BRPI0709049A2 - compound inhibiting the interaction between mdm2 and p53, pharmaceutical composition, process for preparing said pharmaceutical composition, use of the compound, combination, and process for preparing said compound - Google Patents

Abstract

COMPOUND INHIBITOR OF THE INTERACTION BETWEEN MDM2 AND P53, PHARMACEUTICAL COMPOSITION, PROCESS TO PREPARE THE REFERRED PHARMACEUTICAL COMPOSITION, USE OF THE COMPOUND, COMBINATION, AND PROCESS TO PREPARE THE COMPOUND. The present invention relates to compounds of formula (I), their use as an inhibitor of a p53-MDM2 interaction as well as pharmaceutical compositions comprising said compounds of formula (I) in which n, m, p, s, t , R ^ 1 ^, R ^ 2 ^, A and Z have definite meanings.

Description

Report of the Invention Patent for "INHIBITORS OF INTERACTION BETWEEN MDM2 AND P53".

Field of the invention

The present invention relates to compounds and compositions containing said compounds acting as inhibitors of the interaction between MDDM and p53. Furthermore, the present invention provides processes for preparing the described inhibitors, compositions comprising them and methods of using them, for example, as a medicament.

p53 is a tumor suppressor protein that plays a major role in regulating the balance between cell proliferation and cell growth arrest / apoptosis. Under normal conditions, the p53 half-life is very short and consequently the p53 level in cells is low. However, in response to cellular DNA damage or cellular stress (eg, oncogene activation, telomere erosion, hypoxia), p53 levels increase. This increase in p53 levels leads to activation of the transcription of several genes that direct the cell to arrest growth or apoptosis processes. Thus, an important function of p53 is to prevent uncontrolled proliferation of damaged cells and thereby to protect the organism from cancer development.

MDM2 is a fundamental negative regulator of p53 function. It forms a negative self-regulating loop binding to the p53 amino terminal transactivation domain and thus MDM2 likewise inhibits the ability of p53 to activate transcription and targets p53 for degradation. proteolytic. Under normal conditions, this regulating loop is responsible for maintaining low p53 levels. However, in wild type p53 tumors, active p53 equilibrium concentration can be increased by antagonizing the interaction between MDM2 and p53. This will result in the restoration of p53-mediated antiproliferative and pro-apoptotic effects on such tumor cells.

MDM2 is a cellular proto-oncogene. Overexpression of MDM2 was observed in a range of cancers. MDM2 is overexpressed in a variety of tumors due to increased gene amplification or transcription or translation. The mechanism by which MDM2 amplification promotes tumorigenesis is at least partly related to its interaction with p53. In cells that overexpress MDM2, the p53 protective function is blocked and thus cells are unable to respond to DNA damage or cell stress by increasing p53 levels leading to arrest of cell growth and / or apoptosis. Thus, after DNA damage and / or cell stress, cells that overexpress oMDM2 are free to continue to proliferate and assume a tu-morphogenic phenotype. Under these conditions, disruption of the p53 eMDM2 interaction would release p53 and thereby allow normal signs of growth arrest and / or apoptosis to function.

MDM2 may likewise have separate functions beyond the inhibition of p53. For example, it has been shown that MDM2 interacts directly with the pRb E2F1 / DP1-regulated transcription factor. This interaction may be crucial for p53-independent p53 oncogenic activities. An E2F1 domain shows striking similarity to p53's MDM2 binding domain. Since MDM2 interactions with both p53 and E2F1 are located at the same binding site in MDM2, it can be expected that MDM2 / p53 antagonists not only activate cellular p53, but also modulate E2F1 activities, which are generally unregulated. in tumor cells.

Similarly, the therapeutic efficacy of currently used DNA-damaging agents (chemotherapy and radiotherapy) can be limited by MDM2 p53 negative regulation. Thus, if inhibition of p53 MDM2 feedback is discontinued, an increase in functional p53 levels will increase the therapeutic efficacy of such agents by reestablishing the function of wild-type p53 that leads to apoptosis and / or reversal of p53. p53-associated drug resistance. It has been shown that combining MDM2 inhibition and detrimental treatments of inventive DNA leads to synergistic antitumor effects (Vousden K.H., Cell, Vol. 103,691-694, 2000).

Thus, disruption of MDM2 and p53 interaction offers an approach for therapeutic intervention in p53-type tumors, could also exhibit antiproliferative effects on tumor cells that are devoid of functional p53 and, in addition, can sensitize tumorigenic cells. for chemotherapy and radiotherapy.

Background of the invention

JP 11130750, published May 18, 1999, describes among others substituted phenylaminocarbonylindolyl derivatives as 5-HT receptor antagonists.

EP1129074, published May 18, 2000, describes anthranilic acid amides as inhibitors of vascular endothelial growth factor receptors (VEGFR) and useful in the treatment of angiogenic disorders.

EP1317443, published March 21, 2002, discloses tricyclic tert-amine derivatives useful as chemokine receptor CXCR4 or α-CR5 modulators for treating human immunodeficiency virus and feline immunodeficiency virus.

EP1379239, published October 10, 2002, describes N- (2-arylethyl) benzylamines as 5-HT 6 receptor antagonists.

WOOO / 15357, published March 23, 2000, provides piperazine-4-phenyl derivatives as inhibitors of the interaction between MDM2 ep53. EP1137418, published June 8, 2000, provides tricyclic compounds to re-establish conformational stability of a p53 family protein.

EP1443937, published May 22, 2003, describes substituted 1,4-benzodiazepines and their uses as inhibitors of MDM2-p53 interactions.

EP1458380, published June 26, 2003, provides cis-2,4,5-triphenyl-imidazolones which inhibits the interaction of MDM2 protein with p53-like compounds and has antiproliferative activity.

EP1519932, published January 15, 2004, describes bisarylsulfonamide compounds that bind to MDM2 and can be used in cancer therapy. It remains a necessity for effective and potent small molecules that inhibit interactions between MDM2 and p53.

The compounds of the present invention differ from the prior art in structure, pharmacological activity and / or pharmacological potency.

Description of the invention

The present invention provides compounds, compositions and methods for inhibiting interactions between MDM2 and p53 to treat cancer. In addition, the compounds and compositions of the present invention are useful in enhancing the effectiveness of chemotherapy and radiotherapy.

This invention relates to the compounds of formula (I)

<formula> formula see original document page 5 </formula>

an N-oxide form, an addition salt or a stereo-chemically isomeric form thereof, wherein

m is 0, 1, or 2 and when m is 0 then a direct link is desired;

η is 0, 1, 2, 3 or 4 and when η is 0 then a direct link is intended;

ρ is 0, or 1 and when ρ is 0, then a direct link is intended;

s is 0, or 1 and when s is 0 then a direct link is intended;

t is 0 or 1 and when t is 0 then a direct bond is preferred;

R 1 and R 2 are each independently hydrogen, halo, C 1-6 alkyl, C 1-6 alkyloxy,

arylC 1-6 alkyloxy, heteroarylC 1-6 alkyloxy (phenylthio, hydroxyC 1-6 alkylcarbonyl,

C1-6 alkyl substituted with a substituent selected from amino, aryl and heteroaryl; or

C 3-7 Cycloalkyl substituted with one substituent selected from amino, aryl and heteroaryl;

A is a radical selected from

<formula> formula see original document page 6 </formula> <formula> formula see original document page 7 </formula>

on what

R4 and R5 are each independently selected from hydrogen, halo, C1-6alkyl, polyalkylC1-6alkyl, cyano, cyanoC1-6alkyl, hydroxyC1-6alkyl, hydroxy, amino, C1-6alkyloxy, C1-6alkylcarbonyl, methylsulfonyl-min, aryl or heteroaryl;

Z is a radical selected from

formula> formula see original document page 7 </formula>

on what

R 6 or R 7 are each independently selected from hydrogen, halo, hydroxy, amino, C 1-6 alkyl, nitro, polyalkylC 1-6 alkyl, cyano, cyanoC 1-6 alkyl, tetrazoloC 1-6 alkyl, aryl, heteroaryl, arylC 1-6 alkyl, heteroaryl1 6 alkyl, aryl (hydroxy) C 1-6 alkyl, heteroaryl (hydroxy) C 1-6 alkyl, arylcarbonyl, heteroarylcarbonyl, C 1-6 alkylcarbonyl, arylC 1-6 alkylcarbonyl, heteroarylC 1-6 alkylcarbonyl, C 1-6 alkyloxy, C 3-6 alkyl hydroxy) C 1-6 alkyl, C 1-6 alkyloxyC 1-6 alkyloxy, C 1-6 alkyloxyC 1-15 alkyloxyC 1-6 alkyl, C 1-6 alkylcarbonyloxyC 1-6 alkyl, C 1-6 alkyloxycarbonylC 1-6 alkyloxyC 1-6 alkyloxy - 6 alkyl, C 1-6 alkyloxycarbonyl, C 1-6 alkylcarbonyloxy, aminocarbonyl, hydroxyC 1-6 alkyl, aminoC 1-6 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-6 alkyl and - (CH 2) v- (C (= O) r) - (CHR10) u -NR8R9; on what

ν is 0, 1, 2, 3, 4, 5, or 6 and when ν is 0, then a direct bond is intended;

r is 0, or 1 and when r is 0 then a direct link is intended;

u is 0, 1, 2, 3, 4, 5, or 6 and when u is 0 then a direct bond is desired;

R10 is hydrogen or C1-6 alkyl;

R8 and R9 are each independently selected from hydrogen, C1-12 alkyl,

C 1-6 alkylcarbonyl, C 1-6 alkylsulfonyl, arylC 1-6 alkylcarbonyl, C 3-7 cycloalkyl, C 3-7 cycloalkylcarbonyl, - (CH 2) k-NR 11 R 12, C 1-12 alkyl substituted with a substituent selected from hydroxy, hydroxycarbonyl, cyano

C 1-6 alkyloxycarbonyl, C 1-6 alkyloxy, aryl or heteroaryl; or C 3-7 cycloalkyl substituted with one substituent selected from hydroxy, C 1-6 alkyloxy, aryl, amino, arylC 1-6 alkyl, heteroaryl or heteroarylC 1-6 alkyl; or

R 8 and R 9 together with the nitrogen to which they are attached may optionally form a substituted morpholinyl, piperidinyl, pyrrolidinyl, pipe-razinyl, or piperazinyl selected from C 1-6 alkyl, arylC 1-6 alkyl, arylC 1-6 alkyloxycarbonyl, heteroaryl, C3,7 cycloalkyl and C 3,7 cycloC 1-6 alkyl; on what

k is 0, 1, 2, 3, 4, 5, or 6 and when k is 0 then a direct connection is desired;

