WO2010015590A1

WO2010015590A1 - Organotin(iv) complexes with n-acetylcysteine possessing antitumoral activity, process for their production and their use

Info

Publication number: WO2010015590A1
Application number: PCT/EP2009/060006
Authority: WO
Inventors: Claudia Pellerito; Cristina Prinzivalli; Ornella Pellerito; Michela Giuliano
Original assignee: Universita' Degli Studi Di Palermo
Priority date: 2008-08-04
Filing date: 2009-08-03
Publication date: 2010-02-11
Also published as: ITRM20080426A1; IT1392832B1

Abstract

Organotin(IV) compounds with N-acetylcysteine having the general formula (1 ) o (2): wherein: R₁, R₂ and R₃ are each independently selected from the group consisting of : H, an alkyl, alkoxyl, alkenyl or alkynyl group with up to 12 carbon atoms, an isocyclic or heterocyclic aromatic or aralkyl group with up to 12 carbon atoms, and they are preferably chosen from methyl, ethyl, butyl or phenyl, are synthetized by simple procedures from commercial products, and are active as anticancer agents. In particular, the Bu₂Sn-N-acetylcysteinate derivative was found to have a potent and selective anticancer activity in vitro against several cancer cell lines.

Description

ORGANOTIN(IV) COMPLEXES WITH N-ACETYLCYSTEINE POSSESSING ANTITUMORAL ACTIVITY, PROCESS FOR THEIR PRODUCTION AND THEIR USE

Field of the invention The present invention relates to novel organotin(IV) derivatives showing antitumour activity, a process for their production and their use in medicine. In particularl, the invention relates to the synthesis and the study of organotin(IV) derivatives with N-acetylcysteine (NAC) which exhibit excellent selective antitumoral in vitro activities against several cencer cell lines and particularly human hepatocarcinoma HepG2, thus their are suggested for producing drugs for tumors treatment, both alone and mixed with other chemotherapeutic agents. State of the art

Several inorganic salts and metal complexes are used in medicine for many therapeutic applications, but only two metal based complexes have been really successful in clinical practice as antitumoral agents: the cis- diamminedichloroplatinum(ll) (cis-platin) and cis-diammine-(1 ,1 - cyclobutanedicarboxylato)platinum(ll) (carboplatin). The first one was synthesyzed for the first time by Michele Peyrone and pubblished in 1844 and its tumor growth inhibition properties were discovered only in 1965. The second one, a second generation cis-platin analogous (patent US 4140707, published in 1979 to the Research Corp.), obtained Food and Drug Administration approval in 1989 for second-line treatment of ovarian carcinoma.

Some tumors developed immediately resistance to the mentioned compounds that, in addition, show severe side effects, as nefrotoxicity and hematotoxicity, as well as ototoxicity. Particularly in the last period, the research in metal-based antitumorals derivatives was focused on treatment of tumors resistent to treatment with platinum derivatives. In this concern, very interesting examples are anticancer titanium compounds acting against tumors in the gastrointestinal tract, whereas platinum antitumour derivatives are ineffective towards the same tumors.

In the last years, one of the major developments in the field of metal-based antitumour complexes, that have been synthesized and studied, is the focus on organotin(IV) complexes, wherein the metal acceptor is coordinated by a biological organic ligand, containing in particular sulphur ligands, carboxylates, or amine groups.

Organotin(IV) compounds, which are generally very toxic, even at low concentration, starting from the '50s years of the XX century they were used for industrial applications at first as catalyst in PVC then for agricultural applications as fungicides and miticides. One of the major and widespread application of organotin(IV) compounds is the use as active ingredients in antifouling marine paint, for preventing biological growths on submerged surfaces in fresh and sea water.

It is well known that some organotin(IV) derivatives display antitumoral activity. First results date back to the 1972, when Ph₃Sn acetate was evaluated for its inhibitory effects in tumour growth. It is also known that the biological activity is essentially determined by the number and nature of the organic group bound to the central Sn atom and that di-organotin(IV) derivatives show the highest antitumor activity combined with the lowest mammalian toxicity, while tri- organotin(IV) derivatives are generally very toxic.

Many organotin(IV) compounds were assayed against several cancer cell lines and there are several publications in this topic. Probably adducts of the type R₂SnX₂L₂ are the most studied organotin compounds (X= halogen, pseudohalogen; L= ligand containing O, N donors, R= ) but also fluorinated derivatives, bis(stannyl) methanes, compounds containing R₂SnO group, derivatives with carboxylic acids, aminoacids, purines, pyrimidines, and peptides. Some examples of organotin derivatives suggested as antitumoral drug are disclosed in the patent publications EP 0484596 A1 and EP 0677528 A1 (Gielen et al., both with Pharmachemie B. V. as applicant), and EP 0538517 A1 (Boualam et al., Pharmachemie B. V.).

The above-mentioned R₂SnX₂L₂ derivatives show a biological activity whose mode of action differs from that of the similar cis-diammineplatinum(ll) family drugs from which they have been designed.

The c/s-diamminedichloroplatinum(ll), characterized by a Cl-Pt-Cl- bond angle less then 95°, is active in vivo only after that the chloride ion is displaced; that allows the platinum to react with DNA nucleobases. This mechanism of action is theoretically also possible for diorganotin(IV) derivatives, and in fact it is known that organotin(IV) form adducts with purines and pyrimidines. Actually crystallographic data for both active or inactive R₂SnX₂L₂ derivatives revealed an average Cl-Sn-Cl bond angle less then 103° which may implicate an activation mechanism similar to that of cis-diammineplatinum(ll).

It has been revealed that R₂SnX₂L₂ containing N-donor ligands and showing an average Sn-N bond lengths > 239 pm are active complexes, whereas complexes with stronger Sn-N bond lengths < 239 pm are inactive. This implies that the predissociation of N donors groups is more critical then the loss of halogen ions. Recent work demonstrated that the occurrence of relatively stable ligand— Sn bonds, and a low hydrolytic decomposition of Sn-N bond, allow the ligand to interact with the active site of the target before the R₂Sn²⁺ toxin is released. With regard to the data published on new antitumoral agents, in the last years great attention is focused to the study of apoptosis as process that may represent a therapeutic strategy if opportunely activated.

As it is well known, apoptosis is a physiological process of cell death, which is also called "programmed cell death" or PCD because responds to a genetically determined program that is carried out actively by the cells and that requires energy, gene transcription and protein synthesis. Apoptosis, with proliferation, differentiation and cell migration, is a molecular program which is involved in the regulation of the size of tissues and in the maintenance of cell number and cellular homeostasis.

The apoptotic mechanism is also activated as a defense mechanism to protect the integrity of organisms when the cells are subjected to different damages. Thus prevents the propagation of cell population with abnormal genetic and irreversible damages. Apoptosis has biochemical and morphological aspects different to necrosis, another type of cell death. Necrosis does not occur through a specific genetic program, unlike apoptosis which is also called "clean death"; it is not a private event that affects only the cell where it turns on, but necrosis extends to many cells in a diffuse inflammatory event that culminates with the swelling of cellular organelles and cell lysis. During apoptosis, a sequence of events which are common to different cell types occurs and remain constant over the course of phylogenesis. This process begins with a rapid morphological cell transformation, consisting in the reduction of cell volume, cytoskeleton modification, loss of contact between the cells and their separation from original tissue. In the nucleus, the nuclear lamina is degraded and the chromatin is condensed and fragmented and then move to the perinuclear region. Thus the formation of vesicles containing chromatin occurs, which move towards the plasma membrane and are surrounded by the evagination of a plasma membrane. The apoptotic cell takes a look as bubbles, commonly defined blebbing. Exposure of phosphatidylserine on the outer leaflet of the cell membrane close to the cells indicates that it is ready to engulf. In the next step, the cells form spheroidal subunits surrounded by membranes known as "apoptotic bodies" which, released into the environment, disappear through the process of phagocytosis; thus there is not release of cytosolyc content in the environment and therefore does not rise to any secondary flogistic process.

