt(d(GpG)) and (cis-1{,}4-DACH)Pt(d(GGTTT)) adducts indicate that at a temperature close to the physiological one (40 [degree]C) HH1 and [capital Delta]HT1 conformers are present in comparable amounts. In contrast{,} at low temperature (close to 0 [degree]C) the equilibrium shifts dramatically toward the more stable HH1 conformer (for the (cis-1{,}4-DACH)Pt(d(TGGT)) adduct the HH1 conformer is always dominant{,} even at high temperature). Notably{,} (cis-1{,}4-DACH)PtCl2 (Kiteplatin) has been recently reinvestigated and found to be particularly active against colorectal cancer (including oxaliplatin-resistant phenotypes).

The initial aim of the present work was the synthesis of the axial disuccinato Pt(IV) derivative of [PtCl2(cis-1,4-DACH)] (Kiteplatin, 1 in Figure 1 ) (DACH = diaminocyclohexane), which contains an isomeric form of the diamine ligand present in oxaliplatin (i.e., 1R,2R-DACH). The interest in this compound stems from its activity on several cisplatin and oxaliplatin-resistant cell lines. Oxidation of 1 with hydrogen peroxide affords cis,trans,cis-[PtCl2(OH)2(cis-1,4-DACH)] (2) which was treated with succinic anhydride in suitable solvents. To our surprise, in dimethylformamide (DMF) (50-70 °C or under light irradiation) or in dimethylsulfoxide (DMSO) (under light irradiation) the formation of the succinato complex cis,trans,cis-[PtCl2{OC(O)CH2CH2C(O)OH}2(cis-1,4-DACH)] (3) was accompanied by reduction to 1. It was found that solvolysis of 2 and formation of a μ-oxo dinuclear species (5) is the key step. The dinuclear species can then undergo reduction to a 1:1 mixture of 1 and 2 with concomitant elimination of oxygen (1/2 O2 in the form of H2O2). The whole process is fostered by heat and/or light, which could favor solvolysis of 2 as well as decomposition of hydrogen peroxide to water and oxygen so preventing the reoxidation of 1 to 2. Because of its peculiar behavior, compound 5 could be exploited also for the development of a technology for water splitting.

Although the encouraging antitumor activity of [PtCl(2)(cis-1,4-DACH)] (1; DACH = diaminocyclohexane) was shown in early studies almost 20 years ago, the compound has remained nearly neglected. In contrast, oxaliplatin, containing the isomeric 1(R),2(R)-DACH carrier ligand, enjoys worldwide clinic application as a most important therapeutic agent in the treatment of colorectal cancer. By extending the investigation to human chemotherapy-resistant cancer cells, we have demonstrated the real effectiveness of 1 in circumventing cisplatin and oxaliplatin resistance in LoVo colon cancer cells. The uptake of compound 1 by the latter cells was similar to that of sensitive LoVo cells. This is not the case for all other compounds considered in this investigation. Interaction with double-stranded DNA, investigated by a biosensor assay and by quantum mechanical/molecular mechanical geometry optimization of the 1,2-GG intrastrand cross-link, does not show significant differences between 1 and oxaliplatin. However, the DNA adducts of 1 are removed from repair systems with lower efficiency and are more effective in inhibiting DNA and RNA polymerase.