WO2013105037A1 - Inhibitors of p53-mdm2 interaction - Google Patents

Description

 DESCRIPTION P53-MDM2 INTERACTION INHIBITORS

Technical field of the invention

The present invention relates to (thio) xanthones of formula 1 and their analogues and their use as inhibitors of the p53-MDM2 interaction and their use as medicaments in laboratory studies of cellular processes in which this p53-MDM2 interaction involved and in the treatment of cancers expressing a native form of the p53 protein. The present invention provides for an improvement of the characteristics of existing p53-MDM2 interaction inhibiting agents with regard to their efficacy and synthesis process.

This invention is part of the technical field of fine chemicals and cancer medicines.

Background of the Invention

The p53 tumor suppressor protein is a key cellular regulator responsible for maintaining genomic integrity after exposure to stressors. Over the past thirty decades, the effectiveness of p53 protein in tumor suppression has been demonstrated by inducing cell cycle arrest and apoptotic cell death. In some human tumors the protective activity of this protein is suppressed by mutation. In the case of tumors with the native form of protein p53, the activity of this protein It is suppressed by cellular overexpression of the major negative regulators, MDM2 and MDMX (also known as MDM4). MDM2 oncoprotein is an ubiquitin E3 ligase inhibits the activity of p53 protein through two pathways: one involving direct interaction with the p53 protein masking its transactivation domain and hence its transcriptional activity, and another involving the ubiquitination and consequent proteosomal degradation of p53 protein. MDMX oncoprotein mainly inhibits the transcriptional activity of p53 (Di et al. Curr Cancer Drug Targets, 2011, DOI: 10.2174 / 156800911797264789). Inhibiting the interaction of MDM2 protein with p53 protein has been considered a very promising strategy for reactivation of p53 protein in cancers with a native form of p53, such as breast, colorectal, prostate, kidney, lung cancers. , melanoma, osteosarcoma and embryonic nephroma, among others. Consequently, inhibitors of this interaction represent anticancer agents, and in recent years there has been an exhaustive search for p53-MDM2 interaction inhibitor molecules (Di et al. Curr Cancer Drug Targets, 2011, 15: DOI: 10.2174 / 156800911797264789 ).

In order to restore the functions of the TP53 gene, p53-based gene therapy has already been clinically approved in China. However, due to the demonstrated lack of efficacy, it has not yet been approved in the United States and Europe. Other limitations associated with this type of therapy are the difficulty of developing systems to release these agents at the target site (Cheok et al. Nat Rev Clin Oncol, 2011,8: 25-37). Another therapeutic approach is immunization vaccination with p53-derived peptides. However, clinical vaccination trials have shown low efficacy. monotherapy (Speetjens, et al. Clin Cancer Res, 2009, 15: 1086-1095).

 Therefore, small inhibitors of protein-protein interaction have advantages as drug candidates over macromolecules investigated in the therapies discussed above.

 Several small molecules described as inhibitors of the p53-MDM2 interaction are in the preclinical phase and are marketed as fine chemicals, namely for use in laboratory studies of cellular processes, the most important being: nutlins, isoquinolinones, benzodiazepinediones and spiroxindois (Dickens et al. Semin Cancer Biol, 2010, 20 (1): 10-8). Many of these derivatives, initially investigated in preclinical trials, have revealed marked genotoxicity and / or cytotoxicity and / or low bioavailability. P53-MDM2 interaction inhibitor derivatives found in preclinical assays have one or more stereogenic centers, leading to enantiomeric mixtures, namely racemates, such as nutlin (cis-imidazolines), isoquinolinone derivatives PXN727 and PXN822, benzodiazepinedionic derivatives TDP521252 and TDP665759, and spiroxindole MI219. This feature limits the effectiveness of the purification process of these derivatives (Davis and Johnston Chem Sci, 2011, 2: 1076-1079).

There is at least one p53-MDM2 interaction inhibitor in commercially unavailable phase I clinical trials, the cisline imidazole of the nutline series RG7112 or RO5045337 (Khoury et al. Med Chem Commun, 2011, 2: 246-260). This derivative presented as a clinical limitation a reduction in lymphocyte count in some patients (Wasserman, 2nd Annual CMOD Canadian Biomarkers and Surrogate Endpoints Meeting "Biomarkers and Personalized Medicine" May 17, 2010; Beryozkina et al. J Clinical Oncology, 2011 ASCO Annual Meeting Proceedings (Post-Meeting Edition), 2011, 29 (15 suppl May 20 Supplement): 3039). In addition, the nutlin, besides having several stereogenic centers, have limitations associated with their chemical structure namely stability, as well as difficulties inherent to their synthesis, analysis, and purification. The synthesis of nutlin-3A, a commercially available p53-MDM2 interaction inhibitor and used in laboratory studies of cellular processes, comprises nine steps for obtaining the racemate, with the need for further separation of the enantiomers by chiral critical fluid chromatography. . An enantioselective synthesis has been described by Davis and Johnston (Davis and Johnston, Chem Sci, 2011, 2: 1076-1079), involving 6 steps and costly catalysts. Additionally, resistance problems to nutlin derivatives have been described (Cheok et al. Nat Rev Clin Oncol, 2011, 8: 25-37; Bernal et al. Cancer Celi, 2010, 18: 411-422).

Other products commercially available and in preclinical trials, such as RITA and tenovin-6, act in the native form of p53 by other mechanisms. Rita binds directly to p53 blocking its ability to interact with MDM2 but not its ability to activate p53-dependent apoptosis (Grinkevich, et al. Cancer Celi, 2009, 15: 441-453). Tenovin-6 does not inhibit the p53-MDM2 interaction but rather the deacetylase activity of the SirT1 and SirT2 proteins. This small molecule protects MDM2-mediated p53 degradation with virtually no effect on p53 synthesis (Lain et al. Cancer Celi, 2008, 13: 454-463). Another MDM2 inhibitor in phase I clinical trials is JNJ- No. 26854165. JNJ-26854165 or Serdemetan, a tryptamine (4-pyridine) -1,4-diaminophenyl) derivative also marketed as a fine chemical, is described as inhibiting the MDM2-proteasome interaction.

For all the technical problems exposed, inhibition of the p53-MDM2 interaction still lacks molecules that are effective, stable, with viable synthetic processes and with therapeutic application.

The compounds described in the present invention have been found to be able to inhibit the p53-MDM2 interaction efficiently and may be useful in laboratory studies of cellular processes in which this p53-MDM2 interaction is involved and in the treatment of cancers expressing a form. native to p53 protein. It has recently been shown that RITA, a known p53-Mdm2 interaction inhibitor, was also capable of reactivating mutated forms of p53 (Zhao et al. Celi Cycle, 2010, 9: 1847-55). Additionally, nutlin-3A was shown to stabilize p73 protein by activating its transcriptional activity in tumor lines without p53 (Lau et al., Oncogene, 2008, 27: 997-1003). The compounds of the present invention have been found to exhibit inhibitory activities in mutated p53 forms as well as in p53 (p63 or p73) isoforms.