Many drugs which are currently used in anticancer therapies act by activating cytotoxic death by apoptosis. The goal of anticancer research is the design of drugs capable of inducing preferentially apoptosis in tumoral cytotypes without affect non-tumoral cells, and to identify the mechanism of activation of apoptosis and the factors associated with it.

The study of molecular mechanisms of apoptosis has identified two different pathways through which the programmed cell death is achieved: the extrinsic pathway and the intrinsic pathway. Both pathways require the intervention of several proteins, some with pro-apoptotic function that will be activated during the process and others with anti-apoptotic function that will be inhibited. Many of these proteins can be grouped into two broad families: caspase proteins and Bcl-2 family.

The caspases are a family of cytosolyc cysteine proteases which are synthesized in proenzimatic form and then are activated by proteolytic cleavage at a specific aspartate residue within a cascade through which functional enzymes are generated. The caspases can be separated into two functionally different groups: the early caspases, as the caspases 2, 8, 9 and 10, and the effector caspases, as the caspases 3, 6 and 7. The caspase activation may occur not only by proteolytic cleavage but also through association with regulatory proteins, such as the pro-caspase 9. The effector caspases act during the final phase of apoptosis on the level of nuclear lamina, ICAD (inhibitor of caspase activated DNase) and PARP (poly-ADP-ribose polymerase).

The activation of extrinsic pathway is consequent to the linkage of several proteins such as FasL or TNF to death receptors of TNF family (for example Apo/Fas/CD95, TNF-α, DR4 and DR5) which are localized on cell membrane and mediate various physiological responses such as inflammation, proliferation, antiviral activity and cellular death. The receptor-ligand binding induces the receptor activation, that form trimers and contact, specific cytoplasmatic proteins (for example FADD) through the intracellular portion and this culminates with the activation of caspases cascade.

The intrinsic pathway involves the mitochondria and is activated by intracellular events (for example DNA damage, oxidative stress) and also by extracellular events (cytotoxic compound administration, ionizing radiation). These signals converge on mitochondria and cause the dissipation of mitochondrial membrane potential, release in cytosol of cytochrome-c which activates caspase-9 into apoptosome and consequently the executive caspases. It is known that the dissipation of mitochondrial membrane potential may occur as a result of cytoplasmatic ROS (reactive oxygen species) accumulation, after strong oxidative stress, often associated with the mechanism of action of several compounds. During apoptotic activation of mitochondria, several factors of Bcl-2 family are commonly involved. These factors are divided into pro-apoptotic (Bax, Bak, Bok, Bad, Bim, BcIXs, Bid, Noxa, Puma) and anti-apoptotic (Bcl-2, BclX_L, Mcl-1 ) members. The anti-apoptotic factors moves from the cytosol, where they are inactive, to the mitochondria and then they are activated through different mechanisms. In the outer mitochondrial membrane, the pro-apoptotic members facilitate the release of cytochrome-c through the formation of channels and compete with the anti-apoptotic factors, which are opposed to these events.

This invention relates to anti-tumor organometallic complexes, and specifically to organotin(IV) compounds with ligands of biological origin, said compounds inducing apoptosis in tumoral cells, promising as selective drug for cancer therapy. The above compounds are particular interesting for use in the treatment of hepatocellular carcinoma, colorectal cancer, leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. Hepatocellular carcinoma (HCC) is a tumour that develops slowly both in cirrhotic and healthy liver and it is one of the most frequent tumours in the world with unfavourable prognosis: it is estimated that the mortality rate associated with HCC is more then 90%. At this stage, liver resection represent the only radical treatment of HCC. Liver transplantation needs a patient selection: small and liver limited disease without secondary tumours or metastasis are the fundamental requirements. Other palliative and controversial treatments are radiotherapy, that seems to be palliative and controversial, and systemic chemotherapy, using the drugs combination of fluorouracil (5-FU), adriamycin and doxorubicin. Hepatic arterial (HA) infusional chemotherapy possesses a number of constraints not found in systemic chemotherapy and it is complementary to i.v. drug administration. The drug used should have activity in a dose-responsive way without significant hepatic toxicity. Selective drug administration in the hepatic artery allows the delivery of active agents at a higher local concentration while it reduces the peripheral toxic effects of the cytotoxic drug.

The concept of arterial injected metabolic radiotherapy is based on the liver's double vascularization (hepatic artery, portal vein) and on the fact that HCCs are hypervascularized tumours which are mainly fed by the hepatic artery while 80% of the liver unaffected by the tumour is vascularized by the portal vein. Hence by injecting these therapeutic agents intra-arterially we can obtain a high "tumour/unaffected liver" ratio which enables us to administer a considerable tumour targeted dose, preserving the healthy part of the liver as long as possible. The recent advances in imaging technology allow a more selective approaches, producing selective ischemia within the tumour while inducing decreased collateral damage to the tumour free liver parenchyma. That interventistic radiological procedures includes chemoembolization and alcoholization, suitable for cirrhotic patients or for patients with unreasectable HCC with large or multiple lesions.

Those regional therapies inhibit tumor growth, and cause the destruction of small tumor nodules but there are not radical cures so there are still palliative approaches. Recently another regional treatment have been proposed: the radioembolization with arterially injected Lipiodol 1-131 (radioactive iodine) instead of cytotoxic drugs as in chemoembolization.

Colorectal cancer is the third most common form of cancer and the second leading cause of cancer-related death in the Western world and most commonly it spreads to the liver.

There are many therapeutic protocols for later stage colorectal cancer treatment and for hepatocarcinoma: one regimen involves the combination of infusional 5- fluorouracil (5-FU) and leucovorin (LV), methotrexate (toxic for retina), epirubicine, paclitaxel (Taxol, Patene), daunorubicin (cardiotoxic), irinotecan or oxaliplatin. The latter is the complex (R,R)-1 ,2-diaminocyclohexane(ethanedioate-0,0) platinum; its cytotoxicity results from inhibition of DNA synthesis by causing crosslinking of

DNA. When oxaliplatin is administered mixed with 5-FU/LV, usually the 5-FU toxicity increases, with following symptoms diarrhoea, myelotoxicity, mucosity.

It is clear that current drugs used in chemotherapy are toxic but not selective. For example, the 5-FU is an antimetabolite that causes leucopenia, platelets decreasing, hepatotoxicity and pulmonary toxicity, cardiotoxicity, and neurotoxicity.

Also oxaliplatin is toxic for SNC, hematopoietic system, gastrointestinal tract and allergic reactions can occur.

Against progress in over the past decade has led to a major improvement in survival rate in patients with colorectal cancer with hepatic metastasis, however it is still necessary to better evaluate the efficacy of intra-arterial infusion, the landmark of second-line treatments and post-operative adjuvant treatments. It is also necessary to design new selective antitumoral agents.

Summary of the invention This invention provides novel organotin(IV) complexes with the organic ligand N- acetylcysteine, a N-acetyl derivative of the natural occurring sulphur-containing amino acid L-cysteine, of formula:

The compounds of the invention surprisingly show a strong anti-tumour activity, resulting from in vitro cytotoxicity studies on several cancer cellular lines. The compound of the invention showed excellent antitumoral activity in vitro towards hepatoblastoma cells (HepG2), leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney cell lines. In case of hepatoblastoma cells the in vitro cytotoxicitywas also corroborated by a cytofluorimetric analysis of the cell-cycle-dependent DNA content distribution to probe several aspects of drug-induced cytotoxicity, like apoptosis and by other biological studies described later.

The organic ligand used in organotin(IV) complexes, provided by this invention, is a low molecular weight cysteine derivative showing antioxidant activity both in vitro and in vivo, acting as free radicals or hydrogen peroxide scavenger by reduction. The N-acetylcysteine (NAC) is a drug commonly used as a mucolytic agent in the management of acute or chronic bronchopulmonary diseases.

