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Title:
HYDROXYBENZENE DERIVATIVES HAVING A N-ARYL SUBSTITUTE IMINO GROUP AND USE THEREOF IN THE TREATMENT OF SOLID TUMOURS
Document Type and Number:
WIPO Patent Application WO/2018/116158
Kind Code:
A1
Abstract:
The present invention relates to new compounds having hydroxyl, imino and carboxylic functionalities, processes for the production thereof and use thereof as antitumour agents for the treatment of solid tumours.

Inventors:
LONGO PASQUALE (IT)
SATURNINO CARMELA (IT)
ARRA CLAUDIO (IT)
PALMA GIUSEPPE (IT)
MARICONDA ANNALUISA (IT)
SINICROPI MARIA STEFANIA (IT)
PUOCI FRANCESCO (IT)
IACOPETTA DOMENICO (IT)
Application Number:
PCT/IB2017/058132
Publication Date:
June 28, 2018
Filing Date:
December 19, 2017
Export Citation:
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Assignee:
UNIV DEGLI STUDI DI SALERNO (IT)
International Classes:
C07C251/24; C07C249/02
Foreign References:
FR2338246A11977-08-12
EP0586917A11994-03-16
Other References:
ALIZADEH K ET AL: "Experimental and computational study on the aqueous acidity constants of some new aminobenzoic acid compounds", JOURNAL OF MOLECULAR LIQUIDS, ELSEVIER, AMSTERDAM, NL, vol. 149, no. 3, 30 October 2009 (2009-10-30), pages 60 - 65, XP026675143, ISSN: 0167-7322, [retrieved on 20090828], DOI: 10.1016/J.MOLLIQ.2009.08.007
KAUR G ET AL: "Synthesis, structure-activity relationship, and p210 protein tyrosine kinase activity of novel AG 957 analogs", BIOORGANIC & MEDICINAL CHEMISTRY, PERGAMON, GB, vol. 13, no. 5, 1 March 2005 (2005-03-01), pages 1749 - 1761, XP027637696, ISSN: 0968-0896, [retrieved on 20050301]
EL-DIN, NABAWEYA SHARAF; EL-FATATRY, HAMED: "Separation of N-(substituted benzylidene)-p-aminobenzoic acid conformers having biological activity", ALEXANDRIA JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 5, no. 2, 1991, pages 163 - 165, XP009500110
SMIRNOV, E. A.; AGRACHEVA, E. B.; GACHKOVSKII, V. F.: "Structure and luminescence of salicylidene-p-aminobenzoic acid esters with an additional electron-donor group in the aldehyde component", ZHURNAL OBSHCHEI KHIMII, vol. 40, no. 2, 1970, pages 375 - 379, XP009500109
Attorney, Agent or Firm:
BORRINI, Stefano et al. (IT)
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Claims:
CLAIMS 1.

wherein independently of one another indicate:

R H, Na, CH2CH3, adamantyl

Xi H, OH and

X2 H, OH.

2. The compounds according to claim 1, wherein said R, Xi are X2 are selected so as to form the following compounds

3. The compound according to claim 2, wherein R is Na, Xi is OH and X2 is H.

4. The compounds according to at least one of claims 1 to 3, for use in the treatment of solid tumours.

5. The compounds according to claim 4, wherein said solid tumours belong to the class consisting of Triple Negative (MDA.MB231) breast cancer and pancreatic cancer (MiaPaca2).

6. Pharmaceutical compounds comprising a therapeutically acceptable amount of at least one compound as claimed in at least one of claims 1 to 3 and a pharmaceutically acceptable additive.

