Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
  • A61K31/382—Heterocyclic compounds having sulfur as a ring hetero atom having six-membered rings, e.g. thioxanthenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
  • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
  • C07D311/78—Ring systems having three or more relevant rings
  • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
  • C07D311/82—Xanthenes
  • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
  • C07D311/86—Oxygen atoms, e.g. xanthones

Definitions

• 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.
• 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 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).
• p53-based gene therapy 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).
• 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).
• 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.
• 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).
• JNJ- No. 26854165 Another MDM2 inhibitor in phase I clinical trials is JNJ- No. 26854165.
• 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.
• the present invention describes compound of formula 1,
• Y represents oxygen, sulfur, CH 2 or NH
• 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;
• R1-R8 is not hydrogen
• Y represents oxygen, sulfur
• oxyalkylene 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;
• R1-R8 is an amino group and / or amino it contains counter ions such as Cl ⁇ , which is ionically bonded to the amine;
• R1-R8 is not hydrogen
• alkyl 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;
• R1-R8 is not hydrogen
• R1-R8 is an amino group and / or amino it contains counter ions such as Cl ⁇ , which is ionically bonded to the amine;
• R1-R8 is not hydrogen
• 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
• Y represents oxygen or sulfur
• 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
• 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.
• the novel compounds are used in medicine or medicine.
• compositions comprising a pharmaceutically acceptable carrier and a therapeutically active amount of the free compound, salts or esters thereof described above.
• the pharmaceutical composition may be administered topically, orally, parenterally or injectably.
• the pharmaceutical composition may further comprise a chemotherapeutic agent such as etoposide.
• alkyl denotes alkanes, alkenes and alkynes, including branched, isomers and stereoisomers, preferably C1 -C20.
• aryl denotes monovalent or bivalent aromatics preferably phenyl, naphthyl, toluyl, xylyl.
• heteroaryl denotes heterocyclic aromatic ring systems, preferably one to three carbons is substituted by a nitrogen, oxygen or sulfur heteroatom.
• 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.
• halogen denotes fluorine, chlorine, bromine or iodine.
• aldehyde denotes aldehydes, preferably C1 -C3, namely carbaldehyde, acetaldehyde, propaldehyde.
• 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.
• 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.
• 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.
• 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).
• 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 ⁇ . 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).
• DMSO solvent dimethyl sulfoxide
• 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 ⁇ ; 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 ⁇ ; 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
• 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 ⁇ ; 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 ⁇ ; 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.
• p53 protein 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.
• RLU relative light units
• 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 ⁇ 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 ⁇ 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.
• 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 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.
• These molecules have a dibenzo- ⁇ - (thio) pyronic or (thio) xanthonic nucleus or derivative according to general formula 1.
• Y represents oxygen, sulfur, C3 ⁇ 4 or N-H
• 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;
• R1-R8 is not hydrogen
• Y represents oxygen, sulfur
• oxyalkylene 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;
• R1-R8 represents an amino and / or aminoalkyl group it contains counterions such as Cl- which is ionically bonded to the amine;
• R1-R8 is not hydrogen
• oxyalkylene 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;
• R1-R8 is not hydrogen
• R1-R8 represents an amino and / or aminoalkyl group it contains counterions such as Cl- which is ionically bonded to the amine;
• R1-R8 is not hydrogen
• 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-
• 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.
• Sousa and Pinto Suda, ME and Pinto MMM, Curr Med Chem, 2005, 12: 2447-2479
• Y represents oxygen or sulfur
• 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
• Another aspect of the invention relates to the preparation of compounds of formula 1, their salts or esters, wherein
• Y represents oxygen or sulfur
• 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.
• 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.
• 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
• 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.
• a basifying agent for example potassium carbonate in stoichiometric proportions
• a catalyst in catalytic quantity, such as copper iodide.
• Compounds should be dissolved or suspended in inert solvent, preferably N-methylpyrrolidone (NMP).
• NMP N-methylpyrrolidone
• 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.
• R 1 represents an alkylamine
• R 1 represents an alkylamine
• R 1 represents an alkylamine
• R 1 represents an alkylamine
• 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.
• 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.
• 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
• 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.
• 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.
• MLM1 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.
• 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).
• MCF7 breast adenocarcinoma
• HCT116 colon carcinoma cell lines
• p53 + + transfected with the artificial gene pG13 (used as a response element).
• 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).
• 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).
• Example 1 Synthesis of 1-Carbaldehyde-3,4-dimethoxyxanthone. To a solution of 1-Carbaldehyde-4-hydroxy-3-methoxyixantone
• Example 3 Analysis of transcriptional activity of p53 protein: luciferase assay in yeast.
• yeast cells were cultured in selective induction medium and incubated in 96-well plates in the presence of 10 ⁇ of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2-methylamide compounds.
• the transcriptional activity of p53 protein was determined by measuring the luminescence of cultures in the presence of a luminescent luciferase substrate.
• 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
• Example 5 Evaluation of cellular levels of p53, p21 and Bax proteins in human tumor lines.
• 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.

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