Studies concerning this invention showed that new synthesized organotin(IV) complexes with N-acetylcysteine induce apoptosis in HCC HepG2 cells, while they are inactive towards non tumoural cells used as control (Chang liver cells). Hepatocellular carcinoma (HCC) is one of the most frequent tumours in the world, being the 90% of all malignant liver cancer and furthermore it has been proved that the incidence of HCC is increasing remarkably in many countries in the last decades. It is estimated that the incidence of HCC reaches 3-6-% of solid tumors in USA and Europe, and up to 20-40 % in Africa and South-East Asia. In Caucasian populations in industrialized countries and in Japan the hepatocarcinoma HCC is mainly associated with C or B virus cirrhosis (70-80% of patients); usually post hepatic cirrhosis, less frequently alcoholic cirrhosis or secondary to hemocromatosis. Human hepatoma HepG2 cell line is characterized (from a biomolecular point of view) by an alteration of β catenin gene, caused by a point mutation or a deletion of exons 3 and 4 (aa 25-140). This deletion removes the potential site of regulation by glycogen synthase kinase 3 β (GSK-3β) responsible for its phosphorylation. The lack of phosphorylation prevents the ubiquitination and its degradation by the proteasome. Accordingly, β-catenin accumulates in the cytoplasm and moves into the nucleus where, after the association with the transcription factor TCF/LEF, activates genes involved in cell cycle progression and protection from apoptosis. β catenin alteration can contribute to the deregulated proliferation of hepatoma cells, but also to the acquisition of resistance to apoptosis. HepG2 cells represent, therefore, a good hepatoma model to study the biochemical mechanisms which are involved in the apoptotic mechanism. The hepatic Chang liver cells, employed as non tumoral cell model, are immortalized cells in active proliferation, as beyond the genetic program of senescence. This feature allows the maintenance in culture. According to this invention, a specific class of organotin(IV) derivatives of N- acethyl-L-cysteine, that could be synthesized with high yields and low costs by simple reaction of commercial reagents, wherein there are two or three organic groups linked to the tin, show excellent anti-tumor activity inducing apoptosis; at the same time compounds exhibit higher selectivity and lower toxicity then those of the corresponding organotin(IV) parents. In detail, this invention provides diorganotin(IV) and triorganotin(IV) complexes with N-acethylcysteine which exhibit high toxicity against human hepatocarcinoma cell lines, but they are less toxic, being some of them not toxic, towards normal human hepatocyte cell line. They are all also less toxics then the corresponding organotin(IV) parents used as reagents for their synthesis. The toxicity is time and concentration dependent: in detail it seems that biological ligand, constituting the complexes, modulates the toxic effects of the organometallic moiety. The cell toxicity occurs by apoptosis induction, as it has been showed and confirmed by biochemical study on caspases expression. As it will be described later in detail, the activity of the compounds concerning this invention has been demonstrated in vitro by MTT cytotoxicity test on HepG2 hepatoblastoma cell line, and it has been confirmed subsequently by a cytofluori metric analysis of the cell-cycle-dependent DNA content distribution to quantify apoptosis, by Western blotting for probing caspases involvement in apoptosis induced by organotin(IV) compounds, by studying the level expression of PAR4 and pAkt factors, as well as by ROS production evaluation. The use of organotin(IV) derivatives disclosed in this invention could represent a targeted specific anticancer treatment with high selectivity, and affecting poorly the physiology of normal cells.

The compound of the invention showed excellent antitumoral activity in vitro also towards leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney cell lines.

It should be pointed out that although some organotin(IV) complexes with N- acethylcysteine disclosed in this invention were previously described from a chemical point of view (i.e. G. Domazetis et al., in Inorg.Chim.Acta, (1979), Vol.32,L48-L50; G. Domazetis et al., Tri-n-butyltin(IV) derivatives of L-cysteine ethyl ester, N-acetyl-L-cysteine and α-glutamylcysteinyl glicine (Glutathione reduced), J. Organometal.Chem., 173 (1979) 357-376), there have not been ever reported so far biological or pharmacological activities or properties of these compound, and least of all their use as selective antitumoral agents for hepatic and colorectal diseases. Detailed description of the invention

Thus the invention relates to organotin(IV) complexes with N-acetylcysteine of the general formula (1 ) or (2) :

wherein:

R₁₅R₂ and R₃ are each independently selected from the group consisting of: H, an alkyl, alkoxyl, alkenyl or alkynyl group with up to 12 carbon atoms, an isocyclic or heterocyclic, aromatic or aralkyl group with up to 12 carbon atoms, for use as medicinal active ingredients.

In details, as remarked, these active agents are antitumoral agents, and more in detail they are suitable for hepatic and colorectal cancer. According a preferred embodiment of the invention, R-ι,R₂ and R₃ (when present) are each independently selected from the group consisting of: H, an alkyl group with up to 8 carbon atoms, an alkoxyl group with up to 8 carbon atoms, a phenyl group.

Either among diorganotin(IV) complexes with N-acetylcysteine either among triorganotin(IV) complexes with N-acetylcysteine disclosed for medical use according to this invention and having respectively the general formula (1 ) and (2), preferred compounds are those wherein R₁, R₂ and R₃ (when present) are each independently selected in the group consist in methyl, ethyl, propyl, butyl and phenyl groups. Among diorganotin(IV) derivatives of general formula (1 )

wherein R₁ and R₂ are each independently selected from the group consisting of: H, an alkyl, alkoxyl, alkenyl or alkynyl group with up to 12 carbon atoms, an isocyclic or heterocyclic, aromatic or aralkyl group with up to 12 carbon atoms, that are not described in literature for their putative use as antitumoral agents selective for hepatic and colorectal cancer, particularly preferred is dibutyltin(IV) acetylcisteinate complex, of formula (1 a):

Compound of formula 1 a showed the highest antitumoral activity in vitro among all compounds tested within the contest of this invention.

According to the synthetic procedure provided for producing compounds of the present invention, organotin(IV) complexes with N-acetylcysteine of general formula (1 ) and (2) can be synthesized by reaction of N-acetylcysteine and the corresponding organotin(IV) precursors. Further object of the present invention is a process for producing organotin complexes with N-acetylcysteine with general formula (1 ) or (2), as above described, said process including reacting an organotin(IV) derivative wherein R₁, R₂ and R₃ (when present) have the meaning above already said, with N- acetylcysteine in methanol, chloroform or water, in a temperature range comprised between 10 °C and 90 °C, preferably between 25 °C and 75 °C. Preferably, at the end of said the reaction, the reaction mixture is quenched by cooling, and the resulting precipitate containing the desired compound of formula (1 ) or (2) is collected by filtration.

According to this inventions when the aim of the process is the production of organotin(IV)complexes with N-acetylcysteine with general formula (1 ) or (2), that includes preferably the reaction between organotin(IV)derivative with N- acetylcysteine in refluxing methanol. Preferably, at the end of the reaction described, the reaction mixture is quenched by cooling, and the resulting precipitate containing the desired compound of formula (1 ) or (2) is collected by filtration.

Further aspect of the invention is the use of one ore more compounds having general formula (1 ) or (2), as above described, for the production of a pharmaceutical preparation, in detail an anticancer preparation, and preferably as active ingredient in an anticancer preparation for the treatment of hepatic and colorectal tumours as well as for leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. According to the invention, the compounds above described can be advantageously used in cancer therapy, administered in a pharmaceutical preparation as toxic agents by themselves or in combination with other chemotherapeutic agent for enhancing the therapeutic effects, and for reducing pharmacoresistences. This invention relates also to a pharmaceutical preparation, including as active component at least one of the organometallic complexes with N-acetylcysteine with general formula (1 ) or (2) together with one ore more pharmaceutically acceptable adjuvants and/or vehicles. In an even more preferred embodiment, the preparation contains as active component dibutyltin(IV) -N-acetylcisteinate complex, of formula (1 a). According to this invention, the pharmaceutical preparations containing organotin(IV) derivatives with N-acetylcysteine suitables for the therapeutical administration can be designed using conventional methods already known by expertises, by using adjuvants and vehicles pharmaceutically acceptable, for obtaining preparations suitable for parenteral injection (intravenous, intramuscular or subcutaneous), for oral administration of solid or liquid formulations, for transdermal, rectal, intravaginal or topical administration, for instance in oronasal mucosa and similar.