R 11 and R 12 are each independently selected from hydrogen, C 1-6 alkyl, arylC 1-1 alkyloxycarbonyl, C 3-7 cycloalkyl, C 1-12 alkyl substituted with a substituent selected from hydroxy, C 1-6 alkyloxy, aryl, and heteroaryl; and C 3-7 cycloalkyl substituted with a substituent selected from hydroxy, C 1-6 alkyloxy, aryl, arylC 1-6 alkyl, heteroaryl, and heteroarylC 1-6 alkyl; or R 11 and R 12 together with the nitrogen to which they are attached may optionally form a C 1-6 alkyloxycarbonyl substituted morpholinyl, piperazinyl or piperazinyl: aryl is phenyl or naphthalenyl;

each phenyl or naphthalenyl may optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, hydroxyC 1-6 alkyl, C 1-6 alkyl, amino, polyC 1-6 alkyl and C 1-6 alkyloxy; and

each phenyl or naphthalenyl may optionally be substituted with a divalent radical selected from methylenedioxy and ethylenedioxy;

heteroaryl is pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl or tetrahydrofuranyl;

each pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl, or tetrahydrofuranyl may optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, C1-6alkyl amino, polyoxyC 1-6 alkyl, aryl, arylC 1-6 alkyl or C 1-6 alkyloxy; and

each pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, benzofuranyl, or tetrahydrofuranyl may optionally be substituted for a bivalent radical selected from methylenedioxy or ethylenedioxy.

The compounds of formula (I) may likewise exist in their tautomeric forms. Such forms although not explicitly indicated in the above formula are intended to be included within the scope of the present invention.

Various terms used in the foregoing definitions and below are explained below. These terms are sometimes used as such or in compound terms.

When used in the foregoing definitions and hereinafter halo is generic to fluorine, chlorine, bromine and iodine; C1-Balkyl defines straight and branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms such as, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, 1-methylethyl, 2-methylpropyl, 2- methyl butyl, 2-methylpentyl and the like; C 1-6 alkyloxy defines a hydroxy substituent on straight or branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms; triamethyl defines methyl containing three different or identical halo substituents, for example trifluoromethyl; C 3-7 Cycloalkyl includes cyclic hydrocarbon groups having from 3 to 10 carbons such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloeptyl, and the like.

The term "addition salt" includes salts which the compounds of formula (I) may be capable of forming with organic or inorganic bases such as amines, alkali metal bases and alkaline earth metal bases, or ammonium bases. quaternary, or with organic or inorganic acids, such as mineral acids, sulfonic acids, carboxylic acids or phosphorus-containing acids.

The term "addition salt" also encompasses pharmaceutically acceptable salts, metal complexes and solvates and salts thereof which the compounds of formula (I) are capable of forming.

The term "pharmaceutically acceptable salts" means pharmaceutically acceptable acid or base addition salts. Pharmaceutically acceptable acid or base addition salts as mentioned hereinabove are intended to comprise the non-toxic and non-toxic acid base addition salt forms therapeutically active which the compounds of formula (I) are capable of forming. The compounds of formula (I) which have basic properties may be converted to their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Suitable acids include, for example, inorganic acids such as hydroalic acids, for example hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and similar acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic , benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and similar acids.

The compounds of formula (I) which have acidic properties may be converted to their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base. Suitable base salt forms include, for example, ammonium salts, alkaline earth metal and alkali salts, for example lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, for example for example benzatin, N-methyl-D-glycamine, hydrabamine salts, and amino acid salts such as, for example, arginine, lysine and the like.

The terms acid or base addition salt likewise include hydrates and solvent addition forms that the compounds of formula (I) are capable of forming. Examples of such forms are e.g. hydrates, alcoholates and the like.

The term "metal complexes" means a complex formed between a compound of formula (I) and one or more organic or inorganic metal salt or salts. Examples of said organic or inorganic salts include halides, nitrates, sulfates, phosphates, acetates, trifluoroacetates, trichloroacetates, propionates, tartrates, sulfonates, for example methylsulfonates, 4-methylphenylsulfonates, salicylates, benzoates and the like of the second major group of periodic system, for example, magnesium or calcium ossals of the third or fourth major group, for example aluminum, tin, lead as well as the first to eighth transitional groups of the periodic system such as, for example, chromium, manganese, iron, cobalt, nickel, copper, zinc and the like.

The term "stereochemically isomeric forms of compounds of formula (I)" as used herein defines all possible compounds made up of the same atom bonded by the same sequence of bonds but having different non-interchangeable three-dimensional structures as the compounds. of formula (I) may have. Unless otherwise stated or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms that said compound may possess. Said mixture may contain all diastereomers and / or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of formula (I) both in pure form or in admixture with each other are intended to be within the scope of the present invention.

Of special interest are those compounds of formula (I) which are stereochemically pure.

Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as substantially free isomers of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term "stereoisomerically pure" refers to compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of other possible isomers) to a stereoisomeric excess of 100%. % (i.e. 100% of one isomer and none of the other), more particularly compounds or intermediates having a stereoisomeric excess of 90% to 100%, even more particularly having a stereoisomeric excess of 94% to 100% and more. in particular having a stereoisomeric excess of 97% to 100%. The terms "enantiomerically pure" and "diastereomerically pure" should be understood in a similar manner, but then taking into account the enantiomeric excess, respectively the diastereomeric excess of the mixture in question.

Tautomeric forms of the compounds of formula (I) are intended to comprise those compounds of formula (I) wherein, for example, an enol group is converted to a keto group (keto-enol tautomerism).

The / V-oxide forms of the compounds of formula (I) are intended to comprise those compounds of formula (I) wherein one or more nitrogen atoms are oxidized to the so-called / V-oxide, particularly those / V- oxides wherein one or more of the piperidine, piperazine or pyridazinyl nitrogen are N-oxidized.

The compounds of formula (I) may be converted to the corresponding N-oxide forms following the procedures known in the art to convert a trivalent nitrogen into their N-oxide forms. Said N-oxidation reaction may generally be carried out by reacting the starting material of formula (I) with an appropriate organic or inorganic peroxide. Suitable inorganic peroxides include, for example, hydrogen peroxide, alkali metal or alkaline earth metal peroxides, for example sodium peroxide, potassium peroxide; Suitable organic peroxides may comprise peroxy acids such as, for example, substituted benzenecarboperoxic acid or substituted benzenocarboperoxy acids, for example 3-chlorobenzenocarboperoxy acids, peroxoalkanoic acids, for example peroxoacetic acid, alkyl peroperoxides, e.g. -butyl. Suitable solvents are water, for example, lower alcohols, for example ethanol and the like, hydrocarbons, e.g. toluene, ketones, eg 2-butanone, halogenated hydrocarbons, e.g. dichloromethane, and mixtures of such solvents.

The present invention is likewise intended to include any isotope of atoms present in the compounds of the invention. For example, hydrogen isotopes include tritium and deuterium and carbon isotopes include C-13 and C-14.

As used hereinafter, the term "compounds of formula (I)" is intended to include in the same manner the α-oxide forms, the pharmaceutically acceptable base or acid addition salt and all stereoisomeric forms.

A first group of interesting compounds are those compounds of formula (I) wherein one or more of the following restrictions apply:

a) m is 0;

b) η is 0 or 2;

c) ρ is 1;

d) s is 0;

e) t is 0 f) R 1 and R 2 are each independently hydrogen;

g) A is a radical selected from (a-15), (a-21), (a-30), (a-39) or (a-40);

h) R4 and R5 are each independently selected from hydrogen or C1-6 alkyloxy;

i) Z is the radical (b-2); or

j) R6 and R7 are each independently selected from hydrogen.

A second group of interesting compounds consists of those compounds of formula (I) and those compounds of the group described above wherein one or more of the following restrictions apply:

a) m is 0;

b) η is 2;

c) ρ is 1;

d) s0;

e) t is 0;

f) R1 and R2 are each independently hydrogen;

g) A is a radical selected from (a-21), (a-39) or (a-40);

h) R4 and R5 are each independently selected from hydrogen or C1-6 alkyloxy;

i) Z is the radical (b-2); or

j) R6 and R7 are each independently selected from hydrogen.

A group of preferred compounds consists of those compounds of formula (I) or any subgroup thereof, wherein m is 0; η is 0 or 2; ρ is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-15), (a-21), (a-30), (a-39) or (a-40); R 4 and R 5 are each independently selected from hydrogen or C 1-6 alkyloxy; Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen.

A more preferred group of compounds consists of those compounds of formula (I) or any subgroup thereof wherein m is 0; η is 2, ρ is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-21), (a-39) or (a-40);

R4 and R5 are each independently selected from hydrogen or C1-6 alkyloxy;

Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen.

Most preferred compounds are compounds No. 2, compounds No. 3 or compounds No. 5.

<table> table see original document page 15 </column> </row> <table>

The compounds of formula (I), their pharmaceutically acceptable salts and N-oxides and stereochemically isomeric forms may be prepared in conventional manner. The starting materials and some of the intermediates are known compounds and are commercially available or may be prepared according to conventional reaction procedures as generally known in the art.

Various such preparation methods will be described below in more detail. Other methods for obtaining final compounds of formula (I) are described in the examples.

The compounds of formula (I) may be prepared by reacting an intermediate of formula (II) with an intermediate of formula (III) wherein W is an appropriate leaving group such as, for example halo, for example fluorine, chlorine, bromine or iodine, or a sulfonyloxy radical such as methylsulfonyloxy, 4-methylphenylsulfonyloxy and the like. The reaction may be carried out in an inert reaction solvent such as, for example, an alcohol, for example methanol, ethanol, 2-methoxyethanol, propanol, butanol and the like; an ether, for example 4,4-dioxane, 1,1'-oxybispropane and the like; a ketone, for example 4-methyl-2-pentanone; or Ν, Ν-dimethylformamide, nitrobenzene, aceto-nitrile, acetic acid and the like. Addition of a suitable base such as, for example, alkali or alkaline earth metal carbonate or hydrogen carbone, for example triethylamine or sodium carbonate, may be used to recover the acid which is released during the course of reaction. A small amount of an appropriate metal iodide, for example sodium or potassium iodide may be obtained to promote the reaction. Stirring may enhance the rate of the reaction. The reaction may conveniently be carried out at a temperature ranging from room temperature to the reflux temperature of the reaction mixture and, if desired, the reaction may be performed at increased pressure.

