RU2434863C2 - Derivatives of cyclic alkylamines as inhibitors of reaction between mdm2 and p53 - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: invention describes a compound of formula (I) (I), its N-oxide form, addition salt or stereochemically isomeric form, where m equals 0, 1, and m equals 0 denotes a direct bond; n equals 0, 1, 2 or 3, and n equals 0 denotes a direct bond; p equals; t equals 0 or 1, and t equals 0 denotes a direct bond; denotes -CR8=C<, and the dotted line denotes a bond, where R8 denotes hydrogen; R1 and R2 denotes hydrogen; R3 and R4 denote hydrogen; R5 denotes hydrogen; R6 and R7 each is independently selected from hydrogen or C1-6alkyl; Z denotes a radical selected from (a-1) (a-2) (a-4), where R10 and R11 are each independently selected from hydrogen, hydroxy, C1-6 alkylcarbonyl, C1-6 alkylcarbonyloxy C1-6 alkyl, C1-6 alkyloxycarbonyl, C1-6 alkylcarbonyloxy, hydroxy C1-6 alkyl. The invention also describes a pharmaceutical composition for treating cancer, based on the compound of formula I, as well as a method of preparing said composition and use of the compound of formula I, combination thereof with an anticancer agent and preparation method thereof. ^ EFFECT: improved properties of compounds. ^ 14 cl, 6 ex, 2 tbl

Description

Field of Invention

This invention relates to compounds and compositions containing these compounds, acting as inhibitors of the interaction between MDM2 and p53. Moreover, this invention relates to methods for producing the described inhibitors, compositions containing them, and methods for their use, for example, as medicines.

p53 is a tumor suppressor protein that plays a major role in regulating the balance between cell proliferation and cessation of cell growth / apoptosis. Under normal conditions, the half-life of p53 is very short, and therefore the level of p53 in the cells is very low. However, in response to cellular DNA damage or cellular stress (e.g., oncogen activation, telomere erosion, hypoxia), p53 levels increase. This increase in p53 levels leads to the activation of transcription of certain genes, which leads to cell termination of growth or apoptosis. Thus, an important function of p53 is to prevent the uncontrolled proliferation of damaged cells and, therefore, protect the body from cancer.

MDM2 is a key negative regulator of p53 function. It forms a negative autoregulatory loop by binding to the p53 amino-terminal transactivation domain, and thus MDM2 inhibits p53's ability to activate transcription and targets p53 for proteolytic degradation. Under normal conditions, this regulatory loop is responsible for maintaining low p53 levels. However, in tumors with wild-type p53, the equilibrium concentration of active p53 can be increased by antagonizing the interaction between MDM2 and p53. As a result, there will be a restoration of p53-mediated pro-apoptotic and anti-proliferative effects in such tumor cells.

MDM2 is a cellular proto-oncogen. Overexpression of MDM2 is observed in a number of malignant diseases. MDM2 is overexpressed in a variety of tumors due to gene amplification or increased transcription or translation. The mechanism by which the amplification of MDM2 promotes tumor formation at least in part relates to its interaction with p53. In cells overexpressing MDM2, the protective function of p53 is blocked, and therefore the cells are not able to respond to DNA damage or cell stress by increasing levels of p53, leading to cessation of cell growth and / or apoptosis. Thus, after DNA damage and / or cell stress, cells overexpressing MDM2 are free to continue to proliferate and acquire a tumorigenic phenotype. Under such conditions, disruption of the interaction of p53 and MDM2 would facilitate the release of p53 and, accordingly, normal signals of growth termination and / or apoptosis would function.

MDM2 may also have specific functions in addition to inhibition of p53. For example, it has been shown that MDM2 interacts directly with the pRb-regulated transcription factor E2F1 / DP1. This interaction could be crucial for the p53-independent oncogenic activities of MDM2. The E2F1 domain is strikingly similar to the p53 MDM2 binding domain. Since the interactions of MDM2 with both p53 and E2F1 take place at the same binding site on MDM2, it can be assumed that MDM2 / p53 antagonists will not only activate cellular p53, but also modulate E2F1 activities, the regulation of which usually ceases in tumor cells.

In addition, the therapeutic effectiveness of the DNA damaging agents currently used (chemotherapy and radiotherapy) can be limited by downregulating p53 via MDM2. So, if inhibition of p53 in response to MDM2 is interrupted, increasing the functional levels of p53 will increase the therapeutic efficacy of such agents by restoring wild-type p53 function, which leads to apoptosis and / or reverse development of drug resistance associated with p53. It has been demonstrated that combining inhibition by MDM2 and DNA damaging treatments in vivo leads to synergistic antitumor effects (Vousden K.H., Cell, Vol. 103, 691-694, 2000).