7. A process for the production of compounds of general formula (2) comprising the steps of the following Scheme 1

Description:
HYDROXYBENZENE DERIVATIVES HAVING A N-ARYL SUBSTITUTE IMINO GROUP AND USE THEREOF IN THE TREATMENT OF SOLID TUMOURS DESCRIPTION

The present invention relates to new compounds having hydroxyl, imino and carboxylic functionalities, processes for the production thereof and use thereof as antitumour agents for the treatment of solid tumours. The compounds of the present invention have led to the development of a new therapeutic approach for the pharmacological treatment of the Triple Negative (MDA.MB231) breast cancer and of the pancreatic cancer (MiaPaca2). Even the cardiotoxicity thereof was evaluated by comparing it with that of a drug known from the state of art, doxorubicin. State of art

Currently among the antitumour drugs available on the market those based upon metallic compounds (example: cis-platin, carbo-platin, etc.), which are alkylating agents, are particularly important. Cisplatin enters the cells by diffusion wherein it is then converted into its active form. It is believed that the active species, even if it is not determined with absolute certainty, is the monohydrate one. The main function of cisplatin is that of linking to DNA, even if it is capable of interacting with several types of proteins vital for DNA replication and the cell division. However, platinum complexes have several side effects and considerable toxicity.

Cisplatin is nephrotoxic, in fact it can cause renal damages (tubular degeneration, necrosis and mineralization of the tubular epithelial cells). It further shows ototoxicity which appears especially in children, by causing among other things equilibrium loss due to hearing difficulty. The initial symptoms include tinkling (ear whistling). These effects should stop at the end of treatment, but sometimes it happens that some patients lose irreversibly their hearing in the range of high frequencies (> 4KHz). The other toxic symptoms are common to those caused by other antitumour drugs: nausea, vomiting, decrease in red blood cells, neurotoxicity, shown above all with high dosages, with tingling sensation at hands and feet. On the contrary the decrease in blood platelets, facial swelling, dyspnea (shortness of breath), muscle cramps, blurred vision and appetite loss are less common.

Another drug particularly important in the oncological clinic therapy is represented by Doxorubicin which is one of the most effective antitumour agents for the treatment of leukemia and a wide range of solid tumours (1,9). This compound belongs to the family of anthracyclines thereamong there are other three molecules, one thereof has been already on the market for long time: Daunorubicin, and other two already in clinical phase for their antitumour effects: Epirubicin and Idarubicin. All of them have in common 4-ring 7,8,9, 10- tetrahydrotetracene-5,12-quinone polycycle, but they differ for the residues Rl, R2 and R3, as shown in figure 1 (general Formula of anthracyclines). The whole family of anthracyclines shows cardiac toxicity, which in the long term can be fatal to patient.

Among the malignant pathologies most widespread within the population the breast tumour (in particular the Triple Negative) and the pancreatic cancer can be mentioned, tumours therefor the current chemotherapy is not sufficiently effective, it is very toxic and often it is not applicable.

The "Triple Negative" breast cancer, as designated by the name itself, is a particularly aggressive form, difficult to be treated since the wall of the tumour cells has neither the receptors for hormones: estrogens and progesterone, nor the receptors of HER2 {Human Epidermal Receptor). The standard treatment of the Triple Negative tumour is by surgical route thereto an adjuvant therapy is then added generally represented by anthracyclines and by taxanes. The same drugs are used in patients wherein a neoadjuvant chemotherapy is required for reducing the sizes of the tumour until making it operable with conserving surgery.

Even for the pancreatic cancer the most effective treatment is the surgical one, but it is particularly complex and it has many risks especially due to the possible post- surgery complications. The chemotherapy, applied both before and after the surgical operation has provided for many years as single treatment the administration of gemcitabine, followed by cisplatin and capecitabine. Recently three drugs are used in association: 5-fluorouracil, oxaliplatin and irinotecan which has led almost to double the survival of patients. Unfortunately, this treatment, being particularly aggressive, is reserved only to people under good general conditions, without jaundice and biliary prostheses.

Therefore, in the state of art the need is felt for developing new compounds with pharmacological activity against tumours with reduced toxicity and a targeted selectivity only towards the cancer cells, above all applicable in the therapy of these tumour forms.

Now it has surprisingly found that molecules characterized by a common skeleton of imines having two aromatic substituted rings can be used selectively and effectively in the therapies of the breast tumour and of the pancreatic cancer and they do not have the same side effects of the molecules known from the state of art. The molecules according to the present invention have not the quinone portion existing instead in doxorubicin which is probably responsible for its cardiotoxicity.