Summary of the invention

The present invention describes compound of formula 1,

 their salts or esters, in which:

Y represents oxygen, sulfur, CH ₂ or NH;

• Z represents C = 0, CH ₂ , CH-OH, C = NOH, C = NOCH ₃ , NO, NOH,

S = 0 or SO ₂ ;

 • R1-R8

independently represent hydrogen, hydroxy, aldehyde, halogen, trifluoromethyl, acetylene, carboxyl, cyano, nitro, SO ₂ H ₂ , aryl or heteroaryl substituted by halogen or hydroxy or methoxy, amine, aminoalkoxy, aminoaryl, imine, alkyl, cycloalkyl alkyl halogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as aryl-substituted alkyl, furan or dioxane-substituted pyran or pyran, or halogen or hydroxyl-substituted aryl or alkoxy (di) oxyalkyl or alkoxy ;

wherein if one of R1-R8 represents an amino and / or aminoalkyl group it contains counterions such as HCO3 ^" , C0 ₃ ^2" , Cl ^" , NH2C 6H4 S O3 ^" , 1-CH ₃ C ₆ H ₂ -3- 4 OH (CHCH 3) - 6-SC> 3 ^~ that are coordinated or ionically bonded to the amine;

 Wherein at least one of R1-R8 is not hydrogen;

in the treatment of diseases that are positively influenced by inhibition of the p53-MDM2 or p53-MDMX interaction. In particular in the treatment of cancer, preferably in the treatment of cancer, namely cancer of the breast, colorectal, prostate, kidney, lung, melanoma, osteosarcoma or embryonic nephroma, among others. embodiment of the compound of formula 1 wherein:

 Y represents oxygen, sulfur;

 Z represents C = 0;

 R1-R8

 independently of one another represent hydrogen, hydroxy, aldehyde, halogen, amine, aminoalkoxy, alkyl, alkylhalogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl-substituted pyran or pyran, or dioxane or dioxane substituted by aryl, or aryl substituted by halogen or hydroxy or alkoxy or (di) oxyalkylene;

in which one of R1-R8 is an amino group and / or amino it contains counter ions such as ^Cl⁻, which is ionically bonded to the amine;

 wherein at least one of R1-R8 is not hydrogen.

In another embodiment of the compound of formula 1, wherein:

 • Y represents oxygen;

 • Z represents C = 0;

 • R1-R8

independently of one another represent hydrogen, hydroxy, aldehyde, halogen, alkyl, alkyl halogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl-substituted pyran or pyran, or aryl-substituted dioxane or dioxane, or aryl replaced for halogen or hydroxy or alkoxy or alkylene (di) oxyl;

 Wherein at least one of R1-R8 is not hydrogen.

In another embodiment of the compound of formula 1, wherein:

 • Y represents sulfur;

 • Z represents C = 0;

 • R1-R8

 independently of each other represent hydrogen, hydroxy, halogen, amine, aminoalkoxy, alkyl, alkylhalogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as pyran or alkyl-substituted pyran;

in which one of R1-R8 is an amino group and / or amino it contains counter ions such as ^Cl⁻, which is ionically bonded to the amine;

 Wherein at least one of R1-R8 is not hydrogen.

In another embodiment of the compound of formula 1, wherein the compounds may be: 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6 -one; 1,2-dihydroxyixantone; 1- {[2- (diethylamino) ethyl] amino} -4-propoxythioxantone; 1 - {[2- hydrochloride

(diethylamino) ethyl] amino} -4-propoxythioxantone; (2R, 3R) -2- (hydroxymethyl) -3- (8-methoxy-2,2-dimethylchroman-6-yl) -2H- [1,4] dioxino [2,3-c] xanthen-7 (3H ) -one; 1-aminoalkyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone; halogen-4-hydroxy-2,2-dimethyl dihydropyranoxantone; 1-bromo-4-hydroxy-2,2-dimethyl dihydropyranoxantone; carbaldehyde-3,4 dialkoxyxanthone; 1-Carbaldehyde-3,4-dimethoxyxanthone, among others.

Another aspect of the present invention is the description of compound of formula 1, salts or esters thereof, wherein

Y represents oxygen or sulfur;

 • Z represents C = 0;

 • R1-R8 independently of one another represent hydrogen, hydroxyl, aldehyde, halogen, alkyl, alkyl halogen, amine, alkoxy, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl substituted pyran or pyran or dioxane or aryl substituted dioxane, or halogen or hydroxy substituted aryl or alkoxy or (di) oxyalkylene;

 R 1 represents carbaldehyde or methyl halogen or alkylamine, and R 3 and R 4 represent alkoxy; or R 2 and R 3 represent 2,2-dimethyl dihydropyran wherein at least one or more of R 1, R 4 -R 8 represents halogen, methyl halogen, hydrogen or alkylamine

In another embodiment of the compound of formula 1, wherein the compounds may be: 1-aminoalkyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone; halogen-4-hydroxy-2,2-dimethyl dihydropyranoxantone; halogenomethyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone; 1-bromo-4-hydroxy-2,2-dimethyl dihydropyranoxantone; carbaldehyde-3,4-dialkoxyxanthone; halogenomethyl-3,4-dialkoxyxanthone; 1-Carbaldehyde-3,4-dimethoxyxanthone. In a preferred embodiment, the novel compounds are used in medicine or medicine.

Another aspect of the present invention is pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically active amount of the free compound, salts or esters thereof described above. In a preferred embodiment, the pharmaceutical composition may be administered topically, orally, parenterally or injectably.

In another embodiment the pharmaceutical composition may further comprise a chemotherapeutic agent such as etoposide.

The term "alkyl" denotes alkanes, alkenes and alkynes, including branched, isomers and stereoisomers, preferably C1 -C20.

The term "aryl" denotes monovalent or bivalent aromatics preferably phenyl, naphthyl, toluyl, xylyl.

The term "heteroaryl" denotes heterocyclic aromatic ring systems, preferably one to three carbons is substituted by a nitrogen, oxygen or sulfur heteroatom.

The term "alkoxy" denotes any alkyl attached to an oxygen atom, preferably C 1 -C 20, even more preferably methoxy, ethoxy, propoxy or isopropoxy, butyloxy, including multiple chains such as ethoxy ethoxy and substituted alkoxy chains such as diethylaminoethoxy. The term "halogen" denotes fluorine, chlorine, bromine or iodine.

The term "aldehyde" denotes aldehydes, preferably C1 -C3, namely carbaldehyde, acetaldehyde, propaldehyde.

The term "pharmaceutically acceptable carrier" denotes a pharmacologically acceptable diluent, solvent, adjuvant or excipient that is substantially non-toxic to the subject to which it is to be administered.

Additionally, the compounds described in the present invention exhibit elevated activities in p53-MDMX interaction, in mutated forms of p53 as well as in p53 (p63 and p73) isoforms.

The compounds described in the present invention also have advantages over inhibitors described above with respect to the method of obtaining and stability. The described compounds are obtained by a more efficient synthetic process compared to the nutlin derived inhibitors, i.e. their synthesis proceeds through a smaller number of steps (in some examples, carried out in just two steps from the respective ones). commercially available raw materials), some being isolated in the form of salts by a purification process under favorable environmental conditions and yields of up to 85-6.

Accordingly, the substances of the present invention are considered potential pharmacological agents for studies of cellular processes in which this p53-MDM2 interaction is involved and therapeutic agents in the oncological field. Description of the invention

 The present invention relates to (thio) xanthones and their analogues as inhibitors of the p53-MDM2 interaction, and the use of these substances in laboratory studies of cellular processes in which this p53-MDM2 interaction is involved. These compounds activate the native form of the p53 tumor suppressor protein by inhibiting its interaction with the endogenous inhibitor MDM2. These substances further represent potential therapeutic agents in the treatment of cancer since by activating the p53 protein they induce cell cycle arrest and apoptosis in tumor cells and can be used alone or in combination with other therapies, in particular with chemotherapeutic agents such as etoposide. The present invention provides for an improvement over existing p53-MDM2 interaction inhibiting agents in terms of efficacy and attainment, as their synthetic process, which takes place in only four steps and in high yields, is suitable for an industrial scale. This invention also relates to the use of these novel substances in the treatment of cancers that express: a native form of the p53 protein and overexpress the endogenous inhibitor MDMX; p53 isoforms (p63 and p73); or mutated forms of p53.

 This invention is part of the technical field of fine chemicals and cancer medicines.