<formula> formula see original document page 16 </formula>

The compounds of formula (I), wherein ρ is 1, referred to herein as the compounds of formula (la) may be prepared by converting the intermediates of formula (IV) with lithium aluminum hydride to an appropriate solvent such as as tetrahydrofuran.

<formula> formula see original document page 16 </formula>

The compounds of formula (1a) may likewise be prepared by reacting an appropriate carboxaldehyde of formula (VI) 1 with an intermediate of formula (V) in the presence of an appropriate reagent such as sodium borohydride, for example. polymer-supported sodium tetrahydroborate or cyanohydroborate in a suitable solvent such as an alcohol for example methanol.

<formula> formula see original document page 17 </formula>

Similarly the compounds of formula (I) wherein t is 1, herein referred to as compounds of formula (1b), may be prepared by reacting an intermediate of formula (II) with a suitable carboxaldehyde of formula (VII). .

<formula> formula see original document page 17 </formula>

The compounds of formula (I), wherein s is 1, referred to herein as the compounds of formula (1c), may be prepared by reacting an intermediate of formula (VIII) with lithium aluminum hydride in a suitable solvent. such as tetrahydrofuran.

<formula> formula see original document page 17 </formula>

The compounds of formula (I) or intermediates thereof may also be converted to one another by reactions known in the art or functional group transformations. Several such transformations are already described herein above. Other examples are hydrolysis of carboxylic esters to the corresponding carboxylic acid or alcohol; hydrolysis to the corresponding carboxylic acids or amines; hydrolysis of nitriles to the corresponding amides; amino groups in imidazole or phenyl may be substituted by hydrogen by diazotation reactions known in the art and subsequent replacement of the diazo group by hydrogen, alcohols may be converted to esters and ethers; primary amines may be converted to secondary or tertiary amines; double bonds may be hydrogenated to the corresponding single bond; An iodine radical in a phenyl group may be converted to an ester group by insertion of carbon monoxide in the presence of a suitable palladium catalyst.

Intermediates of formula (II), wherein m is 0 and s is 0, referred to herein as intermediates of formula (II-a), may be prepared by a nitro-reduction reaction starting with an intermediate of formula (1). IX), in the presence of a metal catalyst such as Raney Nickel and a suitable reducing agent such as hydrogen, in a suitable solvent such as methanol or ethanol.

<formula> formula see original document page 18 </formula>

Intermediates of formula (X), wherein s is 0, may be prepared by reacting an intermediate of formula (XI) with an intermediate of formula (XII) in the presence of appropriate reagents such as N- (ethylcarbonimidoyl) -N, N-dimethyl-1,3-propanediamine) monohydrochloride (EDC) and -1-hydroxy-1H-benzotriazole (HOBT). The reaction may be carried out in the presence of a base such as triethylamine in a suitable solvent, such as a mixture of dichloromethane and tetrahydrofuran. <formula> formula see original document page 19 </formula>

Intermediates of formula (VI) may be prepared by reacting intermediates of formula (XIII) with lithium aluminum hydride in a suitable solvent such as tetrahydrofuran.

<formula> formula see original document page 19 </formula>

Intermediates of formula (VIII) may be prepared by reacting an intermediate of formula (XIV) with an intermediate of formula (XV) in the presence of 2-Chloro-1-methylpyridinium iodide and triethylamine in a suitable solvent such as acetonitrile. .

<formula> formula see original document page 19 </formula>

Intermediates of formula (IX), where ρ is 1, referred to herein as intermediates of formula (IX-a), may be prepared by reacting an intermediate of formula (XVI) with an intermediate of formula (XVII), wherein Q is a suitable leaving group such as, for example, halo, e.g., fluorine, chlorine, bromine or iodine, or C1-6 alkyloxy, for example methyloxy, in diisopropylethyl amine.

<formula> formula see original document page 20 </formula>

Intermediates of formula (XIV) may be prepared by converting an intermediate of formula (XVIII) in the presence of sodium hydroxide and water in a suitable solvent such as ethanol.

<formula> formula see original document page 20 </formula>

Intermediates of formula (XVIII), wherein m is O, referred to herein as intermediates of formula (XVIII-a), may be prepared by reacting an intermediate of formula (XVI) with an intermediate of formula ( where Q is as defined above in a suitable solvent such as diisopropylethyl amine.

<formula> formula see original document page 20 </formula>

Intermediates of formula (V) wherein m, η and s are O, referred to as intermediates of formula (V-a), may be prepared by converting an intermediate of formula (XX) with hydrochloride solution.

<formula> formula see original document page 20 </formula>

Intermediates of formula (XX) may be prepared by reacting an intermediate of formula (XXI) with an intermediate of formula (III) wherein W is an appropriate leaving group such as, for example, ha. The reaction may be carried out in an inert reaction solvent such as, for example, acetic acid.

<formula> formula see original document page 21 </formula>

The compounds of formula (I) and some of the intermediates may have at least one stereogenic center in their structure. Such a stereogenic center may be present in an R or S configuration.

Some of the compounds of formula (I) and some of the intermediates in the present invention may contain an asymmetric carbon atom. Pure stereochemically isomeric forms of said compounds and said intermediates may be obtained by the application of procedures known in the art. For example, diastereoisomers may be separated by physical methods such as selective crystallization or chromatographic techniques, for example, countercurrent distribution, liquid chromatography and the like. Enantiomers may be obtained from racemic mixtures by first converting said racemic mixtures with suitable resolving agents such as, for example, chiral acids, to mixtures of diastereomeric salts or compounds; then physically separating said diastereomeric salt mixtures or compounds comprising, for example, selective crystallization, supercritical fluid chromatography or chromatographic techniques, for example, liquid chromatography and similar methods; and finally converting said separate diastereomeric salts or compounds to the corresponding enantiomers. Pure stereochemically isomeric forms may likewise be obtained from the pure stereochemically isomeric forms of the appropriate intermediates and starting materials, provided that the intermediate reactions occur stereospecifically.

The compounds of formula (I), pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof have valuable pharmacological properties in which they inhibit the interaction between p53e MDM2.

The term "MDM2" is used herein to mean a protein obtained as a result of mdm2 gene expression. Within the meaning of this term, MDM2 encompasses all mdm2-encoded proteins, mutants thereof, alternative portion proteins thereof, and phosphorylated proteins thereof. Additionally, when used herein, the term "MDM2" includes MDM2 analogs, for example, MDMX, likewise known as MDM4 homologues, and MDM2 and analogs from other animals, for example, human HDM2 homolog or human analog HDMX.

The term "inhibiting interaction" or "interaction inhibitor" is used herein to mean preventing or reducing the direct indirect association of one or more molecules, peptides, proteins, enzymes or receptors; or prevent or reduce the normal activity of one or more molecules, peptides, proteins, enzymes, or receptors.

The term "p53-MDM2 interaction inhibitor" or "p53-MDM2 inhibitor" is used herein to describe an agent that enhances p53 expression in the assay described in C.1. This increase may be caused by, but is not limited to, one or more of the following mechanisms of action:

- inhibition of interaction between p53 and MDM2,

- direct association with MDM2 or p53 protein,

- interactions with upstream or downstream targets, for example kinase or enzyme activities involved in ubiquitination or SUMO modification,

- sequestration or transport of MDM2 and p53 in different cellular compartments,

- modulation of proteins associated with MDM2, for example (but not limited to), p73, E2F-1, Rb1 p21waf1 or cipl,

- down-regulation or interference with MDM2 expression and / or MDM2 activity, for example (but not limited to), impact on cell localization, post-translational modification, nuclear export or ubiquitin ligase activity,

- direct or indirect stabilization of the p53 protein, for example by maintaining its functional structural form or preventing imperfect duplication,

enhancement of p53 expression or expression of p53 family members, for example p63 and p73.

- increased p53 activity, for example (but not limited to), increasing its transcriptional activity and / or

- Increased gene and protein expression of the p53 signaling reaction series, for example (but not limited to) p21waf1, cipl, MIC-1 (GDF-15), PIG-3 and ATF-3.

Accordingly, the present invention describes the compounds of formula (I) for use as a medicament.

Furthermore, the invention likewise relates to the use of a compound for the manufacture of a medicament for treating a mediated disorder through a p53-MDM2 interaction, wherein said compound is a compound of formula (I).

The term "treating" or "treatment" as used herein encompasses any treatment of a disease and / or condition in an animal, particularly a human being, and includes: (i) preventing a disease and / or condition from occurring in an individual who may be predisposed to the disease and / or condition but has not yet been diagnosed as having it; (ii) inhibit the disease and / or condition, i.e. interrupting its development; (iii) alleviate disease and / or condition, ie, causing regression of disease and / or condition.

By the term "a disorder mediated through a dep53-MDM2 interaction" is meant any unwanted or harmful condition resulting in or inhibition of the interaction between the MDM2 protein and p53 or other cell proteins that induce apoptosis, induced cell death, or regulate the cycle. cell phone.

This invention likewise provides a method for treating a mediated disorder through a p53-MDM2 interaction by administering an effective amount of a compound of the present invention to an individual, for example a mammal (and more particularly a human being). -no) in need of such treatment.

The compounds of the invention may have antiproliferative effects on tumor cells, even if such cells are devoid of functional p53. In particular, the compounds of the invention may have antiproliferative effects on wild type p53 tumors and / or overexpressed tumors. MDM2.

Thus, this invention likewise provides a method for inhibiting tumor growth by administering an effective amount of a compound of the present invention to an individual, for example, a mammal (and more particularly a human). in need of such treatment.

Examples of tumors that can be inhibited but not limited to, lung cancer (eg adenocarcinoma and including non-small cell lung cancer), pancreatic cancers (eg pancreatic carcinoma such as pancreatic carcinoma). exocrine cancer), colon cancers (eg, colorectal carcinomas, such as, for example, colon adenocarcinoma and colon adenoma), esophageal cancer, oral squamous carcinoma, tongue carcinoma, gastric carcinoma, nasopharyngeal cancer, lymphoid lineage tumors (e.g., acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), non-Hodgkin's lymphoma, Hodgkin's disease, myeloid leukemia (eg, acute myelogenous leukemia (AML)), thyroid follicular cancer, myelodysplastic syndrome (MDS) , tumors of mesenchymal origin (eg, fibrosarcomas and rhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas, brain tumors, gliomas, benign skin tumor (eg, keratoacanthoma), breast carcinoma (eg, advanced breast cancer), kidney carcinoma, ovarian carcinoma, cervical carcinoma, endometrial carcinoma, bladder carcinoma, including prostate cancer advanced, testicular cancers, osteosarcoma, head and neck cancer and epidermal carcinoma.