The present invention then relates to molecules of the following general formula 2

wherein independently of one another indicate:

R H, Na, CH2CH3, adamantyl

Xi H, OH and

X 2 H, OH

Brief description of the figures

Seven figures are enclosed to the present description wherein:

figures A and B explain a tumour cell growth inhibition assay on SC5, SC6, Sc8, JC1, SCI compounds, solubilized in DMSO.

figures C and D show the activity for inhibiting the proliferation of the molecules soluble in physiological solution SC4 and SC7 compared with doxorubicin solubilized too in physiological solution at the same concentrations.

Figure E shows the modulation of the cell proliferation by SC4 compound, solubilized in physiological solution, on pancreatic tumour cells after 14 days of treatment after one single exposition to two different concentrations Figure F an Evaluation of the cell proliferation by means of Colony Assay

Figure G shows the inhibition results of SC4 on HUMAN TOPOISOMERASE II Figure H shows with tumour growth curve the in vivo effects of SC4, by using a heterotopic model of pancreatic cancer,

Figure I shows a weight curve of heterotopic murine model of pancreatic cancer subjected to treatment with SC4 and its control drug, doxorubicin.

Figure L shows a weight curve of heterotopic murine model of pancreatic cancer subjected to treatment with SC4 and its control drug, doxorubicin.

Figure M shows the weights of the organs lungs and liver as possible organ-specific toxicity index.

Figure N shows the analysis of the metastasis in lung by means of counting on histological preparations, by distinguishing the metastasis in macro and micro depending their size and the effect after treatment.

Figure O shows the analysis of the metastasis in lung on histological preparations, by means of counting the number of metastases based upon the effect after treatment. Figure P shows the impact of SC4 and of its possible toxicity.

Detailed description of the invention

The present invention relates to new compounds of the following general formula 2

wherein independently of one another indicate:

Compounds which represent particular implementations of the present invention are shown in the following table 1 identified by their abbreviations. Table 1 : Abbreviations of the compounds and of the related substituents

Such molecules synthesized in laboratory were in vitro and in vivo tested. In in vitro tests human cell lines of pancreatic cancer (MiaPaca2) and of Triple Negative (MDA-MB231) breast cancer were used. The new molecules the invention relates to were implemented with quick synthetic methods and were in vitro and in vivo studied. Of such new molecules, the cytotoxic action on the two solid cancers taken into consideration was determined. The in vitro studies allowed to determine even DL50. In in vivo tests mice having thymic atrophy were used. Such mice, since they do not develop lymphocytes T, constitute an ideal model for developing oncological pathologies.

The present invention further relates to processes for producing the compounds the present invention relates to as well as their therapeutic application. Such new compounds are constituted by an imine linked to two aromatic rings having one of the hydroxyl groups and the other one a carboxylic, ester function or carboxylate sodium.

The synthesis of these new molecules was implemented by reaction between one dihydroxybenzaldehyde and the /?-aminobenzoic acid. The acid group was then salified or esterified. The following scheme 1 shows the followed synthetic strategy

Scheme 1 : synthetic scheme of the new molecules (Xi = H and X 2 = OH, or Xi = OH and X 2 = H).

The evaluation of the biological activity of the synthetized molecules allowed to show that they have a significant effectiveness in inhibiting the two tumours taken into consideration. The effectiveness of such molecules depends upon the chemical structure of the same. These compounds according to the invention represent a new class of organic molecules provided with antiproliferative activity.

The in vitro experiments carried out on cell cultures of Triple Negative (MDA.MB213) breast cancer and pancreatic cancers (MiaPaca2), allowed to establish that the inhibition of cell growth, obtained with some of the new synthetized molecules, can be referred to antiblastic activity, at depending concentrations with a Lethal Dose 50 (DL50) at low concentrations: of 20 and 12 μΜ), respectively.

The in vivo experiments were carried out on a murine strain genetically identified as nude mouse or hairless mouse that is nu/nu, which shows thymic atrophy; such mice do not develop lymphocytes T, this peculiarity allows the model not to reject the tissues or cells not syngeneic to the mouse itself, then ideal model for the development of murine models for the oncological pathologies.

The model developed in the test was a murine model of pancreatic cancer. Cells of pancreatic cancer were inoculated in the quadriceps femoris and the growth was followed by measuring with digital gauge. The growth defined as tumour growth was calculated with the formula of an ellipsoid: square larger diameter x smaller diameter/2.