Description of the Figures Fig. 1 - General formula 1 of the (thio) xanthonic derivatives and their analogs.

Fig. 2 - Illustrative scheme of the p53 protein transactivation assay in yeast co-expressing the human p53 (native form) and MDM2 proteins. This artificial system utilizes a yeast diploid reporter strain co-transformed with vectors encoding each of human proteins p53 and MDM2 under the control of an inducible promoter (GAL1-10). Additionally, it contains a DNA sequence integrated into the yeast XV chromosome containing the response element (ER; Ex .: PUMA gene); a minimal promoter (Pcycl) and a reporter gene (Luciferase). The transcriptional activity of p53 protein was evaluated by quantifying the luciferase activity used as a reporter gene, which in turn is directly proportional to the amount of light emitted. Luminescence, expressed as relative light units (RLU), was measured using a luminometer.

Fig. 3 - A compound of the present invention, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1), reduces the inhibitory effect of MDM2 protein on p53 protein transcriptional activity in yeast p53 transactivation assays. The commercial inhibitor of p53-MDM2 interaction (nutline-3A) was used as a positive control; The compounds were tested at a concentration of 10 μΜ. The effect of the compounds was evaluated after 16 hours of treatment. The transcriptional activity of p53 protein was evaluated at the PUMA gene level and considered 100% for yeasts incubated only in the presence of solvent dimethyl sulfoxide (DMSO). The results presented correspond to the mean ± standard error of the mean of four independent experiments.

Fig. 4 - A compound of the present invention, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one

(LEM1), reduces the inhibitory effect of MDM2 protein on p53 protein transcriptional activity in p53 transactivation assays in breast adenocarcinoma (MCF7) cell lines. The commercially available p53-MDM2 nutlin-3A interaction inhibitor and the p53 doxorubicin transcriptional activator (DOXO) were used as positive controls; DOXO was tested at a concentration of 1.5 μΜ; nutlin-3A and 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) were tested at a concentration of 10 μΜ; Yeasts incubated only in the presence of solvents (water or DMSO) were used as negative controls. The cell lines used express the native form of the p53 protein and overexpress the MDM2 protein and were transfected with the artificial pG13 gene. The effect of the compounds was evaluated after 16 hours of treatment. The transcriptional activity of p53 protein was evaluated by quantifying the luciferase activity used as a reporter gene, which in turn is directly proportional to the amount of light emitted. Luminescence, expressed as relative light units

(URL) was measured using a luminometer. Results correspond to the mean ± standard error of the mean of two independent experiments.

Fig. 5 - A compound of the present invention, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1), reduces the inhibitory effect of MDM2 protein on p53 protein transcriptional activity in p53 transactivation in colon carcinoma cell lines (HCT116 p53 ^{+ +} ). The commercially available p53-MDM2 nutlin-3A interaction inhibitor and the p53 doxorubicin transcriptional activator (DOXO) were used as positive controls; DOXO was tested at a concentration of 1.5 μΜ; nutlin-3A and 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) were tested at a concentration of 10 μΜ; Yeasts incubated only in the presence of solvents (water or DMSO) were used as negative controls. The cell lines used express the native form of the p53 protein and overexpress the MDM2 protein and were transfected with the artificial pG13 gene. Cell lines that do not express p53 protein (HCT116 p53 ^- ) were used as a negative control. The effect of the compounds was evaluated after 16 hours of treatment. The transcriptional activity of p53 protein was evaluated by quantifying the luciferase activity used as a reporter gene, which in turn is directly proportional to the amount of light emitted. Luminescence, expressed as relative light units (RLU), was measured using a luminometer. Results correspond to the mean ± standard error of the mean of two independent experiments.

Fig. 6 - A compound of the present invention, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1), led to increased expression levels of (A) p21 and (B) Bax proteins in colon carcinoma cell lines (HCT116 p53 ^{+ +} ). The results presented were obtained after 8 hours of treatment with 10 μΜ of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one ( LEM1). For the detection of p21 and Bax proteins polyclonal anti-p21 and monoclonal anti-Bax antibodies were used, respectively. Actin protein was used as charge control. Cells treated only in the presence of solvent (DMSO) were used as a negative control. Immunoblots were developed using a chemiluminescence amplifier kit.

Fig. 7 - A compound of the present invention, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1), led to an increase in p53 protein levels in colon carcinoma cell lines (HCT116 p53 ^{+ +} ). Results shown correspond to 8 and 16 hour treatments with 10 μΜ of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1). For detection of p53 protein an anti-p53 monoclonal antibody was used. Actin was used as charge control. Cells treated only in the presence of solvent (DMSO) were used as a negative control. Immunoblots were developed using a chemiluminescence amplifier kit.

Table 1 below shows the effect of other compounds of formula 1 on inhibiting the p53-MDM2 interaction using the yeast model. Nutlina-3A (commercially available) was used as a positive control. The effect obtained with yeast expressing p53 protein alone was considered as 100%. Results correspond to the mean ± standard error of the mean of five independent experiments. Table 1: Evaluation of the effect of other compounds of formula 1 on inhibition of p53-MDM2 interaction using the yeast model.

Compounds% effect of p53

DMSO 0.2 ± 4.2

Nutlina-3A ™ 67.8 ± 3.3

3,4-dihydro-12-hydroxy-2,2-dimethyl

55.8 ± 5.5 2H, 6H-pyran [3,2-b] xanthen-6-one

 1-Carbaldehyde-3,4-dimethoxyxanthone 100 ± 7.1

1,2-dihydroxyixantone 100 ± 2.7

(2R, 3R) -2- (hydroxymethyl) -3- (8-methoxy-2,2-dimethylchroman-6-yl) -2H-82.8 ± 6.6 [1,4] dioxino [2,3-c] xanthan 7 (3H) -one

 In this assay, the transcriptional activity obtained with yeast expressing only the native form of p53 incubated in the presence of solvent (DMSO) was considered as 100% effect. The transcriptional activity (-0%) obtained with yeast co-expressing the p53 and MDM2 proteins incubated only in the presence of the solvent (indicated in the table as DMSO) indicates that a total reversal of p53 transcriptional activity by the MDM2 protein occurred. Inhibitors of p53-MDM2 interaction are characterized by reestablishing p53 transcriptional activity by at least 50%. Based on this, the 1-carbaldehyde-3,4-dimethoxyxanthone and 1,2-dihydroxixantone compounds fully reestablished the transcriptional activity of p53 (100% effect), finding that they are very potent compounds.

In this invention are described (thio) interaction inhibitor xanthones p53-MDM2 with application in laboratory studies of cellular processes in which this p53-MDM2 interaction is involved and in the treatment of cancers expressing a native form of p53 protein, an improvement in the characteristics of commercially available p53-MDM2 interaction inhibitors is expected with respect to its efficacy. and / or attainment and / or stability.

These molecules have a dibenzo-γ- (thio) pyronic or (thio) xanthonic nucleus or derivative according to general formula 1.

 pharmaceutically acceptable salts or esters in

Y represents oxygen, sulfur, C¾ or N-H;

Z represents C = 0, CH ₂ , CH-OH, C = NOH, C = NOCH ₃ , NO, NOH, S = 0 or SO ₂ ;

 • R1-R8

independently represent hydrogen, hydroxy, aldehyde, halogen, trifluoromethyl, acetylene, carboxyl, cyano, nitro, SO ₂ H ₂ , aryl or heteroaryl substituted by halogen or hydroxy or methoxy, amine, aminoalkoxy, aminoaryl, imine, alkyl, cycloalkyl alkyl halogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as pyran or pyran substituted by alkyl, furan or dioxane or dioxane substituted by aryl, or halogen or hydroxy substituted aryl or alkoxy or (di) oxyl alkylene;

wherein if one of R1-R8 represents an amino and / or aminoalkyl group it contains counterions such as HCl ₃ ^" , C0 ₃ ^2" , Cl ^" , H ₂ C ₆ H ₄ SO 3 ^" , 1-CH ₃ C ₆ H ₂ -3-OH- 4 (CHCH ₃ ) - 6-SO ₃ ^- which are coordinated or ionically bonded to the amine;

wherein at least one of R1-R8 is not hydrogen.