The compounds of the present invention may likewise be used for the treatment and prevention of inflammatory conditions.

Thus, this invention likewise provides a method for treating and preventing inflammatory conditions by administering an effective amount of a compound of the present invention to an individual, for example a mammal (and more particularly a human being) in need of such treatment.

The compounds of the present invention may likewise be used for the treatment of autoimmune diseases and conditions. By term "autoimmune diseases" is meant any disease in which an animal's immune system reacts adversely to a high antigen. By term "high antigen" is meant any antigen that is normally found in the animal's body. Representative autoimmune diseases include, but are not limited to: Hashimoto's thyroiditis, Grave's disease, multiple sclerosis, pernicious anemia, Addison's disease, dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), dermatomyositis , Crohn's disease, Wegener's granulomatosis, Antiglomerular Basement Membrane Disease, Antiphospholipid Syndrome, Dermatitis Herpetiformis 25, Allergic Encephalomyelitis, Glomerulonephritis, Membranous Glomerulo-nephritis, Goodpasture Syndrome, Lambert-Eaton, Penis, Myastheniagia Bullous, Polyendocrinopathies, Reiter's Disease, and Rigid Man Syndrome.

Accordingly, this invention likewise provides a method for the treatment of autoimmune conditions and diseases and the treatment of diseases associated with conformational mal-duplicate or unstable proteins by administering an effective amount of a compound of the present invention. to an individual, for example a mammal (and more particularly a human) in need of such treatment.

The compounds of the present invention may likewise be useful for treating diseases associated with conformational maladopted or unstable proteins.

Examples of diseases associated with maladopted or unstable conformational proteins include, but are not limited to: fibrosecystic (CFTR), Marfan syndrome (fibrilin), Amyotrophic lateral sclerosis (superoxide dismutase), scurvy (collagen), syrup urine disease maple (alpha-ketoacid dehydrogenase complex), osteogenesis imperfecta (pro-collagen pro-collagen typel), Creutzfeldt-Jakob disease (prion), Alzheimer's disease (beta-amyloid), familial amyloidosis (lysozyme), cataracts (crest-linins) ), familial hypercholesterolaemia (LDL receptor), anthritrypsin deficiency, Tay-Sachs disease (betahexosaminidase), retinitis pigmentosa (rhodopsin), and Ieprechaunism (insulin receptor).

Accordingly, this invention likewise provides a method for treating diseases associated with conformational mal-duplicate or unstable proteins by administering an effective amount of a compound of the present invention to an individual, e.g., a mammal (and more). particularly a human being) in need of such treatment.

Due to their useful pharmacological properties, the subject compounds may be formulated into various pharmaceutical forms for administration purposes.

To prepare the pharmaceutical compositions of this invention, an effective amount of a particular compound in the form of a base or acid addition salt as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, the carrier of which may take a wide variety of forms depending on the composition of the invention. desired preparation form for administration. These pharmaceutical compositions are desirably in unit dosage form suitable, preferably for administration orally, rectally, percutaneously, or by parenteral injection. For example, by preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions. , syrups, elixirs and solutions; solid vehicles such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets.

Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.

For parenteral compositions, the carrier will normally comprise sterile water, at least to a large extent, however other ingredients to aid solubility, for example, may be included. Injectable solutions, for example, may be prepared wherein the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may likewise be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In compositions suitable for percutaneous administration, the carrier optionally comprises a suitable penetration enhancing agent and / or wetting agent, optionally combined with suitable additives of any nature in minor proportions, the additives of which do not cause a significant deleterious effect to the skin. Said additives may facilitate administration to the skin and / or may be useful for preparing the desired compositions. These compositions may be administered in various ways, for example as a transdermal patch, as a stain, as an ointment. It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form when used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. . Examples of such dosage unit forms are tablets (including marked or coated tablets), capsules, pills, powder packets, tablets, injectable solutions or suspensions, full teaspoons, full tablespoons and the like, and segregated multiples. of these.

It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form when used in the specification and claims herein refer to physically discrete units suitable as unitary dosages, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including marked or coated tablets), capsules, pills, powder packs, lozenges, injectable or suspension solutions, full teaspoons, full tablespoons and the like, segregated multiples. of these.

The compound of the invention is administered in sufficient amounts to inhibit the interaction between MDM2 and p53 or other cell proteins that induce apoptosis, induce cell morle, or regulate the cell cycle.

The oncogenic potential of MDM2 is not only determined by its ability to suppress p53, but also by its ability to regulate other tumor suppressor proteins, eg retinoblastoma pRb protein and the closely associated E2F1 transcription factor. .

Thus, the compound of the invention is administered in an amount sufficient to modulate the interaction between MDM2 and E2F transcription factors.

Those skilled in the art could easily determine the effective amount of the test results presented below. It is generally considered that a therapeutically effective amount would be from 0.005 mg / kg to 100 mg / kg body weight, and in particular from 0.005 mg / kg to 10 mg / kg body weight. It may be appropriate to administer doserequeride as single, two, three, four or more subdoses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 0.5 to 500 mg, and in particular 10 mg to 500 mg of active ingredient per unit dosage form.

As another aspect of the present invention, a combination of a p53-MDM2 inhibitor with another anticancer agent is considered, especially for use as a medicament, more specifically in the treatment of cancer or related diseases.

For the treatment of the above conditions, the compounds of the invention may be advantageously employed in combination with one or more other medicinal agents, more particularly with other anti-cancer agents. Examples of anticancer agents include, but are not limited to:

platinum coordinating compounds for example cisplatin, carboplatin or oxaliplatin;

taxane compounds for example paclitaxel or docetaxel;

Topoisomerase I inhibitors such as campto-tecine compounds for example irinotecan or topotecan;

topoisomerase II inhibitors such as antitumor epipodophyllotoxins or podophyllotoxin derivatives for example etoposide or teniposide;

vinca antitumor alkaloids for example vinblastine, vincristine or vinorelbine;

antitumor nucleoside derivative for example 5-fluorouracil, leucovorin, gemcitabine or capecitabine;

alkylating agents such as nitrogen mustard or nitro-sourea for example cyclophosphamide, chlorambucil, carmustine, thiotepa, mefa-Ian or lomustine;

antitumor anthracycline derivatives e.g. daunorubicin, doxorubicin, doxyl, idarubicin or mitoxantrone;

molecules targeting the IGF-1 receptor for example picro-podophyllin;

tetracarcin derivatives for example tetrocarcin A;

glucocorticoids, eg prednisone antibodies, eg trastuzumab (HER2 antibody), ritu-ximab (CD20 antibody), gentuzamab, cetuximab, pertuzumab or beva-cizumab;

selective estrogen receptor antagonists or estrogen receptor modulators, for example tamoxifen, iulvestrant, toremiene, droloxy, iaslodex or raloxifene;

aromatase inhibitors such as exemestane, anastrozole, Ie-trazol and vorozol;

differentiating agents such as retinoids, vitamin D or retinoic acid and retinoic acid metabolism blocking agents (RAMBA) for example, acutane;

DNA methyl transasease inhibitors, for example azacytidine or decitabine;

antitolates eg disodium premetrexed;

antibiotics for example antinomycin D, bleomycin, mitomycin C, dactinomycin, carminomycin or daunomycin;

antimetabolites, for example clopharabine, aminopterin, cytosine arabinoside or methotrexate;

apoptosis inducing agents and antiangiogenic agents such as Bcl-2 inhibitors for example YC 137, BH 312, ABT 737, gossypol, HA 14-1, TW 37 or decanoic acid;

tubulin binding agents for example combrestatin, colchicines or nocodazole;

kinase inhibitors, for example flavoperidol, i-matinib mesylate, erlotinib or gefitinib;

arnesyltranserase inhibitors, for example, tipifarnib;

histone deacetylase (HDAC) inhibitors for example sodium butyrate, suberoylanilide hydroxamide acid (SAHA), depsipeptide (FR 901228), NVP-LAQ824, R306465, JNJ-26481585 or trichostatin A;

- Ubiquitin-proteasome reaction series inhibitors, for example, PS-341, MLN .41 or bortezomib;

- Yondelis - Telomerase inhibitors, for example telomestatin;

- Matrix metalloproteinase inhibitors, for example, batimasta te, marimasíate, prinostate or metastate.

As stated above, the compounds of the present invention in the same manner have therapeutic applications in sensitizing tumor cells for chemotherapy and radiotherapy.

Therefore, the compounds of the present invention may be used as "radiosensitizing" and / or "chemosensitizing" or may be produced in combination with another "radiosensitizing" and / or "chemosensitizing".

The term "radiosensitizer" as used herein is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of cells to ionization radiation and / or to promote the treatment of diseases they are treatable with ionization radiation.

The term "chemosensitizer" as used herein is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of cells to chemotherapy and / or promote treatment of diseases are treatable with chemotherapeutic agents.

Several mechanisms for the mode of action of radiosensitizers have been suggested in the literature including: hypoxic cell radiosensitizers (eg 2-nitroimidazole compounds, and benzotriazine dioxide compounds) that mimic oxygen or alternatively behave as agents bioreductors under hypoxia; non-hypoxic cellulosensitizing radiosensitizers (e.g., halogenated pyrimidines) may be analogous to DNA bases and preferably incorporate into the cancer cell DNA, thereby promoting radiation-induced disruption of DNA molecules and / or preventing DNA repair mechanisms. normal; and several other potential mechanisms of action have been hypothesized for radiosensitizers in the treatment of disease. Many cancer treatment protocols currently employ radiosensitizers in conjunction with X-ray radiation. Examples of X-ray activated radiosensitizers include but are not limited to the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, ethanidazole, mitomycin C, RSU1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeutically effective analogues and derivatives thereof.

Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, photophrin, benzoporphyrin derivatives, tin etioporfirin, pheoborbide-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogues and derivatives thereof.

Radiosensitizers may be administered together with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds that promote the incorporation of radiosensitizers into target cells; compounds that control the flow of therapeutics, nutrients, and / or oxygen to target cells; tumor-acting chemotherapeutic agents with or without additional radiation; or other therapeutically effective compounds for treating cancer or other disease.