Three groups were performed: a control one, that is with only the tumour without any treatment, the tumour treated with SC4 at 80 μΜ, and the tumour treated with doxorubicin, as reference drug. The statistical analysis detects a significance for the CTR (control) group vs TRT (treatment) and no significance between DOXO and TRT groups, this designates the pharmacological activity of our molecule. The analysis and the monitoring of the body weight course shows a smaller effect of the group treated with SC4 than the group treated with doxorubicin.

In order to evaluate the possible cardiotoxicity of the molecules the present invention relates to, the treated mice were examined by echocardiography. The echocardiography is the examination which allows to see and detect the pathologies linked to the myocardium, pericardium and the cardiac valves. The echocardiography then takes advantage of using ultrasounds to display the heart's inner structure. After having performed an echocardiographic analysis (similar to that made for human people), a not cardiac toxicity of SC4 compound is deduced.

By way of illustration and not for limitative purposes, examples of synthesis and characterization of the new compounds and of their cytotoxic activity are described hereinafter.

Examples

A series of emblematic examples of the several transformations is described hereinafter, which have not to be considered as limiting the present invention.

Example 1

SCI synthesis provides one single reaction, as shown in scheme 1. In this reaction the 4-aminobenzoic acid (1 g, 7.30 mmol, MW 137.13) and 2,5- dihydroxybenzaldeyde (1 g, 7.24 mmol, MW 138.12) react, at room temperature, in methanol (60 ml) to form (E)-4-(2,5-dihydroxybenzylidene)imino-benzoic acid (SCI). The reaction is reflux performed for 12 hours. At the end of reaction the solvent is removed and the product (E)-4-(2,5-dihydroxybenzylidene)imino-benzoic acid (SCI) is recovered as orange-coloured dusty solid (MW 257.24, 1.75 g, yield 94%) and characterized by spectroscopy of protonic and carbon nuclear magnetic resonance mass spectrometry and elementary

analysis.

Hereinafter signals of SCI compound are shown.

164.2, 153.1, 152.5, 149.7, 130.6, 128.6, 121.7, 121.4, 119.3, 117.3, 116.6. Mass data:

Example 2

SC4 synthesis provides two reaction steps. SC4 compound was obtained by salification of the carboxylic group of SCI . In I step SCI synthesis is performed, as shown in example 1. In II step SCI is made to react with a solution 0.05 M of sodium hydrogen carbonate. 300 mg of SCI and 38 ml of water were introduced into a balloon, provided with magnetic stirrer. The suspension was stirred for 15 minutes and always under stirring 42.3 ml of a solution 0.05 M of NaHC0 3 were dropped slowly. The suspension was filtered and the solvent removed under vacuum. The raw material was washed with methanol. The product (E)-4-(2,5-dihydroxybenzylidene) imino-sodium benzoate (SC4) was recovered as brown-coloured dusty solid (MW 279.22, 0.084 g, yield 28 %).

Formula 4: SC4

1 H NMR (250 MHz, DMSO): δ 8.83 (s, 1H), 7.25 (d, 2H), 7.04 (s, 2H), 6.85 (d, 1H), 6.77 (d, 1H), 6.41 (d, 1H). 13 C NMR (62.5 MHz, DMSO): δ 169.3, 162.7, 153.0, 149.9, 148.7, 138.8, 130.2, 121.1, 120.1, 119.3, 117.1, 116.8. ESI-MS (CH 3 CN,

Example 3

SC5 synthesis provides two reaction steps. SC5 compound was obtained by esterification of the carboxylic group of SCI . In I step SCI synthesis is performed, as shown in example 1. In II step SCI is esterified. For this reaction the use of a catalyst is of fundamental importance, such as the sulphuric acid in ethanol acting a solvent/reagent.

200 mg di SCI, 26.60 mL of CH 3 CH 2 OH and 1 drop of H 2 SO 4 at 98% are introduced into a 50-ml balloon having one neck, provided with magnetic stirrer. The reaction balloon is placed at reflux for 6 h. At the end of reaction, the suspension is filtered. From the soluble fraction the reaction raw material is recovered. The solvent is removed under vacuum and the raw material is washed with ethyl acetate. SC5 product is recovered from the soluble fraction as black-coloured solid (MW 285.29, 0.080 g, yield 40 %).