In one embodiment of the compound of formula 1 wherein:

 Y represents oxygen, sulfur;

 • Z represents C = 0;

 • R1-R8

 independently of one another represent hydrogen, hydroxy, aldehyde, halogen, amine, aminoalkoxy, alkyl, alkylhalogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl-substituted pyran or pyran, or dioxane or dioxane substituted by aryl, or aryl substituted by halogen or hydroxy or alkoxy or (di) oxyalkylene;

 wherein if one of R1-R8 represents an amino and / or aminoalkyl group it contains counterions such as Cl- which is ionically bonded to the amine;

 Wherein at least one of R1-R8 is not hydrogen.

In another embodiment of the compound of formula 1, wherein:

 • Y represents oxygen;

• Z represents C = 0; • R1-R8

 independently of one another represent hydrogen, hydroxy, aldehyde, halogen, alkyl, alkyl halogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl-substituted pyran or pyran, or aryl-substituted dioxane or dioxane, or aryl substituted by halogen or hydroxy or alkoxy or (di) oxyalkylene;

 Wherein at least one of R1-R8 is not hydrogen.

In another embodiment of the compound of formula 1, wherein:

 • Y represents sulfur;

 • Z represents C = 0;

 • R1-R8

 independently of each other represent hydrogen, hydroxy, halogen, amine, aminoalkoxy, alkyl, alkylhalogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as pyran or alkyl-substituted pyran;

 wherein if one of R1-R8 represents an amino and / or aminoalkyl group it contains counterions such as Cl- which is ionically bonded to the amine;

 Wherein at least one of R1-R8 is not hydrogen.

In another embodiment of the compound of formula 1, wherein the compounds may be: 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6 -one; 1,2-dihydroxyixantone; 1- {[2- (diethylamino) ethyl] amino} -4-propoxythioxantone; 1 - {[2- hydrochloride (diethylamino) ethyl] amino} -4-propoxythioxantone; (2R, 3R) -2-

(hydroxymethyl) -3- (8-methoxy-2,2-dimethylchroman-6-yl) -2H- [1,4] dioxino [2,3-c] xanthen-7 (3H) -one; 1-aminoalkyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone; halogen-4-hydroxy-2,2-dimethyl dihydropyranoxantone; 1-bromo-4-hydroxy-2,2-dimethyl dihydropyranoxantone; carbaldehyde-3,4-dialkoxyxanthone; 1-Carbaldehyde-3,4-dimethoxyxanthone, among others.

Still another aspect of the invention relates to the use of these substances of general formula 1 indicated in Fig. 1 in laboratory studies of cellular processes in which the p53-MDM2 interaction is involved.

Synthesis of xanthonic (thio) nucleus compounds can be accomplished by methods described by Sousa and Pinto (Sousa, ME and Pinto MMM, Curr Med Chem, 2005, 12: 2447-2479; Azevedo, CM, Afonso, CM and Pinto, MM (2012), Current Organic Chemistry, 16 (23): 2818-2867. Andreia et al., Biochem Pharmacol, 2012, 83 (1): 57-68).

Another aspect of the present invention is the description of compound of formula 1, salts or esters thereof, wherein

Y represents oxygen or sulfur;

 • Z represents C = 0;

• R1-R8 independently of one another represent hydrogen, hydroxyl, aldehyde, halogen, alkyl, alkyl halogen, amine, alkoxy, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl substituted pyran or pyran or dioxane or aryl substituted dioxane, or halogen or hydroxy substituted aryl or alkoxy or (di) oxyalkylene; R 1 represents carbaldehyde or methyl halogen or alkylamine, and R 3 and R 4 represent alkoxy; or R 2 and R 3 represent 2,2-dimethyl dihydropyran wherein at least one or more of R 1, R 4 -R 8 represents halogen, methyl halogen, hydrogen or alkylamine

In another embodiment of the compound of formula 1, wherein the compounds may be:

1-aminoalkyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone; halogen-4-hydroxy-2,2-dimethyl dihydropyranoxantone;

halogenomethyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone; 1-bromo-4-hydroxy-2,2-dimethyl dihydropyranoxantone;

carbaldehyde-3,4-dialkoxyxanthone; halogenomethyl-3,4-dialkoxyxanthone; 1-Carbaldehyde-3,4-dimethoxyxanthone.

Another aspect of the invention relates to the preparation of compounds of formula 1, their salts or esters, wherein

Y represents oxygen or sulfur;

 • Z represents C = 0;

 • R1-R8 independently of one another represent hydrogen, hydroxyl, aldehyde, halogen, alkyl, alkyl halogen, amine, alkoxy, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl substituted pyran or pyran or dioxane or aryl substituted dioxane, or halogen or hydroxy substituted aryl or alkoxy or (di) oxyalkylene;

R 1 represents carbaldehyde or methyl halogen or alkylamine, and R 3 and R 4 represent alkoxy; or R2 and R3 represent 2,2-dimethyl dihydropyran, wherein at least one or more of R1, R4-R8 represents halogen, methyl halogen, hydrogen or alkylamine.

by introducing carbaldehydes or methyl halogens in 3,4-dialkoxy (thio) xanthones or halogens or methyl halogens or amines in pyran or dioxane (thio) xanthones. In a preferred embodiment, the novel compounds are used in medicine or medicine.

The compounds of formula 1 wherein R1 represents carbaldehyde, and R3 and R4 represent alkoxy may be obtained by alkylation of 1-carbaldehyde-3-alkoxy-4-hydroxy (thio) xanthones which may be obtained from 3-alkoxy-4- hydroxy (thio) xanthones with the addition of a formylating agent such as hexamethylenetetramine in a solvent such as (trifluoro) acetic acid. The reaction should be refluxed for 18 hours, cooled and after addition of an aqueous hydrochloric acid solution heated at reflux for 15 minutes. The reaction mixture should be filtered and the solid thus obtained may be alkylated using an alkylating agent such as methyl sulfate in the presence of a base such as potassium carbonate. The reaction should be carried out at reflux temperature. 3-Alkoxy-4-hydroxy (thio) xanthones should be obtained by selective demethylation of 3,4-dialkoxy (thio) xantone using the aluminum chloride desalcylating agent in a solvent such as anhydrous toluene for a period of 30 minutes. at reflux. The reaction mixture should be poured into crushed ice and extracted with chloroform, followed by sodium carbonate and chloroform again. The organic phases must be combined, dried over anhydrous sodium sulfate and evaporated to dryness. Crystallization of chloroform should give 3-alkoxy-4-hydroxy (thio) xanthones.

 Compounds of formula 1 wherein R 1 represents a methyl halogen may for example be obtained by reaction of 4-hydroxy (thio) xanthones with hydrochloric acid or hydrobromic acid in the presence of an excess of formaldehyde. The reaction mixture is stirred overnight at room temperature. The compound which insolubilizes in the reaction medium is filtered, washed with water and crystallized from ethyl acetate.

 Compounds of formula 1 wherein R 2 and R 3 represent 2,2-dimethyl dihydropyran and wherein at least one or more of R 1, R 4 R 8 represents halogen may be obtained by reaction of a pyran (thio) xanthone with a halogen donor such as N-bromosuccinimide, which should be in excess in ammonium acetate and in an inert solvent such as methanol. The reaction should be carried out at room temperature. The compounds are extracted with non-polar organic solvent, preferably with diethyl ether. The obtained organic solution is washed with water, dried over anhydrous sodium sulfate and filtered. The solvent is evaporated and if necessary, the product thus obtained may be subjected to purification methods such as using silica chromatographic plates, the compounds of the present invention being eluted with different gradients of n-hexane: ethyl acetate.