Chemosensitizers may be administered together with a therapeutically effective amount of one or more other compounds, including, but not limited to: compounds that promote incorporation of chemosensitizers into designated cells; compounds that control the flow of therapies, nutrients, and / or oxygen to target cells; chemotherapeutic agents acting on the tumor or other therapeutically effective compounds to treat cancer or other disease. Calcium antagonist, for example, verapamil, is found to be useful in combination with antineoplastic agents to establish chemosensitivity in tumor-resistant tumor-resistant cells and to enhance the efficacy of such compounds in drug-sensitive malignancies.

Due to their useful pharmacological properties, the combination components according to the invention, that is, the other medicinal agent and the p53-MDM inhibitor may be formulated into various pharmaceutical forms for administration purposes. The components may be formulated separately in individual pharmaceutical compositions or in a unitary pharmaceutical composition containing both components.

The present invention therefore likewise relates to a pharmaceutical composition comprising the other medicinal agent and the p53-MDM inhibitor together with one or more pharmaceutical carriers.

The present invention also relates to the use of a combination according to the invention in the manufacture of a pharmaceutical composition for inhibiting tumor cell growth.

The present invention also relates to a product containing as the first active ingredient a p53-MDM2 inhibitor according to the invention and as a second active ingredient an anticancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer patients. .

The other medicinal agent and p53-MDM2 inhibitor may be administered simultaneously (e.g., in separate or unitary compositions) or sequentially in any order. In the latter case, the two compounds will be administered within a period and in a quantity and manner that is sufficient to ensure that an advantageous or synergistic effect is obtained. It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimens for each component of the combination will depend on the other particular medicinal agent and p53-MDM2 inhibitor which are routinely administered to the particular tumor that is treated and the particular host treated. The ideal method and order of administration and the amounts of dosage and regimen can be readily determined by those skilled in the art using conventional methods and due to the information mentioned herein.

The platinum coordination compound is advantageously administered at a dosage of 1 to 500 mg per square meter (mg / m2) of body surface area, for example 50 to 400 mg / m2, particularly for cisplatin at a dosage of about 75 mg / m2 and carboplatin at about 300 mg / m2 per course of treatment.

The taxane compound is advantageously administered at a dosage of 50 to 400 mg per square meter (mg / m 2) body surface area, for example 75 to 250 mg / m 2, particularly for paclitaxel in a dosage of about 175 to 250 mg / m 2. m2 and docetaxel at about 75 to 150 mg / m2 per course of treatment.

The camptothecin compound is advantageously administered at a dosage of 0.1 to 400 mg per square meter (mg / m2) body surface area, for example, 1 to 300 mg / m2, particularly for irinote cane at a dosage. from about 100 to 350 mg / m2 and for topotecan about 1 to 2 mg / m2 per course of treatment.

The antitumor podophyllotoxin derivative is advantageously administered at a dosage of 30 to 300 mg per square meter (mg / m2) of body surface area, for example, 50 to 250 mg / m2, particularly for etoposide at a dosage of about 35 mg. at 100 mg / m 2 and teniposide at about 50 to 250 mg / m 2 per course of treatment.

The antitumor alkaloid vinca is advantageously administered at a dosage of 2 to 30 mg per square meter (mg / m2) body surface area, particularly for vinblastine at a dosage of about 3 to 12 mg / m2, for vincristine at a dosage about 1 to 2 mg / m 2, and for vinorelbine at a dosage of about 10 to 30 mg / m 2 per course of treatment.

The antitumor nucleoside derivative is advantageously administered at a dosage of 200 to 2500 mg per square meter (mg / m2) of body surface area, for example 700 to 1500 mg / m2, particularly for 5-FU at a dosage of 200 500 mg / m2 for gemcitabine at a dosage of about 800 to 1200 mg / m2 and for capecitabine at about 1000 to 2500 mg / m2 per course of treatment.

Alkylating agents such as nitrogen mustard or nitrosourea are advantageously administered at a dosage of 100 to 500 mg per square meter (mg / m2) body surface area, for example, 120 to 200 mg / m2, particularly for cyclophosamide at a dosage of about 100 to 500 mg / m2, for chlorambucil at a dosage of about 0.1 to 0.2 mg / kg, for carmustine at a dosage of about 150 to 200 mg / m2, and for Iomustine at a dosage of about 100 to 150mg / m2 per course of treatment.

The antitumor anthracycline derivative is advantageously administered at a dosage of 10 to 75 mg per square meter (mg / m2) body surface area, for example 15 to 60 mg / m2, particularly paradoxorubicin at a dosage of about 40 mg / m2. at 75 mg / m2, for daunoru-bicin at a dosage of about 25 to 45 mg / m2, and for idarubicin at a dosage of about 10 to 15 mg / m2 per course of treatment.

The antiestrogen agent is advantageously administered at a dosage of from about 1 to 100 mg daily depending on the particulate agent and the condition being treated. Tamoxifen is advantageously administered orally in a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day, continuing therapy long enough to achieve and maintain a therapeutic effect. Toremifene is advantageously administered orally at a dosage of about 60 mg once daily, continuing therapy long enough to obtain and maintain a therapeutic effect. Anastrozole is advantageously administered orally at a dosage of about 1 mg once daily. Droloxifene is advantageously administered orally in a dosage of about 20- 100 mg once daily. Raloxifene is advantageously administered orally in a dosage of about 60 mg once daily. Exemestane is advantageously administered orally at a dosage of about 25 mg once daily.

Antibodies are advantageously administered at a dosage of about 1 to 5 mg per square meter (mg / m2) of body surface area, or as known in the art, if different. Trastuzumab is advantageously administered at a dosage of 1 to 5 mg per square meter (mg / m2) body surface area, particularly 2 to 4 mg / m2 per course of treatment.

These dosages may be administered, for example, once, twice or more per course of treatment, which may be repeated, for example, every 7, 14, 21 or 28 days.

The compounds of formula (I), pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof may have valuable diagnostic properties in which they may be used to detect or identify an interaction of p53-MDM2 in a sample. comprising comprising detecting or measuring the formation of a complex between a labeled compound and / or p53 and / or MDM2 and or other molecules, peptides, proteins, enzymes or receptors.

Detection or identification methods may use compounds which are labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances, etc. Examples of radioisotopes include 125 I, 131 I 3 H and 14 C. Enzymes are normally made detectable by conjugation of an appropriate substrate which subsequently catalyzes a detectable reaction. Examples thereof include, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably spicy horseradish peroxidase.

Light substances include, for example, luminol, luminol derivatives, luciferin, aequorin and luciferase. Biological samples can be defined as body tissue or body fluids. Examples of body fluids are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva, and the like.

The following examples illustrate the present invention.

Experimental part

Then "DCM" is defined as dichloromethane, "DIPE" is defined as diisopropyl ether, "EtOAc" is defined as ethyl acetate, Έ-tOH "is defined as ethanol," MeOH "is defined as methanol and" THF "is defined. as tetrahydrofuran.

Melting points

For various compounds, indicated by (Kofler), melting points were obtained with a Kofler hot bank consisting of a linear temperature gradient heated plate, a sliding pointer and a temperature range in degrees Celsius.LCMS

General procedure

The HPLC gradient was provided by an Alliance HT2795 (Waters) system comprising a degassing quaternary pump, an automatic sorter and a diode array detector (DAD). Column flow was divided into an MS detector. The EM detector has been configured with an electrospray ionization source. The capillary needle turnover was 3 kV and the source temperature was maintained at 100 ° C in the LCT (Waters Z-Spray Mass Spectrometer Time. Nitrogen was used as the nebulizer gas. was performed with a Waters-Micromass MassLinx-Openlinx data system.

Method A

In addition to the general procedure: reverse phase HPLC was performed on an Xterra-RP C18 column (5 pm, 3.9 χ 150 mm) with a flow rate of 1.0 ml / min at a temperature of 30 ° C. Two mobile phases (mobile phase A: 100% 7 mM ammonium acetate; mobile phase B: 100% acetonitrile); were employed to conduct a gradient condition of 85% A, 15% B (sustained for 3 minutes). ) at 20% A, 80% B within 5 minutes, sustained at 20% A and 80% B for 6 minutes and rebalanced with initial conditions for 3 minutes. An injection volume of 20 11 was used. cone was 20 V for positive and negative ionization mode Mass spectra were acquired by scanning from 100 to 900 in 0.8 seconds using a 0.08 second interlock delay.

In addition to the general procedure: reverse phase HPLC was performed on an Xterra-RP C18 column (5 μιτι, 3.9 χ 150 mm) with a flow rate of 1.0 ml / min at a temperature of 30 ° C. Two mobile phases (mobile phase A: 100% 7 mM ammonium acetate; mobile phase B: 100% acetonitrile); were employed to conduct a gradient condition of 100% A (1 minute hold) to 50% A. 50% B in 4 minutes, 50% A and 50% B hold for 9 minutes and rebalanced with initial conditions for 3 minutes An injection volume of 20 I was used Cone voltage was 20 V for mode positive ionization and 20 V for negative ionization mode Mass spectra were acquired ranging from 100 to 900 in 0.8 seconds using a 0.08 second time delay.

A. Preparation of intermediate compounds

Example A1

Preparation of intermediate 1

<formula> formula see original document page 38 </formula>

A mixture of benzo [b] furan-3-one (400 mg, 0.0029 mol) and N-methoxy-N-methyl (triphenylphosphoranylidene) acetamide (1.19 g, 0.0032 mol) in xylene (10 ml) was stirred at 135 ° C for 35 hours. The reaction was quenched with water and basified with a saturated sodium hydrogen carbonate solution. The mixture was extracted with EtOAc, the organic layer was separated, dried (MgSO 4) 1 filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 pm) (eluent: EtO-Ac / cyclohexane 50/50). The pure fractions were collected and the solvent was evaporated, yielding 210 mg (32%) of intermediate 1 as a brown oil.

1H NMR (300 MHz, CDCl3) δ 7.63 (m, 2H), 7.46 (d, 1H, J = 7.1), 7.25 (m, 2H), 3.84 (s, 2H) 3.70 (s, 3H), 3.22 (s, 3H).

MS (ES +) mlz 220 (M + 1).