The percentage of the active compound in the preparation and the method of tumour treatment could be changed for reaching the optimal dosage. The dosage to administer depends on: route of administration, length of treatment, patient conditions and weight, drug effectiveness and patient reaction. As soon as the right dosage of active compound has been determined, the formulation and the choice of adjuvants and vehicles pharmacologically suitable for each need will be made according to the current knowledge in this field. The specific characteristics of the invention, as well as the advantages either for the methods for synthesizing the suggested compounds, either for their biological and pharmacological activity, will be remarked afterwards or in the detailed illustrative description and experimental results related to this invention. Brief description of the figures

Some experimental results are illustrated in the enclosed pictures, where: the figures 1A and 1 B show the effects induced by the compound Bu₂SnNAC, according to the invention, in comparison with Bu₂SnCI₂ and NAC, on hepatic HepG2 and Chang liver cell viability, respectively after 8 h (A) and 16 h (B) of treatment; the figure 2 shows the influence of NAC on the cytotoxic effect induced by Bu₂SnCI₂ in HepG2 cells according to the invention; the figure 3 shows the involvement of caspases in apoptosis induced by Bu₂SnNAC complex, according to the invention, compared with Bu₂SnCI₂, in HepG2 cells; and the figure 4 shows the effects of the Bu₂SnNAC complex, according to the invention, and the others used for a comparison, on the levels of PAR4 factor and the survival protein pAKT. the figures 5A and 5B show the effect of the compound NAC2 against 60 cancer cell lines at a single dose of 10μM. Experimental section

As already remarked, in what is concerning the synthesis, all the organotin(IV) derivatives N-acetylcysteine described later have been obtained starting from reactants and solvents reagent grade and commercially available. In the experimental chemical session after described, elemental analysis have been carried out at the Department of Inorganic and Analytical Chemistry "S.Cannizzaro" of the University of Palermo, by a Vario EL III (Elementar GmbH) analyzer instrument using the "purge and trap" method, performing double analysis and evaluating the results as average of the two analysis. The FT-IR spectra of the free ligand and of its organotin(IV) complexes were registered, both Nujol mulls and hexachlorobutadiene, on a Mod. Spectrum ONE Perkin-Elmer FT-IR with the appropriated equipped software , between CsI windows, performing 8 scan with a 4 cm^"1 resolution.

The ¹¹⁹Sn Mόssbauer spectra were recorded by maintaining the source at room temperature and by cooling the sample, as fine powder pressed between two aluminium foil, at the temperature of 77.3±0.1 K, using a liquid nitrogen cryostat NRD-1258-MB (Cryo Industries of America inc., Atkinson, NH, USA) and using an ITC 502 Oxford Instrument temperature controller to obtain the temperature control. The multichannel calibration was performed with an enriched iron foil (⁵⁷Fe = 95.2%, thickness 0.06 mm, Ritverc GmbH, St. Petersburg, Russian Federation) at room temperature by using a ⁵⁷Co (10 mCi) source (New England Nuclear, Amersham) while the zero point of the Doppler velocity scale was determined at RT through the absorption spectrum of natural CaSnO3 ([¹¹⁹Sn] = 0.50 mg cm-2 ) as source.

The ¹¹⁹Sn Mόssbauer spectra were recorded in duplicate with a model 209 TAKES (Ponteranica, Bergamo, Italy) multichannel analyzer (4096 channels) and the following MWE (Munchen Wissenschaftliche Elektronik, Gmbh, Starnberg, Germany) apparatus: MR250 driving unit, FG2 digital function generator (moving at linear velocity and constant acceleration in a triangular waveform. )and MA250 velocity transducer, In each point of spectrum velocity , a sufficient number of counts were collected to obtain a well defined spectrum. Data so collected have been elaborated by a personal computer, using appropriate calculations software.

For the solution phase, the complexes have been investigated at 296 K by ¹H,

¹³C(¹HJ and ¹¹⁹Sn(¹HJ NMR using a Bruker AVANCE DRX300 NMR spectrometer, with "probe broad-band" and "inverse detection" respectively, at 300.13, 75.5 and

118.9 MHz. The signals of the solvents were used as internal standard for ¹H and

¹³C(¹HJ spectra, and as external standard the ¹³C signal of TMS (¹³C d= 0.00). For the ¹¹⁹Sn(¹HJ spectra a neat liquid solution of tetramethylstannane (Me₄Sn) was used as external reference (¹¹⁹ Sn d=0.00). Molecular geometries were optimized at the density functional level of theory using the software packages Gaussian '03 (G03) ¹¹² and Turbomole. DFT calculations were performed by the hybrid B3LYP method and using the double-zeta valence plus polarization (DZVP) all electron basis set 6-31 G^** (H, C, O, S). The calculations were performed using density functional theory (DFT). The calculation of chemical shift and coupling constants values were fully optimised at DFT level using the mPW1 PW91 functional with the same basis set.

EXAMPLE 1

MepSnNAC complex synthesis

N-acetylcysteine (NAC) (1 mmol,163.18 mg) was dissolved in 30 ml of methanol or water, then added slowly to a dimethyltin(IV) oxide (Me₂SnO) solution (1 mmol, 164.78 mg), under stirring. After 24 hours at range 25-75 °C, the white solid derivative was recovered after filtration under vacuum and vacuum drying on

P₄O₁₀.

White solid obtained was study by FT-IR analysis. The IR spectrum showed that the reaction proceeded with total conversion of the starting material, as well as all compounds presented as further examples in Table 2.

Analytical data of the obtained compound are reported later in Table 1.

EXAMPLE 2

B UgSn N AC complex synthesis N-acetylcysteine (NAC) (163.18 mg, 1 mmol) was dissolved in 30 ml of methanol or water, then added slowly to a dibutyltin(IV) oxide (Bu₂SnO) solution (1 mmol

,248.93 mg), under stirring. After 24 hours at range 25-75 °C, the white solid derivative was recovered after filtration under vacuum, ricristalyzed from chloroform and vacuum dried on P₄O₁₀₎.

White solid obtained was study by FT-IR analysis. The IR spectrum showed that the reaction proceeded with total conversion of the starting material, as well as all compounds presented as further examples in Table 2. Analytical data of the obtained compound are reported later in Table 1.

EXAMPLE 3

PhgSnNAC complex synthesis

The Ph₂SnNAC complex was obtained as white solid, mixing a diphenyltin(IV) oxide (Ph₂SnO) aqueous suspension and a N-acetylcysteine aqueous solution, in molar ratio1 :1 .

This reaction allowed the complex formation, without unreacted reagents or secondary products.

N-acetylcysteine NAC (163.18 mg, 1 mmol) was dissolved in 30 ml of distillate water, then added slowly to a diphenyltin(IV) oxide (Ph₂SnO) aqueous suspension

(1 mmol ,288.92 mg), under stirring. After 24 hours at range 25-75 °C, the white solid derivative was recovered after filtration under vacuum , and vacuum dried on

P₄O₁₀ P₂O₅).

EXAMPLE 4

(Me₃Sn)^NAC complex synthesis The (Me₃Sn)₂NAC complex was obtained as white solid, mixing a trimethyltin(IV) hydroxide (Me₃SnOH) and a N-acetylcysteine methanolic solutions, in molar ratio

2:1 .