The compounds of formula 1 may be obtained by reacting a pyran (thio) xanthone with a substituted primary or secondary amine, the amine being in excess together with a basifying agent, for example potassium carbonate in stoichiometric proportions, and a catalyst in catalytic quantity, such as copper iodide. Compounds should be dissolved or suspended in inert solvent, preferably N-methylpyrrolidone (NMP). The reaction should be performed in an open microwave reactor vessel. The stirred reaction mixture is irradiated at 400 W for 50 min at final temperatures of 200-205 ° C. The reaction mixture is filtered and the compounds extracted with nonpolar organic solvent, preferably with diethyl ether. The NMP solution may be basified prior to extraction into preferred compounds of formula 1. The obtained organic solution is washed with water, dried over anhydrous sodium sulfate and filtered. The solvent is evaporated and if necessary the product thus obtained may be subjected to purification methods such as using silica chromatography columns (GraceResolv), the compounds of the present invention being eluted with different gradients of n-hexane: ethyl acetate. Salts of the preferred compounds of general formula 1 are obtained by the addition of an acid, preferably hydrochloric acid, to an organic solution of the isolated compounds, preferably of diethyl ether. The salt formed is filtered, washed with organic solvent and dried.

The compounds of formula 1 wherein R 1 represents an alkylamine may, for example, be obtained by reductive amination of 1-carbaldehyde (thio) xanthones using methanol as solvent, by addition of an alkylamine, acetic acid and a reducing agent such as cyanoborohydride. on solid support, it should be stirred at room temperature overnight. The reaction mixture should be filtered and subjected to purification by cationic ion exchange chromatography (Discovery ® DSC-SCX SPE cartridges). Compounds of formula 1 wherein R 1 represents an alkylamine may also be obtained by reaction of 4-hydroxy (thio) xanthones with formaldehyde and an alkylamine in the presence of a solvent such as methanol at reflux for 10 minutes. hours The final suspension should be filtered and the solid washed with methanol. The compound will be obtained by crystallization from ethyl acetate.

 The novel derivatives of formula 1 of Fig. 1 may also be obtained by aromatic nucleophilic substitution reactions in the (thio) xanthone nucleus by methods described (Shargui et al., J Iran Chem Soe, 2008, 5, S33-S39, Xu and Wolf, Chem Comm Chem Commun, 2009, 1715-1717).

Still another aspect of the invention relates to the use of compounds of formula 1 indicated in Fig. 1 as inhibitors of p53-MDM2 interaction.

In the works described herein, the antiproliferative effect of a library of xanthonic derivatives on various tumor lines, namely MCF7 and MDA-MB-231 breast adenocarcinoma cell lines, was examined. The results obtained revealed that preferred compounds of the general formula 1 indicated in Fig. 1, namely 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen -6-one (LEM1), are potent inhibitors of MCF7 line proliferation, having a much lower effect on MDA-MB-231 line (Paiva et al., Med. Chem. Res. 2011, DOI 10.1007 / s00044-011- 9562-Z; Castanheiro et al., Bioorg Med. Chem., 2007, 15: 6080-6088). Based on the major cellular differences between these two types of breast adenocarcinomas, these results showed that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanten-6-one (LEM1) was more potent in tumor lines with a native form of p53 and MDM2 protein overexpression (MCF7) than in tumor lines with a mutated form of p53 (MDA-MB-231). In order to elucidate the molecular mechanism of action of compounds of formula 1 indicated in Fig. 1, the effect of these compounds on the p53-MDM2 interaction was evaluated. To this end, a yeast cell transactivation assay co-expressing the human proteins p53 (native form) and MDM2, implemented for the large-scale screening of p53-MDM2 interaction inhibitors as described (Andreotti et al. ., Pios One 2011, 6: e20643). In this assay, MDM2 protein, as observed in mammalian cells, inhibits the transcriptional activity of p53 protein (Fig. 2). An inhibitor of p53-MDM2 interaction is characterized by being a compound capable of inhibiting the effect of MDM2 protein by restoring the transcriptional activity of p53 protein. In this assay, as well as the commercial p53-MDM2 interaction inhibitor, nutlin-3A, preferred compounds of the general formula 1 indicated in Fig. 1 which include 3,4-dihydro-12-hydroxy-2,2-dimethyl 2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1), 1-carbaldehyde-3,4-dimethoxyixantone, 1,2-dihydroxyixantone, 1- {[2- (diethylamino ) ethyl] amino} -4-propoxythioxantone, (2R, 3R) -2- (hydroxymethyl) -3- (8-methoxy-2,2-dimethylchroman-6-yl) -2H- [1,4] dioxino [ 2,3-c] xanthen-7 (3H) -one, 1-chloro-4-propoxy-9H-thioxanten-9-one, 1- (isobutylamino) -4-propoxy-9H-thioxanten-9-one and 1- (isopropylamino) -4-propoxy-9H-thioxanthen-9-one and its hydrochloride salts, in particular, were able to reverse the inhibitory effect of MDM2 protein on p53 protein activity. The results obtained in these assays thus indicated that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) was a potential inhibitor of p53-MDM2 interaction of potency similar to that of the nutlina-3A compound. In order to confirm the results obtained in the yeast cell screening assay, activity in human tumor cells was secondly analyzed. To this end, p53 protein transactivation assays were performed in human tumor cell lines expressing the native form of the p53 protein and overexpressing the MDM2 protein, namely in breast adenocarcinoma cell lines (MCF7) and carcinoma cell lines. of colon (HCT116 p53 ^{+ +} ). The results obtained in the MCF7 line confirmed that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) reduced similar to the nutline-3A inhibitor and activator of p53 doxorubicin transcriptional activity (DOXO), the effect of the MDM2 protein on the level of p53 transcriptional activity. At the level of the HCT116 p53 ^{+ + line,} it was found that the increased transcriptional activity of p53 protein obtained with 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2 -b] xanthen-6-one (LEM1) was much higher than that obtained with the commercial inhibitor nutlina-3A. These results confirmed that compounds of formula 1 indicated in Fig. 1, namely 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6 -one (LEM1) was able to inhibit MDM2 protein activity by increasing the transcriptional activity of p53 protein in human tumor cells. The activating effect on the transcriptional activity of p53 protein was further confirmed by analyzing cell expression levels of proteins encoded by genes whose transcription is regulated by p53 protein, such as p21 proteins (protein involved in the cell cycle) and Bax (pro-apoptotic protein), in HCT116 p53 ^{+ +} lines. The results obtained showed that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanten-6-one (LEM1) led to an increase in cellular levels of these proteins, confirming the results of p53 transactivation assays in this tumor line.

Finally, since MDM2 protein not only inhibits the transcriptional activity of p53 protein, but also leads to its proteosomal degradation, an inhibitor of p53-MDM2 interaction should also lead to an increase in p53 protein cell levels. Thus, the effect of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) on the levels of p53 expression in HCT116 p53 ^{+ / +} lines. 3,4-Dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) led to an increase in protein levels of p53 such as as expected .