Thus, the disruption of the interaction of MDM2 and p53 is an approach for therapeutic intervention in wild-type p53 tumors, possibly even revealing antiproliferative effects in tumor cells that lack functional p53, and, moreover, can sensitize tumorigenic cells to chemotherapy and radiotherapy.

State of the art

JP 11130750, published May 18, 1999, describes, among other things, 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 (VEGFRs) and as useful in the treatment of angiogenic disorders.

EP1317443, published March 21, 2002, discloses tricyclic tert-amine derivatives useful as CXCR4 or CCR5 chemokine receptor modulators against human immunodeficiency virus and feline immunodeficiency virus.

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

WO00 / 15357, published March 23, 2000, proposes piperazine-4-phenyl derivatives as inhibitors of the interaction between MDM2 and p53. EP1137418, published June 8, 2000, proposes tricyclic compounds to compensate for the conformational stability of the p53 family protein.

EP 1443937, published May 22, 2003, describes substituted 1,4-benzodiazepines and their use as inhibitors of MDM2-p53 interactions.

EP1458380, published June 26, 2003, provides cis-2,4,5-triphenyl-imidazolones that inhibit the interaction of the MDM2 protein with p53-like peptides and have antiproliferative activity.

EP1519932, published January 15, 2004, discloses bisarylsulfonamide compounds that bind to MDM2 and may be useful in cancer therapy.

However, there remains a need for effective and strong small molecules that inhibit the interactions between MDM2 and p53.

The compounds of this invention differ from the compounds of the prior art in structure, in their pharmacological activity and / or in pharmacological efficacy.

Description of the invention

This invention relates to compounds, compositions and methods for inhibiting the interactions between MDM2 and p53 for the treatment of cancer. In addition, the compounds and compositions of this invention are useful for enhancing the efficacy of chemotherapy and radiotherapy.

This invention relates to compounds of formula (I)

to its N-oxide form, additive salt or stereochemically isomeric form, where

m is 0, 1 or 2, and when m is 0, then a direct relationship is implied;

n is 0, 1, 2 or 3, and when n is 0, then a direct relationship is implied;

p is 0 or 1, and when p is 0, then a direct relationship is implied;

t is 0 or 1, and when t is 0, then a direct relationship is implied;

означает -CR⁸=C<, и тогда пунктирная линия представляет связь, -C(=O)-CH<, -C(=O)-N<, -CHR⁸-CH< или -CHR⁸-N<; где

means —CR ⁸ = C <, and then the dashed line represents the bond, —C (= O) —CH <, —C (= O) —N <, —CHR ⁸ —CH <, or —CHR ⁸ —N <; Where

each R ⁸ independently represents hydrogen or C _1-6 alkyl;

R ¹ and R ² are each independently selected from hydrogen, halogen, C _1-6 alkyl, C _1-6 alkyloxy, aryl C _1-6 alkyloxy, heteroaryl C _1-6 alkyloxy, phenylthio, hydroxy C _1-6 alkylcarbonyl, C _1-6 alkyl substituted with a substituent selected from amino, aryl and heteroaryl; or C _3-7 cycloalkyl substituted with a substituent selected from amino, aryl and heteroaryl;

R ³ and R ⁴ are each independently selected from hydrogen, halogen, C _1-6 alkyl, polyhalo C _1-6 alkyl, cyano, cyano C _1-6 alkyl, hydroxy, amino or C _1-6 alkyloxy; or

R ⁴ and R ⁵ together may optionally form a divalent radical selected from methylenedioxy or ethylenedioxy;

R ⁵ represents hydrogen, C _1-6 alkyloxycarbonyl or C _1-6 alkyl;

R ⁶ and R ⁷ are each independently selected from hydrogen, C _1-6 alkyloxy C _1-6 alkyl, or C _1-6 alkyl; or

R ⁶ and R ⁷ together may optionally form a divalent radical selected from

- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ - (b-1),

- (CH ₂ ) ₂ -NR ⁹ - (CH ₂ ) _2- (b-2),

where R ⁹ represents hydrogen, C _1-6 alkyloxy-C _1-6 alkyl or C _1-6 alkyl;