This reaction allowed the complex formation, without unreacted reagents or secondary products. N-acetylcysteine NAC (163.18 mg, 1 mmol) was dissolved in 30 ml of methanol and then added slowly to a trimethyltin(IV) hydroxide (Me₃SnOH) methanolic solution (2 mmol, 180.82 mg), under stirring. After 24 hours at range 25-75 °C, the solution volume was reduced to 10ml by a rotating evaporator.

The reaction mixture was quenched at fridge temperature, and after 24 hours the resulting white desired precipitate was collected by filtration, washed with chloroform and then vacuum dried on P₄Oi₀ . White solid obtained was study by FT-IR analysis. The IR spectrum showed that the reaction proceeded with total conversion of the starting material, as well as all compounds presented as further examples in Table 3. Analytical data of the obtained compound are reported later in Table 1.

EXAMPLE 5 (BUc₁Sn)₉NAC complex synthesis

The (Bu₃Sn)₂N-acetylcysteinate complex was obtained as gel mixing a tributhyltin(IV) hydroxide ((Bu₃Sn)₂O) and a N-acetylcysteine methanolic solutions, in molar ratio 2:1.

N-acetylcysteine NAC (163.18 mg, 1 mmol) was dissolved in 30 ml of methanol, then added slowly to a tributhyltin(IV) hydroxide ((Bu₃Sn)₂O methanolic solution (1 mmol; d=1.171 g/ml; 0.51 ml), under stirring. After 24 hours at range 25-75 °C, the solution volume was reduced to 10ml by a rotating evaporator. The reaction mixture was quenched at fridge temperature, but after 24 hours no solid was formed. The solution was dried by a rotating evaporator. The resulting gel desired was collected and dried on P₄O₁₀

FT-IR analysis of the obtained material correspond to that of the desired compound, as well as for all compounds presented as further examples in Table 3. Analytical data of the obtained compound are reported later in Table 1.

EXAMPLE 6

(Ph₃Sn)₉NAC complex synthesis

The (Ph₃Sn)₂ N-acetylcisteinate complex was obtained as white solid, mixing a triphenyltin(IV) hydroxide (Ph₃SnOH) and a N-acetylcysteine chloroformic or methanolic solutions, in molar ratio2:1.

This reaction allowed the complex formation, without unreacted reagents or secondary products. N-acetylcysteine (NAC) (163.18 mg, 1 mmol) was dissolved in 30 ml of chloroform (chloroform or methanol) , then added slowly to a Ph₃SnOH chloroformic solution (2 mmol; 367.01 mg), under stirring. After 24 hours at range 25-75 °C, the solution volume was reduced to 10ml by a rotating evaporator.

The reaction mixture was quenched at fridge temperature, and after 24 hours the resulting white desired precipitate was collected by filtration, washed with chloroform and then vacuum dried on P₄O₁₀ .

FT-IR analysis of the obtained material correspond to that of the desired compound, as well as for all compounds presented as further examples in Table 3.

Analytical data of the obtained compound are reported later in Table 1.

Table 1

Elemental analysis analytical data [experimental (calculated), (%)] for R₂Sn(IV)NAC (R = Me, n-Bu, Ph) and (R₃Sn(IV))₂NAC derivatives (R = Me, n-Bu, Ph); NAC^2"= [N-acethlcysteinate]^2"

Table 2

Assignment of more relevant absorption bands of N-acetyl-L-cysteine (NAC), of its R₂Sn(IV)NAC (diorganotin) derivatives (AIk = Me, n-Bu, Ph), in the range 4000- 250 cm^"1

* Signals are hidden by those of Ammide II

Table 3

Assignment of more relevant absorption bands of N-acetyl-L-cysteine (NAC), of its (R₃Sn(IV))₂NAC(triorganotin(IV) ) derivatives (R = Me, n-Bu, Ph), in the range 4000-250 cm^"1.

* Signals are hidden by those of Ammide II

¹¹⁹Sn Mόssbauer characterization

By the Mόssbauer spectroscopy, and in detail by the isomer shift parameter, δ, we obtained useful data on the valence state of tin in complexes and on the structure and the bonding in the tin environment. The isomer shift values δ, mms^"1, reflect the meaning of the parameter, increasing their value according to the inductive effects of the organic groups, so they decrease on going from dialkyl to diphenyl derivatives.

In order to verify the correctness of the interpretation, the experimental nuclear quadrupole splitting parameters have been rationalized according to the point charge model formalism. Theorical data of nuclear quadrupole splitting |Δ_Θxp| were calculated by the point-charge model formalism and compared with experimental data.

For the diorganotin (IV) derivatives of formula (1 ) the C-Sn-C angles (θ, deg) were calculated via Eq. (1 ),

where {R} is the p.q.s. of the alkylic and phenylic group in an idealised octahedral configuration (-1.03 mms^"1 and -0.95 mms^"1, respectively) and Δ is the measured quadrupole splitting:

For all compounds, even if the other groups bonded to the tin have not been taken in account, the C-Sn-C angles values were calculated with an error of ± 13°, however the values were useful for evaluating the distorted configuration around the tin atom. The values of the experimental isomer shift δ and of the calculated and experimental nuclear quadrupole splitting [|Δ_Θxp| (mm s^"1) and |Δ_caic| (mm s^"1) ], are summarized in Table 4. In details, the table contains Mossbauer parameters of the organometallic compounds in object, as isomer shift, δ,in mm s \ together with the and experimental nuclear quadrupole splitting [|Δ_Θxp| (mm s^"1)], and the C-Sn-C angles values measured bat the temperature of the liquid nitrogen, and the calculated nuclear quadrupole splitting [|Δ_ca|_C| (mm s^"1) according to the point charge model formalism for idealised tetraedral and trigonal bipyramidal configuration. Furthermore, in table the suggested geometry for synthesized compounds has also been reported: Mossbauer data strongly suggested a trigonal bipyramidal configuration for R₂SnNAC ( R= Me, Bu, Ph) derivatives, where alkyl and phenyl groups lie in equatorial positions.

For derivatives triorganotin(IV) NAC with general formula (2), Mossbauer data indicated more than one environment for the tin atom in these. The obtained |Δ_Θxp| values were in the range of a trigonal bipyramidal and tetrahedral configuration around the tin atom, within the experimental error of ±0.4 mm/s. Table 4

Experimental Mossbauer parameters³, isomer shift δ and nuclear quadrupole splitting |Δ_Θxp| (mm s^"1) measured at liquid N₂ temperature, calculated nuclear quadrupole splittings Δ_caicd, according to the point charge formalism, and C-Sn-C angles(of synthesized compounds)

a Sample thickness ranged between 0.50 and 0.60 mg Sn cm^" ; isomer shift, δ ± 0.03, mms^" with respect to room temperature BaSnOs; Fl and F2 values are the full width at half height of the resonant peaks, respectively, at greater and lower velocity respect to the centroid of the Mossbauer spectra; nuclear quadrupole splittings, |zfexp| ± 0.02 mms ¹. b NAC^2' = N-acethylcysteinate^2'.