Taken together, the experimental results described in this patent confirm that compounds of formula 1 indicated in Fig. 1, which preferably include 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1), 1-carbaldehyde-3,4-dimethoxyixantone, 1,2-dihydroxyixantone, 1- {[2- (diethylamino) ethyl] amino} 4-propoxythioxantone, (2R, 3R) -2- (hydroxymethyl) -3- (8-methoxy-2,2-dimethylchroman-6-yl) -2H- [1,4] dioxino [2,3-c ] xanthen-7 (3H) -one, 1-chloro-4-propoxy-9H-thioxanten-9-one, 1- (isobutylamino) -4-propoxy-9H-thioxanten-9-one and 1- ( isopropylamino) -4-propoxy-9H-thioxanthen-9-one and its hydrochloride salts, namely 1- ((2- (diethylamino) ethyl) amino) -4-propoxy-9H-thioxanten-9-one hydrochloride , are inhibitors of the p53-MDM2 interaction having the potency of 1-carbaldehyde-3,4-dimethoxyxanthone and 1,2-dihydroxixantone being superior to that of nutlin-3A in the yeast assay. Although, in general, the potency of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) has been revealed similar to that of the commercial inhibitor nutlina-3A in colon carcinoma cell lines (HCT116 p53 ^{+ +} ), compound LEM1 revealed a much higher potency than that of nutlina-3A. These results indicate that for some types of human tumors, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1 ), 1-Carbaldehyde-3,4-dimethoxy-xantone and 1,2-dihydroxy-xantone may represent advantageous pharmacological alternatives to the compound nutlin-3A.

 Based on the foregoing, the substances of the present invention have pharmacological applications either in laboratory studies of cellular processes in which p53-MDM2 interaction is involved, or as therapeutic agents in the treatment of cancers expressing a native form of p53 protein.

The substances of the present invention also have advantages over the commercial inhibitors described above with respect to the method of obtaining. The substances of the present invention are obtained by a more efficient synthetic process compared to the described inhibitors such as nutlin-3A and RITA, ie their synthesis takes place in a smaller number of steps (in some instances, performed in only two steps). from their commercially available raw materials). Additionally, some substances of the present invention are isolated in the form of salts by a purification process under favorable environmental conditions, with yields of up to 85-6 ° C.

Accordingly, another aspect of the invention relates to the process for the preparation of a pharmaceutical composition consisting of the combination of at least one compound of general formula 1 indicated in Fig. 1 with an inert vehicle or excipient. The pharmaceutical composition may consist of the combination of at least one compound of formula 1 indicated in Fig. 1 with an anticancer agent and an inert carrier or excipient.

 The pharmaceutical composition may be presented as injectables, tablets, capsules, creams with a pharmaceutically acceptable carrier or excipient. Adjuvants may correspond to one or more substances acting as diluents, flavors, sweeteners, solubilizers, lubricants, suspending agents, binders or disintegrating agents and may further comprise an incapsulant agent.

 The compounds of the present invention may be administered internally, for example, orally, parenterally, intraperitoneally, or extracorporeally, for example, topically.

The present invention describes (thio) xanthones of general formula 1 and their analogues with inhibitory activity of p53-MDM2 interaction and / or p53-MDMX interaction.

The antiproliferative effect of a library of xanthonic (thio) derivatives was analyzed at the level of several tumor lines, namely the MCF7 and MDA-MB-231 breast adenocarcinoma cell lines, by the sulforodamine-B cell growth assay. The results obtained revealed that preferred compounds of the general formula 1 indicated in Fig. 1, namely 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen (LEM1), 1-Carbaldehyde-3,4-dimethoxyxanthone, 1,2-dihydroxyixantone, 1- {[2- (diethylamino) ethyl] amino} -4- propoxythioxantone and (2R, 3R) -2- (hydroxymethyl) -3- (8-methoxy-2,2-dimethylchroman-6-yl) -2H- [1,4] dioxino [2,3-c] xanthene 7 (3H) -one are potent inhibitors of MCF7 line proliferation, notably 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthene. 6-one (LEM1) a value of IC5 ₀ (concentration required to produce 50% inhibition of cell growth) of 5.3 ± 0.7 μΜ after 48 hours of treatment; in contrast, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) had a much lower effect on the MDA line. -MB-231 with an IC5 ₀ value of 29.0 ± 3.6 μΜ after 48 hours of treatment, about six times greater than that obtained in the lines MCF7

(Paiva et al., Med. Chem. Res. 2011, DOI 10.1007 / s00044-011- 9562-z). Although in the work of Paiva et al. (2011) if we try to explain this difference in effects of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one

 (LEM1) through the presence and absence of estrogen receptors in the MCF7 and MDA-MB-231 lines respectively, it is found that unlike the MCF7 line which has a native form of p53 protein and an overexpression of MDM2 protein, the MDA line -MB-231 shows a mutated form of p53 protein. Thus, it is considered in the present invention that a possible explanation for this difference in effects of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen -6-one (LEM1) may be based on a possible activation of protein p53 by 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthenyl-6-one. (LEM1) by inhibiting its interaction with the overexpressed endogenous inhibitor MDM2 in the MCF7 lines.

In order to elucidate the molecular mechanism of action of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) , the effect of this compound on the p53-MDM2 interaction was evaluated. Initially, transactivation assays were performed with Saccharomyces cerevisiae yeast cells co-expressing human proteins p53 (native form) and MDM2, implemented for large-scale screening of p53-MDM2 interaction inhibitors (Andreotti et al. , Pios One 2011, 6: e20643) (Fig. 2). In this assay, MDM2 protein, as observed in mammalian cells, inhibits the transcriptional activity of p53 protein.

 In this p53 protein transactivation assay in yeast it was confirmed that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1 ), similar to the commercial inhibitor nutlina-3A, at a concentration of 10 μΜ, reduced the inhibitory effect of MDM2 protein on the level of p53 protein transcriptional activity (Fig. 3).

Taken together, the results obtained from the yeast screening assays indicated that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6 -one (LEM1) was a potential inhibitor of p53-MDM2 interaction of similar potency to the commercial compound nutlina-3A.

In a second step, the activity of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) was confirmed. as an inhibitor of p53-MDM2 interaction in human tumor cell lines. For this purpose p53 protein transactivation assays were performed in human tumor cell lines expressing the native form of p53 protein and overexpressing the MDM2 protein, namely breast adenocarcinoma (MCF7) and colon carcinoma cell lines (HCT116). p53 ^{+ +} ), transfected with the artificial gene pG13 (used as a response element). The effect of the compounds 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) and nutlin-3A, at a concentration of 10 μΜ, on the level of transcriptional activity of p53 protein in MCF7 and HCT116 lines (p53 ^{+ +} and p53 ^{_ ~} ) was evaluated after 48 hours of treatment. The results obtained in the MCF7 line confirmed that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) reduced similar to that observed with the compound nutlina-3A and the transcriptional activator of p53 doxorubicin (DOXO), the inhibitory effect of the MDM2 protein on the transcriptional activity of the p53 protein (Fig. 4).

With the HCT116 p53 ^{+ + line} , it was found that the increased transcriptional activity of p53 protein obtained with 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2 -b] xanthen-6-one (LEM1) was very similar to that obtained with the commercial inhibitor nutlina-3A (Fig. 5). The specificity of the effect of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) on the p53-MDM2 interaction HCT116 p53 ⁺ lines was confirmed by testing the compound in a cell line lacking p53 HCT116 p53 ^{_ ~.} In these lines, the increased transcriptional activity of p53 protein was completely negated (Fig. 5).

The increased transcriptional activity of p53 protein by 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) has also been confirmed. by analyzing the cellular expression levels of proteins encoded by genes whose transcription is regulated by p53 protein, such as p21 and Bax proteins, in HCT116 p53 ^{+ / +} lines treated with 10 μΜ of 3,4-dihydro Hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1). The results obtained demonstrated that after 8 hours of treatment, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H- pyran [3,2-b] xanten-6-one (LEM1) induced an increase in cellular levels of p21 (Fig. 6A) and Bax (Fig. 6B) proteins, confirming the results of p53 transactivation assays in HCT116 lines p53 ^{+ / +} .