Example A2

a) Preparation of intermediate 2 <formula> formula see original document page 39 </formula>

A mixture of 1 H-pyrrolo [2,3-b] pyridine-3-ethanamine (0.014 mol), 1-fluoro-4-nitro-ben2ene (0.015 mol) and DIPE (0.048 mol) was stirred at 210 ° C for 30 min. minutes DIPE was evaporated. The precipitate was dissolved in DCM / MeOH. The organic layer was washed with 10% potassium carbonate, dried (MgSO4), filtered and the solvent was evaporated. The residue (2g) was purified by silica gel column chromatography (15-40 pm) (eluent: DCM / MeOH / NH40H 97/3 / 0.3). The pure fractions were collected and the solvent was evaporated, yielding 0.152 g (28%) of intermediate 2, 2010C melting point (Kofler).

b) Preparation of intermediate 3

<formula> formula see original document page 39 </formula>

A mixture of intermediate 2 (0.004 mol) and Raney Nickel (1g) in MeOH (20ml) was hydrogenated at room temperature for 2 hours under a pressure of 0.3 mPa (3 bar), then filtered over celite. with MeOH. The filtrate was evaporated yielding 0.92g (100%) of intermediate 3.

Example A3

a) Preparation of intermediate 4

A mixture of imidazo [1,2-a] pyridine-3-acetonitrile (0.028 mol) and 5% Rh / Al203 (4.5g) in EtOH (45ml) and MeOH / NH3 (12.5ml) was hydrog-20. at room temperature for 72 hours under a pressure of 0.3 mPa (3 bar), then filtered over celite. Celite was washed with DCM / EtOH. The filtrate was evaporated. The residue was dissolved in DCM. The organic layer was washed with 10% potassium carbonate, dried (MgSO 4), filtered and the solvent was evaporated, yielding: 3.4g (73%) of intermediate 4.25 b) Preparation of intermediate 5 <formula> formula see original document page 40 </formula>

A mixture of intermediate 4 (0.006 mol), 1-fluoro-4-nitro-benzene (0.007 mol) and DIPE (0.021 mol) was heated at 21 ° C for 30 minutes. DIPE was evaporated. The crude oil was diluted in DCM and EtOH (90/10). The organic layer was washed with 10% potassium carbonate, dried (MgSO4), filtered and the solvent was evaporated. The residue (1.3 g) was purified by silica gel column chromatography (20-45 pm) (eluent: DCM / MeOH 98/2). The pure fractions were collected and the solvent was evaporated. The residue (0.36 g) was crystallized from acetonitrile. The precipitate was filtered off and dried, yielding 0.199 g of intermediate 5, melting point 1719C (Kofler).

c) Preparation of intermediate 6

A mixture of intermediate 5 (0.003 mol) and Raney Nickel (1g) in MeOH (20 ml) was hydrogenated at room temperature for 3 hours under a pressure of 0.3 mPa (3 bar), then filtered over celite.15 Celite was washed with MeOH. The filtrate was evaporated, yielding 1g (> 100%) of intermediate 6.

Example A4

a) Preparation of intermediate 7

A mixture of 1-fluoro-4-nitro-benzene (0.0083 mol), 1H-indol-5-amine (0.0076 mol) and DIPE (0.0166 mol) was stirred at 210 ° C for 18 hours. ras, then taken into DCM. The organic layer was washed with 3N hydrochloric acid, then with NaHCO3, dried (MgSO4) 1, and the solvent was evaporated. The residue was taken up in EtOH. The precipitate was filtered, washed with diethyl ether and dried, yielding 1.08 g (65%) of intermediate 7, melting point 180 ° C.

b) Preparation of intermediate 8 <formula> formula see original document page 41 </formula>

A mixture of intermediate 7 (0.0039 mol) and raney nickel (1g) in MeOH (20ml) was hydrogenated at room temperature for 2 hours under a pressure of 0.3 mPa (3 bar), then filtered over celite. was washed with DCM / MeOH. The filtrate was evaporated, yielding 1 g (> 100%) of intermediate 8.

Example A5

Preparation of intermediate 9

<formula> formula see original document page 41 </formula>

1,1'-Carboxydiimidimidazole (315 mg, 0.0019 mol) was added dropwise in a solution of (6-methoxy-benzofuran-3-yl) -acetic acid (400 mg, 0.0019 mol) in DCM (10 ml). The mixture was stirred at room temperature for 3 hours. N, N-Dimethylhydroxylamine hydrochloride (190 mg, 0.0019mol) was added and the mixture was stirred at room temperature for 4 hours. The reaction was quenched with ice and basified with a 4N solution of sodium hydroxide. The mixture was extracted with DCM, the organic layer was separated, dried (MgSO 4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 pm) (eluent: EtOAc / cyclohexane 50/50). The pure fractions were collected and the solvent was evaporated, yielding 410 mg (85%) of intermediate 9 as a colorless oil.

1H NMR (300 MHz, CDCl3) δ 7.54 (m, 2H), 6.99 (s, 1H), 6.88 (d, 1H, J = 6.4), 3.83 (s, 3H) 3.79 (s, 2H), 3.69 (s, 3H), 3.21 (s, 3H).

Example A6

a) Preparation of intermediate 10

<formula> formula see original document page 41 </formula>

A mixture of (3-iodo-2-thienyl) carbamic acid, 1,1-dimethylethyl ester (0.0105 mol), 4-bromo-2-butenoic acid, ethyl ester (0.0158mol) and potassium carbonate NaCl (0.021 mol) in α, β-dimethylformamide (100ml) was stirred at room temperature for 16 hours. Triphenylphosphine (0.001mol) and palladium acetate (0.0005mol) were added. The mixture was stirred at 70 ° C for 8 hours, then washed with water. The organic layer was separated with EtOAc, dried (MgSO 4) and the solvent was evaporated. The residue was purified by silica gel column chromatography (eluent: E-tOAc / cyclohexane 10/90) yielding 2.3 g (70%) of intermediate 10.

b) Preparation of intermediate 11

<formula> formula see original document page 42 </formula>

A solution of intermediate 10 (0.0016 mol) in THF (10ml) was stirred at -78 ° C under argon. A solution of N10 -dimethylhydroxylamine hydrochloride (0.0004 mol) in THF (40ml) was stirred at -78 ° C under argon. Butyllithium (1.6 M in hexane) (0.016 mol) was added dropwise at -78 ° C to the α, β-dimethylhydroxylamine hydrochloride (0.0004 mol). The mixture was stirred at room temperature for 20 minutes, then cooled to -78 ° C. Intermediate 10 was added dropwise. The mixture was stirred at -10 ° C for 2 hours, poured into saturated NH 4 Cl at -70 ° C and extracted with EtOAc. The organic layer was separated, dried (MgSO 4), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (eluent: EtOAc / cyclohexane 10/90 to 30/70). Pure fractions were collected and the solvent was evaporated, yielding 0.4 g (77%) of intermediate 11.

c) Preparation of intermediate 12

<formula> formula see original document page 42 </formula> Intermediate 11 (0.0012 mol) was dissolved in DCM and absorbed on silica gel. The mixture was stirred at 60 ° C for 24 hours in vacuo. The residue was purified by silica gel column chromatography (eluent: E-tOAc / cyclohexane 80/20). Pure fractions were collected and the solvent was evaporated, yielding (62%) of intermediate 12.

d) Preparation of intermediate 13

<formula> formula see original document page 43 </formula>

Lithium aluminum hydride (0.0008 mol) was slowly added at 0Â ° C in a solution of intermediate 12 (0.0008 mol) in THF (4 ml, dry). The mixture was stirred at 0 ° C for 1 hour, poured into ice, washed with 5% KHSO 4 and extracted with EtOAc. The organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated, yielding intermediate 13. This product was directly used in the next reaction step.

B. Preparation of Final Compounds

<formula> formula see original document page 43 </formula>

Example B1

Preparation of compound 1

A solution of 1 N lithium aluminum hydride in THF (0.65 ml) was added dropwise at 0 ° C to a suspension of intermediate 1 (162 mg, 0.00065 mol) in THF (4 ml). The mixture was stirred for 1 hour at 0 ° C. The reaction was quenched at 0 ° C by slowly adding a solution of 5% potassium bisulfate in water and extracted twice with EtOAc. The organic layer was separated, dried (MgSO 4), filtered, and the solvent was evaporated to yield benzofuran-3-yl acetaldehyde. In a solution of N-4-pyridinyl-1,4-benzenediamine in MeOH (3 ml) and acetic acid (4 drops) was added sodium cyanoborohydride (57 mg, 0.00091 mol) and the pre-dissolved aldehyde in MeOH (1 ml). ). The mixture was stirred at room temperature for 18 hours. The solvent was evaporated, the residual oil taken up in EtO-Ac and washed with saturated sodium hydrogen carbonate solution. The organic layer was separated, dried (MgSO 4), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (40-63 pm) (eluent: DCM / MeOH 95/5). Pure fractions were collected and the solvent was evaporated, yielding 41 mg (20%) of compound 1, melting point 152 ° C - 154 ° C.

1H NMR (300 MHz, MeOH-d4) δ 7.98 (d, 2H, J = 6.6), 7.59 (m, 2H), 7.43 (d, 1H, J = 7.4), 7.24 (m, 2H), 7.00 (dd, 2H, J = 8-7, J = 3.3), 6.68 (m, 4H), 3.45 (t, 2H, J = 7 , 2), 2.98 (t, 2H, J = 7.2).

MS (ES +) mlz 330 (M + 1).

Example B2

Preparation of Compound 2

<formula> formula see original document page 44 </formula>

A mixture of intermediate 3 (0.004 mol) and Î ± -bromo-pyridine hydrochloride (0.004 mol) in acetic acid (5ml) was stirred at 140Â ° C for 15 minutes, then evaporated. The residue was dissolved in DCM / MeOH. The organic layer was washed with 10% potassium carbonate, dried (MgSO4), filtered and the solvent was evaporated. The residue (1g) was purified by silica gel column chromatography (15-40 pm) (eluent: DCM / MeOH / NH40H 90/10/1). The pure fractions were collected and the solvent was evaporated. The residue (0 - 74 g, 63%) was crystallized from acetonitrile. The precipitate was filtered off and dried, yielding 0.541 g of compound 2, melting point 182 ° C (Kofler).

1H NMR (DMSO-d6) δ 2.96 (2H, t, J = 7.7 Hz), 3.30 (2H, t, J = 7.7 Hz), 5.6 (1H, br, t, J = 7.6 Hz), 6.58 (4H, m), 6.95 (2H, d, J = 7.7 Hz), 7.03 (1H, m), 7.33 (1H, m), 7.96 (1H, d, J = 7.7Hz), 8.03 (2H, d, J = 7.6Hz), 8.17-8.21 (1H, m), 8.22 (1H , br, s), 11.38 (1H, br, s).