Computational studies for R₂Sn complexes with N-acetylcysteine(R = Me, Bu) with general formula (1)

The computational studies are consistent with the experimental data. The geometries of the Me₂SnNAC and Bu₂SnNAC complexes, previously synthetized, were optimized and tvibrational frequencies for the two species were also computed. For Bu₂SnNAC complex,two structural isomers ((A) and (B)) have been found, whose energy values showed that the most stable isomer is the one presenting the sulfur atom in the equatorial position (A), in agreement with the Mossbauer data. The bond angles and lengths have been compared with literature data of the captopril, a ligand that coordinates dimethyltin(IV) unit similarly to the NAC

The vibrational frequencies have been compared with IR data. The good agreement between the frequencies and the calculated and experimental bond angles and length confirmed the structural hypothesis previously suggested. Study of anticancer activity of organotin (IV) with N-acetylcysteine complexes

The activity of organotin(IV) with N-Acetylcysteine complexes according to the invention was evaluated at the Department of Biochemical Sciences of University of Palermo, Policlinico "Paolo Giaccone", by using MTT assay to evaluate their cytotoxicity, cytofluorimetric analysis to identify cell cycle distribution for apoptosis quantification and to assess ROS production, Western blotting analysis to evaluate the involvement of caspases, pAKT and Par4 in apoptotic mechanism induced by the compounds. Cell cultures

Chang liver and HepG2 hepatoma cells were cultured in polystyrene 75 cm² flasks and grown in RPMI 1640 medium supplemented with 10% (v/v) heat- inactivated foetal calf serum, 2.0 mM L-glutamine, 1.0 mM sodium pyruvate and antibiotic antimycotic solution (100 U/ml penicillin, 100 Dg/ml streptomycin and 250 ng/ml amphotericin B) and incubated at 37 °C in a humidified, 5% CO₂ atmosphere, 95% air.

Cell viability was determined after detachment of cells from flask by Trypsin - EDTA solution and seeding of 6.5x10³ Chang liver cells and 10⁴ HepG2 cells (on 200 μl of medium) in 96-well cell culture plates (0.3 cm diameter).

Cytofluorometric studies were carried out seeding 1.5x10⁵ Chang liver cells and 2.0x10⁵ HepG2 cells in 6-well cell culture plates (3 cm diameter), while Western blotting analysis was performed seeding 1 O⁶ CeIIs in Petri dishes (10 cm diameter). Stock solutions of compounds were prepared in dimethyl sulfoxide (DMSO) obtaining a 1 mM solution which was opportunely diluted in culture medium to the desired final maximum test concentration. The final concentration of DMSO never exceeded 0.04%, a concentration beyond which there may be phenomena of toxicity. Control cells were cultured in the presence of vehicle alone. MTT Cell viability assay MTT assay is a quantitative colorimetric assay that employs the 3-[4,5- dimethylthiazolyl-2] 2,5-diphenyl-tetrazolium bromide (MTT) (T Mosmann, J.Immunol. Methods, 1983, 65, 55-63). Tetrazolium salt MTT is cleaved to blue formazan by mitochondrial dehydrogenases of vital cells , which belongs to the mitochondrial respiratory chain. Since this enzymatic system is active only in viable (metabolically active) cells, this assay exclusively detects viable cells. The intensity of staining formed is directly proportional to the number of living cells and is measured by means of spectrophotometer. After the treatments, MTT stock solution was added to each well (final concentration 1 mg/ml) for 2 hours at 37 °C. The medium was then removed and 0.2 ml of lysis buffer (obtained by dissolving 20% (p/v) SDS (sodium dodecilsulphate) in 50% N,N-dimethylformamide, pH 4.7) was added to dissolve the formazan product. Finally, after 16 hours of incubation at 37 °C, the absorbance at 570 nm (test wavelength) and at 630 nm (reference wavelength) was measured using an ELISA microplate reader (OPSYS MR, Dynex Technologies). Lysis; buffer was employed as the reference test to reset the instrument.

Data were the mean ± S. E. of three independent experiments involving triplicate assays. The percentage of viable cells is calculated as percentage of viability compared to untreated control cells by assumpting the value of 100% for untreated cells.

In initial experiments, we evaluated the sensitivity of HepG2 and Chang liver cells to all the complex and organotin derivatives. The results showed that organotin parents were highly toxic to both the two cell lines, while the organotin (IV) complexes, in particular the dibutyltin(IV) N-Acetylcysteinate resulted the most active. Then, to evaluate the sensitivity of hepatic HepG2 and Chang liver cells to dibutyltin(IV)dich\or\de (Bu₂SnCI₂) and d/&ufy/f/n(IV)-N-acetylcysteinate (Bu₂Sn- N-Acetylcysteinate), we carried out time- and dose-dependent experiments. The results were reported in Figure 1A and 1 B. MTT colorimetric assay was employed as previously reported.

Cells were incubated in 96-well cell culture plates in presence of the NAC ligand (control) or of the Bu₂SnCI₂ and Bu₂SnNAC at the mentioned concentrations. After the treatment cell viability was evaluated by MTT colorimetric assay. Data were reported as the percentage of viable cells, calculated as percentage of viability compared to untreated control cells, mean D S. E. of three independent experiments involving triplicate assays. The results indicated that the compounds exert different effects in Chang liver and HepG2 cells

In particular, 10 μM Bu₂SnCI₂ induced after 8 hours of treatment a reduction of HepG2cell viability equal to 53%, against a value of 28% observed in the presence of 10 μM Bu₂Sn-N-Acetylcysteinate. The cytotoxicity increased with the time reaching the maximum after 48 hours of treatment (-90%) with the two compounds employed at the same dose (10 μ M).

Interestingly, the studies showed that both the compounds, Bu₂SnCI₂ and Bu₂Sn N-Acetylcysteinate, had toxic effects on hepatoma HepG2cell line. However, after 8 hours of incubation 10 μM Bu₂SnCI₂ was much more toxic than Bu₂SnN Acetylcysteinate employed at the same dose. This can be explained with the ability of N-Acetylcysteine, conjugated in the complex, to modulate, during the initial phase of treatment (8 hours), the high toxic activity of Bu₂SnCI₂.

In order to exclude a protective effect induced by the presence of the free ligand N-Acetylcysteine in the solution, as hydrolysis product of the complex, and to support the hypothesis of the modulating effect of N-Acetylcysteine, cytotoxicity studies on HepG2 cells pre-treated with 10 μM N-Acetylcysteine for 1 hour were performed and then cells were incubated in the presence of 10 μM Bu₂SnCI₂ for 8 hours. The results are shown in Figure 2. Cell viability was tested using a procedure based on the MTT assay. Values are expressed as percentage of viable cells compared to control.

As indicated in the graph, the toxicity induced by 10 μM Bu₂SnCI₂ added to the cells after the pre-incubation with NAC is significantly greater than that observed in the presence of Bu₂Sn N-Acetylcysteinate complex. Thus, it is possible to conclude that the binding with N-Acetylcysteine significantly modulates the cytotoxic effect of the organometallic portion, probably without that any degradation occurs within the cell.

In immortalized Chang liver cells, after 8 hours of treatment with the compounds, were not observed changes in cell viability and even after longer times of incubation (16 and 24 hours) the death came on the very low values (19%) after treatment with 10 μM Bu₂SnCI₂ and 17% after treatment with 10 μM Bu₂Sn N-Acetylcysteinate. Characterization of apoptosis

Cytofluorometric study of cell distribution along the phases of proliferative cycle: quantification of apoptosis.

Quantification of apoptosis was carried out by staining the cells with propidium iodide (Pl) , a fluorescent dye that intercalates to DNA, according to the protocol of Carollo et al. (M. Carollo, L. Parente, N. D'Alessandro, Oncol. Res., 1998, 10, 245-54). This method allows to evaluate the cell distribution along the phases of proliferative cycle. The presence of a peak of lower fluorescence intensity indicates the presence of cells with fragmented DNA. After treatment with the compounds, cells were resuspended in hypotonic solution of fluorochrome consists in 50 μg/ml propidium iodide, 0.1 % sodium citrate, 0.1 % Nonidet P-40 and 100 μg/ml RNAse, and incubated overnight in the dark at a temperature of 4 °C. Cell distribution along the phases of proliferative cycle was evaluated using Beckman Coulter Epics XL cytofluorimeter and Expo32 software for data processing. Fluorescence emitted from the cells was analyzed as frequency of histograms of individual parameters and the percentage of cellular population located in sub-diploid region (subG0/G1 ) was considered as an indicator of programmed cell death. Data obtained were comparable to those observed with MTT assay. As shown in Table 5 (see below) the reduction of HepG2 cell viability was accompanied with a peak of 59.5% (control 1.4%) of cells in subG0/G1 phase of the cell cycle, indicating DNA fragmentation after 8 hours of treatment with 10 μM Bu₂SnCI₂.