Since MDM2 protein not only blocks the transcriptional activity of p53 protein but also leads to its proteosomal degradation, an inhibitor of p53-MDM2 interaction should also cause an increase in p53 protein cell levels. Thus, the effect of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) on protein levels was analyzed. of p53 on p53 ^{+ +} HCT116 tumor lines. The results described show that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) at a concentration of 10 μΜ , leads to a marked increase in p53 protein levels after 16 hours of treatment (Fig. 7).

Taken together, the experimental results presented in the present invention show that 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1 ) is a potent inhibitor of p53-MDM2 interaction with pharmacological applications in laboratory studies of cellular processes in which p53-MDM2 interaction is involved, or as a therapeutic agent in the treatment of cancers expressing a native form of p53 protein. The results obtained further indicated that despite the compounds 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) and nutlin 3A inhibitors of p53-MDM2 interaction with similar potencies in some cancers (such as breast), their activities may differ in other cancers, as in the case of colon cancer. The highest potency of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) in the colon tumor indicates that in this type of tumor 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) may represent a advantageous pharmacological alternative to the compound nutlina-3A.

The experiments presented in the present invention will be described below in more detail.

Results were statistically analyzed using the SigmaStat 3.5 software. Differences between means were tested using the unpaired Student's t-test (P <0.05).

Examples

 For an easier understanding of the invention, the following are examples of preferred embodiments of the invention which, however, are not intended to limit the scope of the present invention.

Example 1: Synthesis of 1-Carbaldehyde-3,4-dimethoxyxanthone. To a solution of 1-Carbaldehyde-4-hydroxy-3-methoxyixantone

(0.67 mmol) in acetone (10 mL) was added potassium carbonate (1.2 mmol) and then methyl sulfate (0.12 mL). It was heated at reflux with magnetic stirring for 6 hours. After reaction, the suspension was filtered, the residue was washed with acetone and the solvent was evaporated. The obtained residue was dissolved in ethyl ether (20 mL), and washed successively with 5% sodium hydroxide (3x15 mL) and water.

(2 x 15 mL). After dehydration with anhydrous sodium sulfate, the solvent was evaporated to give an oily residue. brown. The residue was crystallized from ethyl acetate-n-hexane to yield 1-brown-white crystals of 1-carbaldehyde-3,4-dimethoxyixantone (80%).

 IR ν max (cm -1) (KBr): 3433, 2927, 1684, 1649, 1580, 1464, 1320, 1132, 758; 1H NMR (CDCl3): 511.20 (s, 1H, CHO), 8.30

(dd, 1H, J = 8.0Hz and J = 1.6Hz, H-8), 7.76 (ddd, 1H, J = 8.5Hz, J = 7.5Hz and J = 1.6Hz, H- 6), 7.59 (d, 1H, J = 8.5Hz, H-5), 7.54 (s, 1H, H-2), 7.42 (dd, 1H, J = 7.5Hz and J = 7.5Hz, H-7), 4.10 (s, 3H, OCH 3), 4.04 (s, 3H, OCH 3) ppm; 13 C NMR (CDCl 3): δ 192.7 (CHO); 177.8 (C-9), 156.3 (C-3), 155.4 (C-4b), 150.8 (C-4a), 140.6 (C-4), 134.7 (C -6), 133.5 (Cl), 126.7

(C-8), 124.4 (C-7), 122.0 (C-8b), 117.8 (C-5), 116.1 (C-8a),

108.4 (C-2) ppm; MS (EI): 284 (100), 299 (45), 342 (35) m / z; High resolution mass spectrometry (ESI): 388.394452 m / z.

Example 2: Synthesis of 5-bromo-12-hydroxy-2,2-dimethyl-3,4-dihydropyran [3,2-b] xant-6 (2H) -one.

 A mixture of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xant-6-one (0.132 mmol), N-bromosuccinimide (0.276 mmol) and ammonium acetate (0.013 mmol) in methanol (10 mL) was stirred at room temperature for a period of 5 hours. The crude reaction product was purified by preparative chromatography (Si02; hexane / ethyl acetate 5: 5). Crystallization from acetone gave white crystals for the compound (10%, 5mg): IHRMN (CDCl3): δ 8.28 (IH, dd, J = 7.97 and 1.57 Hz, H-7), 7, 78 (1H, ddd, J = 7.75, 6.98 and 1.67 Hz, H-9), 7.70 (1H, dd, J = 8.3 and 0.96 Hz, H-10), 7.50 (1H, ddd, J = 7.50, 7.02 and 1.24 Hz, H-8), 2.59 (2H, t, J = 6.57, H-4), 1.82 (2H, t, J = 6.60, H-3), 1.43 (6H, s, H-1a and H-1b) ppm; 13 C NMR (CDCl 3): 155.0 (C-10a), 154.4 (C-12a),

151.5 (C-1a), 135.1 (C-9), 126.7 (C-7), 126.4 (C-8), 119.8 (C-5a), 119.1 (C-6a), 117.2 (C-5), 78.8 (C-2), 31.3 (C-3), 26.7 (C-4) , 16.6 (C-1a and C-1b) ppm.

Example 3: Analysis of transcriptional activity of p53 protein: luciferase assay in yeast.

 For the transactivation experiments a diploid strain containing the yLFM-PUMA reporter genes was used.

(Firefly luciferase associated with the p53 protein-induced PUMA gene) and chromosomally integrated RFM-M2 (Renilla luciferase, control) as described in (Andreotti et al., PLoS ONE 2011, 6: e20643). This yeast was transformed by the lithium acetate method with plasmids encoding human p53 proteins.

(native form) and MDM2 and the selection of the co-transformed yeasts was done by cell growth in selective minimal medium as described above.

For luciferase assays, yeast cells were cultured in selective induction medium and incubated in 96-well plates in the presence of 10 μΜ of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2-methylamide compounds. 2H, 6H-pyran [3,2-b] xanthen 6-one (LEM1) or nutlin-3A, or only in the presence of solvent (0.1% DMSO). The transcriptional activity of p53 protein was determined by measuring the luminescence of cultures in the presence of a luminescent luciferase substrate.

(Firefly luciferase Bright Glo; Promega). Control luciferase activity (Renilla) was determined using the Luciferase AssayReagent substrate (Promega) followed by the addition of Stop & Glow buffer (Promega). Luminescence, expressed in relative light units (URLs), was measured using a plate reader.

Example 4: Analysis of p53 protein transcriptional activity: luciferase assay in cell lines. Analysis of transcriptional activity of p53 protein by luciferase assay was performed on MCF7 breast adenocarcinoma cell lines (InterLab Celi Line Collectlon, ICLC, Genoa, Italy) and on HCT116 colon carcinoma cell lines (p53 + / + ) and its HCT116 (p53 ^{_ ~)} derived offered by B. Vogelstein (the Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA). Cells were transfected with a plasmid yLFM-pG13 (Firefly luciferase associated with artificial gene pG13, induced by p53 protein) and RFM-M2

(Renilla luciferase, control) at approximately 80% confluence using Myrus LT-1 transfection reagents (Tema Ricerca, Milan, Italy). Following transfection, the cell lines were incubated in the presence of 1.5 μΜ doxorubicin (DOXO), 10 μ nut nutlin-3A or 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H- pyran [3,2-b] xanten-6-one

(LEM1), or just the solvent (water or DMSO) for 16 hours. Cells were harvested and luciferase assayed similarly to that described in yeast cells.

Example 5: Evaluation of cellular levels of p53, p21 and Bax proteins in human tumor lines.