LCMS (ES +) mlz 330 (M + 1), Rt = 7.10, Method A.

Example B3

Preparation of compound 3 <formula> formula see original document page 45 </formula>

4-Bromo-pyridine hydrochloride (0.004 mol) was added to a solution of intermediate 6 (0.004 mol) in acetic acid (10 ml). The mixture was heated at 140 ° C for 15 minutes in a microwave oven. Acetic acid was evaporated. The crude oil was dissolved in DCM / EtOH (90/10). The organic layer was washed with potassium carbonate, dried (MgSO4), filtered and the solvent was evaporated. The residue (1 g) was purified by silica gel column chromatography (15-40 pm) (eluent: DCM / MeOH / NH40H90 / 10 / 0.5 to 90/10/1). Pure fractions were collected and the solvent was evaporated, yielding 0.63 g (48%) of compound 3.

1H NMR (DMSO-d6) δ 1.75-1.93 (4H, m), 2.65-2.78 (4H, m), 3.32 (2H, s), 3.70 (2H, t , J = 6.1 Hz), 5.62 (1H, br, t, J = 7.6 Hz), 6.58-6.53 (5H, m), 6.95 (2H, d, J = 7.7 Hz), 8.05 (2H, d, J = 7.6 Hz), 8.24 (1H, s).

LCMS (ES +) mlz 334 (M + 1), R, 5.18, Method B.

Melting point 256 ° C (Kofler).

Example B4

Preparation of compound 4

<formula> formula see original document page 45 </formula>

4-Bromo-pyridine hydrochloride (0.002 mol) was added to a solution of intermediate 8 (0.0022 mol) in acetic acid (2.8 ml). The mixture was heated at 110 ° C for 30 minutes, poured into ice water and basified with 10% potassium carbonate. The precipitate was filtered off and dried. The residue (0 - 739 g) was purified by column chromatography on kromasil (5Mm) (eluent: DCM / MeOH / NH40H 96/4 / 0.4 to 88/12 / 1.2). The pure fractions were collected and the solvent was evaporated, yielding 0.018 g of compound 4.

1H NMR (DMSOd6) δ 6.32 (1H, br s), 6.68 (1H, d, J = 5.6Hz), 6.9 (1H, dd, J = 10.2Hz, J = 2.5 Hz), 6.55 (2H, d, J = 10.2 Hz), 7.0 (2H, d, J = 10.2 Hz), 7.27 (1 Η, m), 7.32 (1 Η, d, J = 10.2 Hz), 7.7 (1 Η, br s), 8.17 (1Η, dd, J = 10.2 Hz, J = 2.5 Hz) , 8.37 (1 Η, brs), 10.9 (1 Η, brs) .LCMS (ES +) m / z 301 (M + 1), R, 7-73, Method A.

Example B5

Preparation of compound 5

<formula> formula see original document page 46 </formula>

A solution of 1N lithium aluminum hydride in THF (1.0 ml) was added dropwise at OcC to a suspension of intermediate 9 (249mg, 0.0010 mol). The mixture was stirred for 1 hour at 0 ° C. The reaction was quenched at 0 ° C by slowly adding a solution of 5% potassium bisulfate in water and extracted twice with EtOAc. The organic layer was dried, dried (MgSO 4), filtered, and the solvent was evaporated to yield (6-methoxy-benzofuran-3-yl) -acetaldehyde. To a solution of N-4-pyridinyl-1,4-benzenediamine in MeOH (5 ml) and acetic acid (5 drops) was added sodium cyanoborohydride (90 mg, 0.0014 mol) and the previous aldehyde dissolved in MeOH (1 ml). ). The mixture was stirred at room temperature for 18 hours. The solvent was evaporated, the residual oil taken up in EtOAc and washed with a saturated sodium hydrogen carbonate solution. The organic layer was separated, dried (MgSO 4), filtered and the solvent was evaporated. The residue was washed with MeOH and dried, yielding 125 mg (34%) of compound 5, mp 184-186 ° C.

1H NMR (300 MHz, DMSO-d6) δ 8.23 (s, 1H), 8.01 (dd, 2H, J = 6.3, J = 1.5), 7.71 (s, 1H), 7.51 (d, 1H, J = 8.5), 7.14 (d, 1H, J = 2.1), 6.92 (d, 2H, J = 8.6), 6.85 (dd 1H, J = 8.5, J = 2.2), 6.58 (m, 4H), 5.64 (t, 1H, J = 5.6), 3.77 (s, 3H), 3 .30 (t, 2H, J = 7.0), 2.48 (t, 2H, J = 7.0).

MS (ES +) mlz 360 (M + 1).

Example B6

Preparation of compound 6 <formula> formula see original document page 47 </formula>

Acetic acid (a few drops), then intermediate 13 (0.0004 mol) was added dropwise to a solution of Ν-4-pyridinyl-1,4-benzenediamine (0.0008 mol) and sodium cyanoborohydride (0.001 mol ) in MeOH (4 ml). Intermediate 13 (0.0004 mol) was dissolved in MeOH (4 mL).

The mixture was stirred at room temperature for 20 hours, poured into water, washed with saturated NaHCO 3 and extracted with EtOAc. The organic layer was separated, dried (MgSO 4), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (eluent: DCM / MeOH 85/15). Pure fractions were collected and the solvent was evaporated, yielding 0.086 g (34%) of compound 6.

Table F-1 lists the compounds that were prepared in one of the above examples.

Table F-1

<table> table see original document page 47 </column> </row> <table> C. Pharmacological Example:

The ability of the compounds to preserve p53 in A2780 cells was measured with the p53 enzyme-linked immunosorbent assay. The p53 assay is a sandwich enzyme immunoassay employing two polyclonal antibodies. A polyclonal antibody specific for dep53 protein was immobilized on the surface of the plastic wells. Any p53 present in the sample to be analyzed will bind to the capture antibody. The biotinylated detector polyclonal antibody similarly recognizes p53 protein, and will bind to any p53; which was retained by the capture antibody. The detector antibody is successively bound by spicy horseradish peroxidase conjugated streptavidin. Horseradish peroxidase catalyzes the conversion of o-phenylene diamine to chromogenic substrate, the intensity of which is proportional to the amount of protein binding of p53 to the plate. The colored reaction product is quantified using a spectrometer. Quantification is obtained by constructing a standard curve using known concentrations of purified p53 targeted recombinant HIS protein (see example C.1.).

Cell activity of the compounds of formula (I) was determined in U87MG tumor cells using a colorimetric assay for cell toxicity or survival (see example C.2).

U87MG cells are wild type human comp53 glioblastoma cells. In this cell line MDM2 tightly controls p53 ex-pressure.

C.1. P53 ELISA

A2780 (ATCC) cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and gentamicin at 37 ° C in a 5% CO2 moistened incubator.

A2780 cells were seeded into 20,000 cells per well in a 96-well plate, cultured for 24 hours and treated for 16 hours at 37 ° C in a moist incubator. After incubation, cells were washed once with phosphate-buffered saline and 30 µl per well, low salt RIPA buffer (20 mM iris, pH 7.0, 0.5 mM EDTA, 1% Nonidet P40, 0.5% DOC, 0.05% SDS, 1 mM PMSF, 1 μg / m I aprotinin and 0.5 μ / ml leupeptin) were added. Plates were placed on ice for 30 minutes to complete the lysis. P53 protein was detected in lysates using the sandwich ELISA described below.

High binding polystyrene 96-well EIA / RIA plates (Costar 9018) were coated with capture antibody pAb1801 (Abcamab28-100) at a concentration of 1 μg / ml in coating buffer (0.1 M NaHCO3 pH 8, 2), 50 μΙ per well. The antibody was allowed to adhere overnight at 4 ° C. Coated plates were washed once with Phosphate Buffered Saline (PBS) / 0.05% Tween 20 and 300 μamp blocking buffer (PBS, 17 ° C for added bovine serum albumin (BSA)) over a period of 2 hour incubation at room temperature. Dilutions of purified recombinant HIS labeled p53 protein, ranging from 3-200 ng / ml, were prepared in blocking buffer and used as standards. Plates were washed twice with PBS / 0.05% Tween 20 and blocking buffer or standards were added at 80μΙ / well. To the standards, 20 μΙ of Iise buffer was added. Samples were added to the other wells at 20 μΙ Iisa-do / well. Following an overnight incubation at 4 ° C, the plates were washed twice with PBS / 0.05% Tween 20. 100 µl aliquots of p53 secondary polyclonal antibody (FL-393) (Tebubio, sc-6243 ) at a concentration of 1 μg / ml blocking buffer were added to each well and allowed to adhere for 2 hours at room temperature. Plaques were washed three times with PBS / 0.05% Tween 20. Anti-antibody HRP detection antibody (sc-2004, Tebubio) at 0.04 µg / ml in PBS / 1% BSA was added and incubated for 1 hour at room temperature. Plates were washed three times with PBS / 0.05% Tween 20 and 100 μΙ substrate buffer was added (substrate buffer was prepared shortly before use by adding 1 tablet of 10 mg dia-phenylene). Sigma (OPD) and 125 μΙ 3% H2O2 to 25 ml OPD buffer: 35mM citric acid, 66mM Na2HPO4, pH 5.6). After 5 to 10 minutes, color reaction was stopped by adding 50 μΙ Stop Buffer (1M H2SO4) per well. The absorbance at dual wavelengths of 490/655 nm was measured using a Biorad microplate reader and the results were then analyzed. For each experiment, control (no drug containing) and a null incubation (no cell or drug) were conducted in parallel. The blank value was subtracted from all control and sample values. For each sample, the value of p53 (absorbance units) was expressed as the percentage of the parap53 value present in the control. Percentage preservation higher than 140% was defined as significant. Here, the effects of test compounds are expressed as the lowest dose producing at least 140% of the p53 value present in the control (LAD) (see table F-2).

C.2. Proliferation Assay

All compounds tested were dissolved in DMSO and further dilutions were made in culture medium. Final DMSO concentrations never exceeded 0.1% (v / v) in cell proliferation assays. Controls contained no compound U87MG and DMSO cells and nulls contained DMSO, but no cells.