Example 4

Procedure used for the synthesis of the molecule JCl . JCl compound was obtained by esterification of the carboxylic group of SCI .

The compound JCl was obtained by esterification of the carboxylic group of SCI with 1-adamantanol. The synthesis of this product is constituted by 3 reaction steps. In I step SCI synthesis is performed.

In II step SCI is chlorinated with thionyl chloride (the product is not insulated). In III step the acid chloride is esterified in situ with 1-adamantanol.

100 mg of SCI and 20 mL of SOCl 2 are inserted into a 50-ml balloon with one neck, provided with magnetic stirrer (in inert atmosphere). The reaction balloon is placed at reflux for 2 h. The solvent is removed under vacuum. 10 mL of THF, 0.13 mL of pyridine and 82 mg of 1-adamantanol between 0-5°C are inserted into the same reaction balloon. The resulting suspension is stirred for 16 h at room temperature. Subsequently a filtration is performed and the insoluble filtered material is washed with ethyl acetate. The solvent is removed under vacuum and the product is recovered as light yellow-coloured dusty solid (MW 391.46, 0.053 g, yield 35 %).

Formula 6: JC 1

Example 5

SC6 synthesis provides one single synthesis step. 0.5 g of 3,5-dihydroxybenzaldeyde (MW 138.12, 7.24 mmol), 0.5 g of 4-aminobenzoic acid (MW 137.13, 7.3 mmol) and 35 ml of methanol are inserted into a balloon having one neck, provided with magnetic stirrer. The reaction balloon is placed at reflux for 24 h. At the end of reaction, the solvent is removed and about 0.34 g of sample are recovered which are put inside an extractor for solid materials. Such extraction is performed by using chloroform as solvent at a temperature of 90°C for 5 hours. The insoluble product is dissolved in methanol. The solvent is removed and the product (E)-4-(3,5- dihydroxybenzylidene)imino-benzoic acid (SC6) is recovered as light brown- coloured dusty solid (MW 257.24, 0.17 g, yield 17%).

Formula 7: SC6

¾ NMR (250 MHz, DMSO): δ 9.60 (s, 1H), 8.40 (s, 1H), 7.94 (d, 2H), 7.25 (d, 2H), 7.80 (d, 2H), 6.37 (d, 1H). 13 C NMR (62.5 MHz, DMSO): δ 167.5, 167.1, 162.6, 158.7, 155.5, 137.5, 131.2, 130.6, 127.8, 121.0, 112.5, 108.6, 107.0, 106.2. ESI-MS

Example 6

SC7 synthesis provides two reaction steps. SC7 compound was obtained by salification of the carboxylic group of SC6. In I step SC6 synthesis is performed as shown in example 5. In II step, 50 mg of SC6 and 12.5 ml of water are inserted into a one-neck balloon, provided with magnetic stirrer. The suspension is stirred for 15 minutes and still under stirring 2.86 ml of a 0.05 M solution of NaOH are dropped slowly. It is left under stirring for 5 minutes, it is filtered and the solvent is removed. The product is recovered after washing with chloroform. The (E)-4-(3,5- dihydroxybenzylidene)imino-benzoate sodium (SC7) product is recovered as dark red-coloured vitreous solid (MW 279.22, 0.019 g, yield 35 %).

Formula 8: SC7

Example 7

SC8 compound was obtained by esterification of the carboxylic group of SC6.

In I step SC6 synthesis is performed as shown in example 5. In II step SC6 is esterified. 20 mg of SC6, 10.4 μΐ, of SOCl 2 and 2 mL of CH 3 CH 2 OH are inserted into a two-neck balloon (in inert atmosphere), provided with magnetic stirrer. The solution is stirred for 5 minutes. The reaction balloon is placed at reflux for 10 min. The solvent under vacuum is removed and the (E)-ethyl 4-(3,5- dihydroxybenzylidene)imino benzoate (SC8) product is recovered as a dark brown- coloured solid (MW 285.29, 0.014 g, yield 70 %).