Following incubation of colon carcinoma cell lines (HCT116 p53 ^{+ / +} ) with 10 μΜ of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one (LEM1) or only in the presence of solvent (DMSO) for 8 and 16 hours, protein extraction was performed. Protein detection was performed by Western blot using mouse anti-p53 (DO-1), rabbit polyclonal anti-p21 (C19) and rabbit monoclonal anti-Bax monoclonal antibodies. Secondary antibodies were conjugated to a horseradish peroxidase which upon addition of a substrate chemiluminescent allowed the detection of proteins by exposure to a photographic film. The intensity of the bands was quantified using the Bio-Profil Bio-1D ++ program and normalized to a standard protein (actin).

Example 6: Evaluation of the effect of other compounds of formula 1 on inhibition of p53-MDM2 interaction using the yeast model. Nutline-3A was used as a positive control. The effect obtained with yeast expressing p53 protein alone was considered as 100%. Results correspond to the mean ± standard error of the mean of five independent experiments.

The present invention is, of course, not in any way restricted to the embodiments described herein and a person of ordinary skill in the art may foresee many possibilities for modification thereof without departing from the general idea of the invention as defined in the claims.

The preferred embodiments described above are obviously combinable with each other. The following claims further define preferred embodiments of the present invention.

Claims

1. compound of general formula 1,

 salts or esters, where: Y represents oxygen, sulfur, CH ₂ or NH; Z represents C = 0, CH ₂ , CH-OH, C = NOH, C = NOCH ₃ , NO, NOH, S = 0 or SO ₂ ;  R1-R8 independently of one another represent hydrogen, hydroxy, aldehyde, halogen, trifluoromethyl, acetylene, carboxyl, cyano, nitro, SO ₂ NH ₂ , aryl or heteroaryl substituted by halogen or hydroxy or methoxy, amine, aminoalkoxy, aminoaryl, imine, alkyl, cycloalkyl, alkylhalogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as aryl-substituted alkyl, furan or dioxane-substituted pyran or pyran, or halogen or hydroxy-substituted aryl or (di) alkoxy or alkoxy-substituted aryl oxyl; wherein if one of R1-R8 represents an amino and / or aminoalkyl group it contains counterions such as HCO ₃ ^" , CO ₃ ^3" , Cl ^" , NH ₂ C ₆ H ₄ SO 3 ^" , 1-CH ₃ C ₆ H ₂ -3-OH-4 (CHCH ₃ ) -6-SC> 3 ^- which are coordinated or ionically bonded to the amine; wherein at least one of R1-R8 is not hydrogen; for the treatment of diseases that are positively influenced by inhibition of the p53-MDM2 or p53-MDMX interaction. A compound according to claims 1, wherein Y represents oxygen, sulfur;  • Z represents C = 0;  • R1-R8  independently of one another represent hydrogen, hydroxy, aldehyde, halogen, amine, aminoalkoxy, alkyl, alkyl halogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl-substituted pyran or pyran, or substituted dioxane or dioxane aryl, or aryl substituted with halogen or hydroxy or alkoxy or (di) oxyalkylene; The in which one of R1-R8 is an amino group and / or amino it contains counter ions such as ^Cl⁻, which is ionically bonded to the amine;  Wherein at least one of R1-R8 is not hydrogen. A compound according to claims 1, wherein • Y represents oxygen;  • Z represents C = 0;  • R1-R8 independently of one another represent hydrogen, hydroxy, aldehyde, halogen, alkyl, alkyl halogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as alkyl-substituted pyran or pyran, or aryl-substituted dioxane or dioxane, or halogen or hydroxy substituted aryl or alkoxy or (di) oxyl alkylene;  Wherein at least one of R1-R8 is not hydrogen. A compound according to claim 1 wherein • Y represents sulfur;  • Z represents C = 0;  • R1-R8  independently of one another represent hydrogen, hydroxy, halogen, amine, aminoalkoxy, alkyl, alkylhalogen, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as pyran or alkyl-substituted pyran; The in which one of R1-R8 is an amino group and / or amino it contains counter ions such as ^Cl⁻, which is ionically bonded to the amine; Wherein at least one of R1-R8 is not hydrogen. A compound according to claims 1, wherein Y represents oxygen, sulfur;  • Z represents C = 0;  • R1-R8, independently of one another, represent hydrogen, hydroxyl, aldehyde, halogen, alkyl, alkyl halogen, amine, alkoxy, or together an aminoalkyl, alkoxy, alkylene (di) oxyl group such as pyran or alkyl-substituted pyran, or dioxane or dioxane substituted by aryl, or aryl substituted by halogen or hydroxy or alkoxy or alkylene (di) oxyl; R 1 represents carbaldehyde or methyl halogen or alkylamine, and R 3 and R 4 represent alkoxy; or R2 and R 3 represents 2,2-dimethyl dihydropyran wherein at least one or more of R 1, R 4 -R 8 represents halogen, methyl halogen, hydrogen or alkylamine. A compound according to any one of the claims 1-3, be 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H, 6H-pyran [3,2-b] xanthen-6-one. A compound according to any one of the claims 1-3, be 1,2-dihydroxyixantone. A compound according to any one of the claims 1-2 or 4, be 1- {[2- (diethylamino) ethyl] amino} -4-propoxythioxantone. A compound according to any one of the claims 1-2 or 4, be 1 - {[2- (diethylamino) ethyl] amino} -4-propoxythioxantone hydrochloride. A compound according to any one of claims 1-3 being (2R, 3R) -2- (hydroxymethyl) -3- (8-methoxy-2,2-dimethylchroman-6-yl) -2H- [1,4] dioxino [2,3-c] xanthen-7 (3H) -one. A compound according to any one of claims 1-3,5 being 1-aminoalkyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone. A compound according to any one of claims 1-3,5 being halogen-4-hydroxy-2,2-dimethyl dihydropyranoxantone. A compound according to the preceding claim being 1-bromo-4-hydroxy-2,2-dimethyl dihydropyranoxantone. 14. A compound according to any one of claims 1 ^_ 3, 5, be 3-carbaldehyde, 4- dialcoxixantona. A compound according to the preceding claim being 1-carbaldehyde-3,4-dimethoxyixantone. A compound described in the preceding claim for treating cancer, preferably treating cancer. A compound described in the preceding claim for the treatment of breast, colorectal, prostate, kidney, lung, melanoma, osteosarcoma or embryonic cancer. 18. compound of formula 1,

their salts or esters, where  Y represents oxygen or sulfur;  • Z represents C = 0; • R1-R8, independently of one another, represent hydrogen, hydroxyl, aldehyde, halogen, alkyl, alkyl halogen, amine, alkoxy, or together a group aminoalkyl, alkoxy, (di) oxyalkylene such as alkyl-substituted pyran or pyran, or aryl-substituted dioxane or dioxane, or halogen or hydroxy-substituted aryl or alkoxy (di) oxyl alkylene; R 1 represents carbaldehyde or methyl halogen or alkylamine, and R 3 and R 4 represent alkoxy; or R 2 and R 3 represent 2,2-dimethyl dihydropyran, wherein at least one or more of R 1, R 4 -R 8 represents halogen, methyl halogen, hydrogen or alkylamine. A compound according to claim 18 being 1-aminoalkyl-4-hydroxy-2,2-dimethyl dihydropyranoxantone. A compound according to claim 18 being halogen-4-hydroxy-2,2-dimethyl dihydropyranoxantone. A compound according to the preceding claim being 1-bromo-4-hydroxy-2,2-dimethyl dihydropyranoxantone. A compound according to claim 18 being carbaldehyde-3,4-dialkoxyxanthone. A compound according to the preceding claim being 1-carbaldehyde-3,4-dimethoxyixantone. Compounds described in any of claims 18-23 for use in medicine or medicaments. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically active amount of the free compound, salts thereof or esters described in any one of claims 1-24. The composition of the preceding claim which is administered topically, orally, parenterally or injectably. A composition according to any one of claims 25-26 further comprising a chemotherapeutic agent. The composition of the preceding claim wherein the chemotherapeutic agent is etoposide.