After incubation with 10 μM Bu₂Sn N-Acetylcysteinate, instead, the peak is equal to 2.7%. Prolonging the incubation time up to 16 hours, it was observed that both the compounds, Bu₂SnCI₂ and Bu₂Sn N-Acetylcysteinate, have a similar effect on HepG2 cells, confirming the results observed with cell viability assay. In fact, treatment with 10 μM or 20 μM of both compounds for 16 h induced a significant fragmentation of DNA with about 80% of cells in subG0/G1 phase of cell cycle. Instead after 8 hours of incubation even doubling the concentrations of the compounds (20 μM) was observed the appearance of subG0/G1 peak equal to 65.4% in the presence of Bu₂SnCI₂ while after treatment with Bu₂Sn N- Acetylcysteinate the amount of peak was equal to 27.5%. The compounds showed very modest effects on Chang liver cell distribution along the proliferative phases of the cycle, both prolonging the time of incubation that using increasing concentrations of the compounds; in fact the percentage of cells in subG0/G1 phase did not exceed ever the 10-16%. Table 5

Cytofluorimetric analysis of HepG2 (A) and (B) and Chang liver (C) cells distribution along the phases of the proliferative cycle.

Assessment of ROS production

The production of reactive oxygen species (ROS) was measured using 2', 7'-dichlorofluorescein diacetate (H₂DCFDA), a ROS probe, which is converted to fluorescent compound after the removal of acetate groups by the intracellular esterases and after oxidation dependent on the presence of substantial amounts of ROS. After incubation with compounds, cells were washed gently with warm buffer (37 °C) HBSS/Ca/Mg and incubated at 37 °C for 30 min with 5 μM H₂DCFDA dye. Then cells were detached with trypsin solution, resuspended in buffer and analyzed by flow cytometry using the excitation wavelength of 488 nm and the emission wavelength of 525 nm. Data were analysed using Expo32 software. The percentage of cells showing a higher fluorescence, which reflects ROS production, was determined by comparison with untreated controls.

The study of ROS production showed that 10 μM Bu₂SnCI₂ is able to induce, after 8 hours of treatment, ROS accumulation in HepG2 cells, as demonstrated by the appearance of a peak of fluorescence equal to 84% (vs 8.5% in control cells). Interestingly, in cells incubated with 10 μM Bu₂Sn N- Acetylcysteinate, ROS production is smaller with a peak to fluorescence equal to 47.5%.

Studies on the involvement of caspases in apoptotic mechanism induced by the compounds in HepG2 cells: western blotting analysis Cell lysates preparation

After treatment with the compounds, cells (seeded in 10 cm culture plates) were trypsinized and centrifuged for 20 min at 800 rpm. Then, cells were incubated for 30 min on ice in denaturating RIPA buffer, constituted by 1 % NP-40, 0.5% Na-deoxycholate and 0.1 % SDS, containing a protease inhibitor cocktail (25 μg/ml aprotinine, 1 mM PMSF, 10 mM sodium orthovanadate, 10 mM Sodium- fluoride, 25 μg /ml leupeptine and 0.2 mM Sodium-pyrophosphate). Cells were sonicated three times for 10 sec and protein concentration was evaluated by means of Lowry method (O. H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randali, J.Biol. Chem. 1951 , 193, 265-275). Protein electrophoresis on polyacrylamide gel and blotting on nitrocellulose membrane

Equal amounts of protein samples (60 μg/lane) were diluted in 1 X Laemmli buffer (constituted by 0.0625 M Tris, pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue) (U.K. Laemmli, 1970, Nature, 227, 680- 685), denatured at 90 °C for 5 minm, and subjected to SDS-PAGE in running buffer 1 x (25 mM Tris, 0.1 % SDS, 1.44% glycine, pH 9.0) for about 2 hours at 150 V.

Then, cells were electrotransferred to a nitrocellulose membrane for the detection with specific antibodies. Bands were quantified by densitometric analysis using SMX Image software.

Western blotting analysis indicated that treatment of HepG2 cells with Bu₂SnCI₂ decreased the levels of inactive pro-enzymatic isoforms of caspase-8, caspase-9 and caspase-3. These three proteins mediate, at different levels, signals that converge in the final mechanism of apoptosis. Caspase-8 and caspase-9 intervene in the initial caspase cascade activation and therefore are referred as "initiators", instead caspase-3 acts in the final stage of apoptosis causing the degradation of the nuclear lamina, of ICAD and PARP proteins. On the contrary Bu₂Sn-N-Acetylcysteinate induced the recovery in the levels of caspase- 8, caspase-9 and caspase-3, as shown in Figure 3. This figure illustrates the involvement of caspases in apoptosis induced by Bu₂SnNAC and Bu₂SnCI₂ in HepG2 cells used in the study. Cells were incubated for 8 and 16 h at the indicated compounds concentrations. After treatment, cell lysates were prepared and western blotting was conduced using specific antibody to caspase-8, caspase-9 and caspase-3. The normalization of protein amounts was performed, for each experiment, through actin immunoblotting. The bands were quantified by densitometric analysis using SMX Image software. Experiments performed for the evaluation of caspase-8 indicated that

Bu₂SnCI₂ induced the decrease of the inactive form of protein (57 KDa), but the appearance of the active form of enzyme, indicated as a band at lower molecular weight, was not observed. The decrease of pro-caspase-8 (which was considered as expression of activation in caspase-8) is observed after 8 hours of incubation with the compound and it is more visible after 16 hours. Similar effects were induced also in executioner caspase-9 and caspase-3 proteins.

Treatment with Bu₂Sn-N-Acetylcysteinate, in same experimental conditions, induced the recovery of pro-caspase form levels, compared with Bu₂SnCI₂ parent, supporting the ability of the complex to modulate the apoptotic effect of parent compound.

Mechanism of cellular survival

Recently, it has been demonstrated the existence of an elaborate mechanism of reciprocal regulation between pAKT, one of the most important survival factor, and PAR-4 (Prostate-apoptosis-response-gene-4) proteins. PAR-4 protein is a product of pro-apoptotic gene which induces apoptosis in several tumor cytotypes. This function is induced after phosphorylation in Thr163 by PKA with the consequent migration to the nucleus, where it is able to induce the expression of factors that promote apoptosis (for example through FasL-Fas- FADD-caspase axis) and inhibit the expression of factors which are opposed to it (for example Bcl2 and NF-kB proteins). The inhibition of pro-apoptotic action of PAR-4 occurs as a consequence of the phosphorylation on the residue Ser 249 by pAKT protein, which sequesters it in the cytoplasm. PAR-4 also appears to modulate pAKT through PTEN activation, a fosfatydilinositol-3-phosphate phosphatase which has a strong inhibitory effect on PI-3 kinase by reverting the phosphorylation status and determine accordingly the lack of activation of survival factor p A KT Effect of compounds on the levels of pAKT factor.

To evaluate the involvement of pAKT, one of the most important survival factor and PAR4 in apoptotic pathway induced by the compounds, first were performed experiments to assess the effect of the compounds on pAKT levels.

In particular, we wanted to assess whether the different effect of the two compounds observed after 8 hours of treatment was due to different mechanisms which are induced within the cell in an early phase of treatment in attempt to escape from apoptotic mechanism.

The results of the experiments are shown in Figure 4. The cells were incubated with the indicated concentrations of compounds for 8 and 16 hours. Then western blotting analysis on cell lysates using specific antibodies for pAKT, phospho-PAR4 (Thr163) and PAR4 were performed. Normalization of protein was performed by immunoblotting for actin. The bands were quantified by densitometry analysis using SMX Image software. The results shown in the figure indicate that after 8 hours of treatment with Bu₂SnCI₂ the content of active survival factor pAKT decrease in dose-dependent manner, whereas the levels of pAKT recover after incubation with Bu₂SnNAC.