IN VITRO STUDIES

After having performed the synthesis and the characterization of molecules one tested the in vitro pharmacological activity thereof in particular on the pancreatic cancer and on the Triple Negative breast cancer. For the in vitro studies for these types of cancers the MDA.MB213 human cancer cell lines (human cell line of Triple Negative breast cancer, ATCC-American Tissue Cell Culture - ATCC® HTB-26™), and MiaPaca2 (human cell line of pancreatic cancer, ATCC-American Tissue Cell Culture - ATCC® CRL-1420™) were used;

The molecules were tested by means of a colorimetric assay, MTT assay. MTT assay is a measurement of sensitivity of cell proliferation based upon the decrease in the tetrazolium salt, 3, [4,5-dimethylthiazole-2-] -2,5-diphenyl bromide (MTT).

The tests performed in vitro were differentiated according to the solubility of the synthetized molecules; SC4 and SC7 (soluble in water) were tested by solubilizing them in a physiological solution. SCI, SC5, JCl, SC6 and SC8 compounds showed poor solubility in water, therefore they were tested by using DMSO (dimethyl sulphoxide) as solvent (Figures A-B). The disadvantage of using DMSO as solvent is that it results to be toxic for about 10-15% on the cell viability. The activity of the molecules soluble in water (SC4 e SC7) was compared to doxorubicin solubilized too in physiological solution (Figures C-D).

Moreover, the modulation of the cell proliferation (Figure E) was evaluated by SC4 compound on cells of pancreatic cancer after 14 days of treatment after one single exposure to two different concentrations was evaluated. The graph shows the high inhibitory activity of C4, since at 40 μΜ there is a clear inhibition of the cell proliferation.

IN VITRO BIOAVAILABILITY STUDIES

The in vitro bioavailability studies were performed in solutions simulating the gastric and intestinal environment through a modified version of the most traditional technique of the dialysis membranes. [1] The method of the dialysis membranes is divided into two phases of enzymatic digestion: a first phase wherein there is the pepsin action and a second one wherein even pancreatin takes part.

Digestion by Pepsin. 3.0 mg of SC4 compound were placed into 1.0 mL of a solution of HC1 0.85 N containing 24000 U/mL of pepsine and 3 mL of a solution of sodium azide 0.5% (p/v). The so obtained mixture was inserted into a dialysis membrane (Spectrum Laboratories Inc., MWCO: 12-14,000 Dalton, USA) carefully closed at the two ends and dipped into a vial containing 10 mL of the solution of HC1 0.85 N (pH 1.0). The vial then was placed into a thermostated water bath at 37±0.5°C for 2 h with the purpose of simulating the temperature physiological conditions.

Digestion by Pancreatin. After 2 h, the dialysis membrane was opened and 11 mg of amylase, 11 mg of esterase and 1.3 mL of a solution of NaHC03 (0.8 M) containing 22.6 mg of pancreatin per mL were added. The dialysis membrane was sealed again and inserted into a vial containing 10 mL of a buffer solution at pH 7.0. The vial was placed again in a bath thermostated at 37±0.5°C for further 4 h.

For evaluating the bioavailability of SC4 compound, 2 mL of the solution included in the vials were taken after 2 and 6 h and the samples were analysed by means of UV-Vis spectrophotometer. The quantity of SC4 was determined by using the calibration curves previously obtained from standard solutions of the compound at pH 1.0 and at pH 7.0, respectively. The correlation coefficient (R2), the angular coefficient and the intercept were calculated by means of the method of least squares.

The experiments were carried out in triplicate and the bioavailability was expressed as percentage.

Results

In vitro bioavailability studies The bioavailability is defined as the analyte percentage recovered in the bioaccessible fraction, after in vitro digestion, in relation to the not digested original sample and it is calculated by means of the following Equation (1):

(bioaccessible fraction/total content) x 100% (1)

The obtained results are shown in Table 2.