U87MG cells were seeded in 96-well cell culture plates in 3000 cells / well / 100 μΙ. 24 hours later, the changed medium and the compound and / or solvent were added in a 200 μΙ volume. Following 4 days of incubation, the medium was replaced with 200μΙ fresh medium and cell growth was evaluated using an MTT-based assay. Therefore, 25 μΙ of MTT solution (0.5% Serva MTT research grade in phosphate buffered saline) was added to each well and the cells were also incubated for 2 hours at 37 ° C. The medium was then carefully aspirated and the blue MTT-formazane product was dissolved by adding to each well 25 μΙ of 0.1 M glycine and 100 μΙ DMSO. The plates were shaken for another 10 minutes on a microplate shaker before reading absorbance at 540 nm by a Biorad microplate reader. Within one experiment, the results for each experimental condition are the means of 3 replica cavities. For the purpose of initial evaluations, compounds were tested at a single fixed concentration of 10 - 5 M. For active compounds, experiments were repeated to establish total concentration response curves. For each experiment, controls (not containing drug) and an incubation. (containing no cell or drug) were conducted in parallel.The null value was subtracted from all control and sample values.For each sample, the mean value for cell growth (absorbance units) was expressed as a percentage of the value. Control cell growth IC50 values (drug concentration required to reduce cell growth by 50% of control) were computed using categorized unit analysis (Finney, DJ, Probit Analyzes, 2nd Ed. Chapter 10, GraDed Responses, Cambridge University Press, Cambridge 1962.) Here, the effects of test compounds are expressed as plC50 (negative value of Yog value from IC50 value) (see table F-2).

In some of the experiments the proliferation assay was adapted for use in 384-well culture plates (see table F-2).

<table> table see original document page 51 </column> </row> <table>

D. Composition Example: Film-Coated TabletsP Tablet Core Preparation

A mixture of 100 g of a compound of formula (I), 570 g of lactose and 200 g of starch is well mixed and thereafter moistened with a solution of 5 g of sodium dodecyl sulfate and 10 g of polyvinyl pyrrolidone in about 200 ml of water. The wet powder mixture is sieved, dried and sieved again. Then 100 g of microcrystalline cellulose and 15 g of hydrogenated vegetable oil are added. The total is well-blended and compressed into tablets to produce 10,000 tablets, each comprising 10 mg of a compound of formula (I).

Coating

In a solution of 10 g of methyl cellulose in 75 ml of denatured ethane, a solution of 5 g of ethyl cellulose in 150 ml of dichloromethane is added. Then, 75 ml of dichloromethane and 2.5 ml of 1,2,3-propanethriol are added, 10 g of polyethylene glycol is melted and dissolved in 75 ml of dichloromethane. The latter solution is added to the above and then 2.5 g of magnesium octadecanoate, 5 g of polyvinyl pyrrolidone and 30 ml of concentrated color suspension are added and the total is homogenated. Tablet cores are coated with the mixture thereof obtained in a coating apparatus.

Claims (10)

1. Compound of formula (I) 1 <formula> formula see original document page 53 </formula> a / V-oxide form, an addition salt or a stereochemically isomeric form thereof, wherein 0, 1, or 2 and when m is 0, then a direct bond is intended; η is 0, 1, 2, 3 or 4 and when η is 0 then a direct bond is intended; ρ is 0 or 1 and when ρ is 0 then a direct bond is intended; s is 0, or 1 and when s is 0 then a direct bond is intended; t is 0 or 1 and when t is 0 then a direct bond is R 1 and R 2 are each independently hydrogen, halo, C 1-6 alkyl, C 1-6 alkyloxy, arylC 1-6 alkyloxy, heteroarylC 1-6 alkyloxy, phenylthio, hydroxyC 1-6 alkylcarbonyl, C 1-6 alkyl substituted with a substituent selected from amino aryl and heteroaryl; orC3-7Cycloalkyl substituted with one substituent selected from amino, aryl and heteroaryl; A is a radical selected from <formula> formula see original document page 53 </formula> <formula> formula see original document page 54 </formula> R 4 and R 5 are each independently selected from hydrogen, halo, C 1-6 alkyl, polyalkylC 1-6 alkyl, cyano, cyanoC 1-6 alkyl, hydroxyC 1-6 alkyl, hydroxy, amino, C 1-6 alkyloxy, C 1-6 alkylcarbonyl, methylsulfonylamino, aryl or heteroaryl; Z is a radical selected from <formula> formula see original document page 55 </formula> wherein R 6 or R 7 is each independently selected from hydrogen, halo, hydroxy, amino, C 1-6 alkyl, nitro, polyalkyl. 6 alkyl, cyano, C 1-6 alkyl, tetrazoloC 1-6 alkyl, aryl, heteroaryl, arylC 1-6 alkyl, heteroarylC 1-6 alkyl, aryl (hydroxy) C 1-6 alkyl, heteroaryl (hydroxy) C 1-6 alkyl, arylcarbonyl, heteroarylcarbonyl, aryl 6alkylcarbonyl, arylCvealkylcarbonyl, h C1-6 alkylcarbonyl, C1-6 alkyloxy, C3-7Cycloalkylcarbonyl, C3-7cycloalkyl (hydroxy) C1-6alkyl, arylC6-6alkyloxyC1-6alkyloxyC1-6alkyloxyC6-6alkyloxy 6 alkyl, hydroxyC 1-6 alkyloxyC 1-6 alkyl, C1-6 alkyloxycarbonylC 1-6 alkyloxy, C1-6 alkyloxyoxy alkyl, C1-6 alkyloxycarbonyl, aminocarbonyl, hydroxyC 1-6 alkyloxy, amino-C 1-6 alkyl (C (= O) r) - (CHR10) u-NR8R9, where v is 0, 1, 2, 3, 4, 5, or 6 and when v is 0, then a direct bond is intended; is 0, or 1 and when r is 0 then a direct bond is desired, u is 0, 1, 2, 3, 4, 5, or 6 and when u is 0 then a direct bond is intended R 10 is hydrogen or C 1-6 alkyl R 8 and R 9 are each independently selected from hydrogen, C 1-12 alkyl, C 1-6 alkylcarbonyl, C 1-6 alkylsulfonyl, arylC 1-6 alkylcarbonyl, C 3.7Cidoalkyl, C 3-7 Cycloalkylcarbonyl, - (CH 2) k -NR 11 R 12, C 1-12 alkyl substituted with a substituent selected from hydroxy, hydroxycarbonyl, cyano, C 1-6 alkyloxycarbonyl, C 1-6 alkyloxy, aryl or heteroaryl; substituted with a substituent selected from hydroxy, C 1-6 alkyloxy, aryl, amino, arylC 1-6 alkyl, heteroaryl or heteroarylC 1-6 alkyl; or R 8 and R 9 together with the nitrogen to which they are attached may optionally form a substituted morpholinyl, piperidinyl, pyrrolidinyl, pipe-razinyl, or piperazinyl selected from C 1-6 alkyl, arylC 1-6 alkyl, arylC 1-6 alkyloxycarbonyl, heteroarylC 3 -alkyl, 7 cycloalkyl and C 3-7 cycloC 1-6 alkyl; wherein k is O, 1, 2, 3, 4, 5, or 6 and when k is O, then a straight bond is desired, R 11 and R 12 are each independently selected from hydrogen, C 1 -C 4 -alkyl arylC 4; ealkyloxycarbonyl, C 3-7 cycloalkyl, C 1-12 alkyl substituted with a substituent selected from hydroxy, C 1-6 alkyloxy, aryl, and heteroaryl; C 3-7 cycloalkyl substituted with a substituent inte selected from hydroxy, C 1-6 alkyloxy, aryl, arylC 1-6 alkyl, heteroaryl, and heteroarylC 1-6 alkyl; or R 11 and R 12 together with the nitrogen to which they are attached may optionally form a C 1-6 alkyloxycarbonyl substituted morpholinyl, piperazinyl or piperazinyl, aryl is phenyl or naphthalenyl, each phenyl or naphthalenyl may optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, hydroxyCvealkyl, Cvealkyl, amino, PolyalkylC 1-6 alkyl and C 1-6 alkyloxy; Each phenyl or naphthalenyl may optionally be substituted with a bivalent radical selected from methylenedioxy and ethylenedioxy: heteroaryl is pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl, tetrahydridyl, pyridyl, imidazoline, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl, or tetrahydrofuranyl may optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, C 1-6 alkyl, amino, polyalkylC 1-6 alkyl, aryl, arylC 1-6 alkyl or C 1-6 alkyloxy; Each pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, benzofuranyl, or tetrahydrofuranyl may optionally be substituted for a bivalent radical selected from methylenedioxy or ethylenedioxy. A compound according to claim 1 wherein m is 0; η is 0 or 2; ρ is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-15), (a-21), (a-30), (a-39) or (a-40), R4 and R5 are each independently selected from hydrogen. or C1-6 alkyloxy; Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen. A compound according to claim 1 or 2, wherein 0 is; η is 2; ρ is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-21), (a-39) or (a-40), R 4 and R 5 are each independently selected from hydrogen or C 1-6 alkyloxy; Z is the radical (b-2) ); or R6 and R7 are each independently selected from hydrogen. A compound according to claim 1, 2 or 3, wherein the compound is compound No. 2, compound No. 3 or compound No. 5. <table> table see original document page 57 </column> </ row > <table> A compound according to claim 1, 2, 3 or 4 for use as a medicament. Pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound according to claims 1 to 4. A process for preparing a pharmaceutical composition as defined in claim 6, wherein the pharmaceutically acceptable carriers and a compound as defined in claims 1 to 4 are intimately mixed. Use of a compound as defined in claim 1, 2, 3 or 4 for the manufacture of a medicament for treating a p53-MDM2 interaction mediated disorder. A combination of an anticancer agent and a compound as defined in claim 1,2,3 or 4. A process for preparing a compound as defined in claim 1, wherein: a) reacting an intermediate of formula (II) with an intermediate of formula (III) wherein W is an appropriate outlet such as, for example, halo (b) converting an intermediate of formula (IV) into compounds of formula (I), where ρ is 1, herein referred to as compounds of formula (la), in the presence of hydride c) reacting an appropriate carboxaldehyde of formula (VI) with an intermediate of formula (V) in the presence of an appropriate reagent in a suitable solvent. (d) reacting an intermediate of formula (II) with an appropriate carboxaldehyde of formula (VII) with the formation of a compound of formula (I) wherein t is 1, referred to herein as compounds of formula (1b), or <formula> e) reacting an intermediate of formula (VIII) with lithium aluminum hydride in a suitable solvent with the formation of a compound of formula (I) wherein s is 1, referred to herein as compounds of formula (lc), <formula> formula see original document page 60 </formula>