Recent data indicate that the phosphorylation on Thr163 of PAR4 determines its activation as a pro-apoptotic factor and subtracts it to the phosphorylation on Ser 249 by pAKT, an event that would render PAR4 inactive (J.W. Lee, K. F. Lee, H. Y. Hsu, L.P. Hsu, W.L. Shin, YC. Chu, WT. Hsiao, P.F. Liu, Cancer Lett, 2007, 257(2), 252-62). This mechanism probably explains why, when the cells were incubated for 8 hours with Bu₂SnNAC, pAKT increased (compared to HepG2 cells incubated with Bu₂SnCI₂ in the same experimental conditions) and simultaneously phospho-PAR4 (Thr163) levels decreased. Then it is expected that, after treatment with Bu₂Sn-N- Acetylcysteinate, anti-apoptotic phospho-PAR4 (Ser249) levels increase following pAKT activity, and this would induce a delay, at least during early hours of treatment, in the activation of caspases, and consequently in apoptosis. Instead in cells incubated with Bu₂SnCI_2, after 8 hours of incubation, the decrease in pAKT levels and the increase in pro-apoptotic phospho-PAR4 (ThM 63) were responsible for the greater sensitivity observed in these cells to apoptotic effects induced by Bu₂SnCI₂.

Study on antitumoral activity of NAC2 complex (dibuthyltin N.acetylcysteinate) performed at the National Cancer Institute. NAC2 activity was assayed against 60 different human tumor cell lines, representative for leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The aim was to evaluate a selective growth inhibition or cell killing of particular tumor cell lines induced by NAC2. The screening is a two-stage process, beginning with the evaluation of the compound against the 60 cell lines at a single dose of 10 uM. The output from the single dose screen is reported as a mean graph and is available for analysis by the COMPARE program. We report the results of this one dose test. The compound exhibited significant growth inhibition. Methodology of The In Vitro Cancer Screen

The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% foetal bovine serum and 2 mM L-glutamine. Cells were inoculated into 96 well microtiter plates in 100 μl_ at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drug were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin. Aliquots of 100 μl of this drug dilution was added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentration.

Following drug addition, the plates were incubated for an additional 48 h at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 μl of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4 % (w/v) in 1 % acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1 % acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80 % TCA (final concentration, 16 % TCA). Using the three absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the 10-5 M concentration level (Ti)], the percentage growth was calculated as: [(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz [(Ti-Tz)/Tz] x 100 for concentrations for which TkTz.

Interpretation of One-Dose Data: The One-dose data are reported as a mean graph of the percent growth of treated cells . The number reported for the One- dose assay is growth relative to the no-drug control, and relative to the time zero number of cells. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition. A value of 40 would mean 60% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of -40 would mean 40% lethality. A value of -100 means all cells are dead. Information from the

One-dose mean graph is available for COMPARE analysis. http://dtp.nci.nih.gov/branches/btb/ivclsp.html

The figure 6 shows the results on 60 different human tumor cell lines. The compound NAC2 showed excellent antitumoral activity in vitro towards leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney.

This invention has been described with reference to some forms of specific realizations, but it is intended that variations and changes can be made to it by specialists alone to escape of relative scope of protection.

Claims

1. Organotin(IV) compounds with N-acetylcysteine having the general formula (1 ) o (2):

wherein:

R-I, R₂ and R₃ are each independently selected from the group consisting of:

H, an alkyl, alkoxyl, alkenyl or alkynyl group with up to 12 carbon atoms, an isocyclic or heterocyclic aromatic or aralkyl group with up to 12 carbon atoms, for use as medicinal active ingredients.

2. Organotin(IV) compounds with N-acetylcysteine according to claim 1 , wherein the said medicinal active ingredients are anticancer agents.

3. Organotin(IV) compounds with N-acetylcysteine according to claim 2, wherein the said anticancer agents are agents for the treatment of hepatic and colorectal tumours, leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney.

4. Organotin(IV) compounds with N-acetylcysteine according to any one of claims 1 -3, wherein R₁, R₂ and R₃ are each independently selected from:

H, an alkyl group with up to 8 carbon atoms, or an alkoxyl group with up to 8 carbon atoms or a phenyl group.

5. Diorganotin(IV) compounds with N-acetylcysteine according to claim 4 having the general formula (1 ), wherein R-i and R₂ are each independently selected from methyl, ethyl, propyl, butyl and phenyl.

6. Triorganotin(IV) compounds with N-acetylcysteine according to claim 4 having the general formula (2), wherein R-, , R₂ and R₃ are each independently selected from methyl, ethyl, propyl, butyl and phenyl.

7. A diorganotin(IV) compound with N-acetylcysteine having the general formula (1 )

wherein:

R-i, R₂ are each independently selected from: H, an alkyl, alkoxyl, alkenyl or alkynyl group with up to 12 carbon atoms, an isocyclic or heterocyclic aromatic or aralkyl group with up to 12 carbon atoms, for use as a medicinal active ingredient.

8. A compound according to claim 7, wherein R-i and R₂ are each independently selected from methyl, ethyl, propyl, butyl and phenyl.

9. A compound according to claim 8 wherein R-, and R₂ represent butyl, having the formula (1 a):

10. A triorganotin(IV) compound with N-acetylcysteine having the general formula (2)

wherein: R₁, R₂ R₃ are each independently selected from:

H, an alkyl, alkoxyl, alkenyl or alkynyl group with up to 12 carbon atoms, an isocyclic or heterocyclic aromatic or aralkyl group with up to 12 carbon atoms, for use as a medicinal active ingredient.

11. A compound according to claim 10, wherein R-, and R₂ are each independently selected from methyl, ethyl, propyl, butyl and phenyl.

12. A process for producing the organotin compounds with N-acetylcysteine having the formula (1 ) or (2) as defined in claims 1 , 4, 5 or 6, comprising the step of reacting an organotin derivative wherein R-i, R₂ and R₃ have the meaning recited in claim 1 with N-acetylcysteine in methanol, or in chloroform, or in water, in a temperature range comprised between 10°C and 90 °C.

13. A process according to claim 12, wherein at the end of the said reaction in methanol or in water or in chloroform the reaction mixture is quenched, and the resulting precipitate containing the desired compound of formula (1 ) or (2) is collected by filtration.

14. A process for producing the compound dibutyltin(IV)-N acetylcysteinate of the general formula (1 a), as defined in claim 9, comprising the step of reacting

N-acetylcysteine with dibutyltin oxide in methanol, or in water or in chloroform, in a temperature range comprised between 10°C and 90 °C.

15. A process according to claim 14, wherein at the end of the said reaction in methanol or in water or in chloroform the reaction mixture is quenched, and the resulting precipitate containing the desired compound of formula (1 a) is collected by filtration.

16. Use of one or more organotin(IV) compounds with N-acetylcysteine of the general formula (1 ) or (2), as defined in claims 1 , 4, 5 o 6, for the production of a pharmaceutical preparation.

17. Use according to claim 16, wherein the said pharmaceutical preparation is an anticancer preparation.

18. Use according to claim 17, wherein the said pharmaceutical preparation is an anticancer preparation for the treatment of hepatic and colorectal tumours, leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney.

19. A pharmaceutical preparation comprising, as active ingredient, at least one of the organotin(IV) compounds with N-acetylcysteine of the general formula (1 ) or (2), as defined in claims 1 , 4, 5 or 6, together with one or more pharmacologically acceptable adjuvants and/or vehicles.

20. A pharmaceutical preparation according to claim 19, comprising as an active ingredient the compound dibutyltin(IV)-N-acetylcysteinate of the formula (1 a), as defined in claim 9.