Table 2

TEST ON TOPOISOMERASES HUMAN TOPOISOMERASE I TEST

The relaxation tests were performed in a final volume of 20 μΐ and put together as explained hereinafter. 0.25 μg of pHOT 1 supercoiled in TE buffer were added to a solution containing water (variable volume) and IX of assay buffer. Subsequently, SC4 or a carrier were added and the reactions were started by adding a recombinant human topoisomerase I. The reactions took place by incubating the whole thing at 37°C for 30 minutes and terminated by adding a 5X (stop) buffer. The samples were then subjected to digestion with Proteinase K (50 μg/ml) at 37° for 30 minutes, followed by the extraction the volume with chloroform being equal: isoamyl alcohol (24: 1), stirred and centrifuged for 30 seconds. The upper aqueous phase was removed, loaded in a 1% agarose gel containing IX of TAE buffer without ethidium bromide. After the stroke the gel was coloured with IX of TAE buffer containing ethidium bromide (0.5 μg/ml) for 30 minutes and decolourised with distilled water for 15 minutes, then it was displayed by using UV.

HUMAN TOPOISOMERASE II TEST

The decantation tests were performed in a final volume of 20 μΐ, as described hereinafter. 0.3 μg of kinetoplast DNA (k DNA) were added to a solution containing water, IX of assay buffer, and ImM of ATP. "SC4 or the carrier were added and the reactions were started by adding 3 U of human topoisomerase II and the whole thing incubated at 37°C for 30 minutes. In the end, a 5X stop buffer was added and the samples were treated as described in the preceding paragraph. The aqueous phase was loaded on a 1% agarose gel containing a TAE IX buffer with ethidium bromide (0.5 μg/ml) and displayed by using UV (Figure G) Panel A: human topoisomerase I relaxation assay. Lane 1, super coiled (sc) DNA; lane 2, relaxed DNA marker; lane 3, vehicle, lane 4, SC4 100 μΜ.

Panel B: human topoisomerase II decatenation assay. Lane 1, decatenated DNA marker; lane 2, catenated kDNA; lane 3, vehicle, lane 4, SC4 100 μΜ.

IN VIVO STUDIES

In vivo studies were performed on a murine strain identified as nude mouse that is Nu/Nu, such strain shows thymic atrophy; since such mice do not develop lymphocytes T, they are preferred murine model for the development of oncological pathologies.

A murine model of pancreatic cancer was developed, by inoculating 2xl0 6 human cells of pancreatic cancer (MiaPaca2), in lOOul of physiological solution, in the mouse quadriceps femoris; the growth thereof was monitored by measurement with digital gauge. The growth is defined as Tumour Growth and it was calculated with the formula of an ellipsoid: (larger diameter x smaller diameter)2/2. Three groups were performed: a control one, with tumour only without any treatment, a group with the tumour treated with SC4 at 80 μΜ and a group with tumour treated with doxorubicin, as reference drug. Figure H shows a tumour growth curve: The Tumour Growth shows an effect of inhibiting the tumour growth after 12 days of treatment with SC4.

Figure I shows a weight curve highlighting an absence of systematic toxicity attributable directly to SC4 action.

METASTATIS MURINE MODEL (LUNG COLONIZATION)

The capability of the compound under study of inhibiting the formation of secondary lesions in the lung was evaluated by means of a metastasis murine model (Lung Colonization).

5xl0 5 cells of B16F10 murine melanoma were inoculated in 6/8-week-old female C57/BL6J mice in the caudal vein. 2 groups were developed: a control one and a second group treated twice a week with 80uM of SC4.

The body weight during the whole treatment was evaluated (Fig. ); by highlighting an absence of systemic toxicity attributable to SC4. At time of euthanasia the lungs were taken for the histological analysis; the weight of the lungs highlights an absence of differences (Fig. ).

The histological analysis (Fig A and B) highlights both a smaller number and a smaller size of metastases in the group of mice treated with SC4; thus highlighting a role inhibiting SC4 tumour growth and confirming the molecular action on the mouse isomerases.

CARDIOTOXICITY

The echocardiography is the examination which allows to see and detect the pathologies linked to the myocardium, pericardium and cardiac valves. The echocardiography then uses ultrasounds to display the heart inner structure. After having performed an echocardiographic analysis, a not cardiac toxicity of SC4 compound results.

Figure P shows a in vivo evaluation, on oncological murine models, of the toxic impact of SC4 on heart by means of echocardiography, in comparison to a traditional chemotherapeutic drug (doxorubicin). The analysis shows a smaller or absent toxicity in our molecule with respect to doxorubicin as shown in the following table 3.