Z represents a radical selected from

Where

R ¹⁰ and R ¹¹ are each independently selected from such as hydrogen, halogen, hydroxy, amino, C _1-6 alkyl, nitro, polyhalogen C _1-6 alkyl, cyano, cyano C _1-6 alkyl, tetrazolo C _{1- 6} alkyl, aryl, heteroaryl, aryl C _1-6 alkyl, heteroaryl C _1-6 alkyl, aryl (hydroxy) C _1-6 alkyl, heteroaryl (hydroxy) C _1-6 alkyl, arylcarbonyl, heteroarylcarbonyl, C _1-6 alkylcarbonyl , aryl C _1-6 alkylcarbonyl, heteroaryl C _1-6 alkylcarbonyl, C _1-6 alkyloxy, C _3-7 cycloalkylcarbonyl, C _3-7 cycloalkyl (hydroxy) C _1-6 alkyl, aryl C _1-6 alkyloxy C _{1- 6} alkyl, C _1-6 alkyloxy C _1-6 alkyloxy C _1-6 alkyl, C _1-6 alkylcarbonyl Loksey C _1-6 alkyl, C _1-6 alkyloxycarbonyl C _1-6 alkyloxy C _1-6 alkyl, hydroxy C _1-6 alkyloxy C _1-6 alkyl, C _1-6 alkyloxycarbonyl C _2-6 alkenyl, C _1-6 alkyloxy C _1-6 alkyl, C _1-6 alkyloxycarbonyl, C _1-6 alkylcarbonyloxy, aminocarbonyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, hydroxycarbonyl, hydroxycarbonyl C _1-6 alkyl and - (CH ₂ ) _v - (C (= O) _r ) - (CHR ^l7 ) _u -NR ¹³ R ¹⁴ ; Where

v is 0, 1, 2, 3, 4, 5, or 6, and when v is 0, then a direct relationship is implied;

r is 0 or 1, and when r is 0, then a direct relationship is implied;

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

R ¹⁷ represents hydrogen or C _1-6 alkyl;

R ¹² represents hydrogen, C _1-6 alkyl, C _3-7 cycloalkyl, C _1-6 alkyl substituted with a substituent selected from hydroxy, amino, C _1-6 alkyloxy and aryl; or C _3-7 cycloalkyl substituted with a substituent selected from hydroxy, amino, aryl and C _1-6 alkyloxy;

R ¹³ and R ¹⁴ are each independently selected from hydrogen, C _1-12 alkyl, C _1-6 alkylcarbonyl, C _1-6 alkylsulfonyl, aryl C _1-6 alkylcarbonyl, C _3-7 cycloalkyl, C _{3- 7} cycloalkylcarbonyl, - (CH ₂ ) _k -NR ¹⁵ R ¹⁶ , 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 a substituent selected from hydroxy, C _1-6 alkyloxy, aryl, amino, aryl C _1-6 alkyl, heteroaryl or heteroaryl C _1-6 alkyl; or

R ¹³ and R ^14, together with the nitrogen atom to which they are attached, may optionally form morpholinyl, piperidinyl, pyrrolidinyl, piperazinyl or piperazinyl substituted with a substituent selected from C _1-6 alkyl, aryl C _1-6 alkyl, aryl C _{1- 6} alkyloxycarbonyl, heteroaryl C _1-6 alkyl, C _3-7 cycloalkyl and C _3-7 cycloalkyl C _1-6 alkyl; Where

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

R ¹⁵ and R ¹⁶ are each independently selected from, for example, hydrogen, C _1-6 alkyl, aryl C _1-6 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, aryl C _1-6 alkyl, heteroaryl and heteroaryl C _1-6 alkyl; or

R ¹⁵ and R ^16, together with the nitrogen atom to which they are attached, may optionally form morpholinyl, piperazinyl or piperazinyl substituted with C _1-6 alkyloxycarbonyl;

aryl means phenyl or naphthalenyl;

each phenyl or naphthalenyl optionally can be substituted with one, two or three substituents, each independently selected from halogen, hydroxy, C _1-6 alkyl, amino, polyhalo C _1-6 alkyl and C _1-6 alkyloxy; and

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

heteroaryl means 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 of which is independently selected from halogen, hydroxy, C _1-6 alkyl, amino, polyhalogen C _1-6 alkyl, aryl, aryl C _1-6 alkyl or C _1-6 alkyloxy; and

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

Compounds of formula (I) may also exist in their tautomeric forms. Such forms, although not shown in detail in the above formula, of course, should be included in the scope of the claims according to this invention.

Some of the terms used in the previous definitions are explained hereinafter. These terms are sometimes used as such or in complex terms.

As used herein in the preceding definitions and hereinafter, the term halogen is common to fluorine, chlorine, bromine and iodine; C _1-6 alkyl means straight or 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- methylbutyl, 2-methylpentyl and the like; hydroxy C _1-6 alkyl means straight or branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms; trihalomethyl is methyl containing three identical or different halogen substituents, for example trifluoromethyl; C _3-7 cycloalkyl includes cyclic hydrocarbon groups having from 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and the like.

The term “addition salt” contains salts that the compounds of formula (I) are capable of forming with organic or inorganic bases, such as amines, alkali metal bases and alkaline earth metal bases or quaternary ammonium bases, or with organic or inorganic acids, such as mineral acids, sulfonic acids, carboxylic acids or phosphorus acids.

The term “addition salt” further comprises pharmaceutically acceptable salts, metal complexes and solvates and their salts, which the compounds of formula (I) are capable of forming.

The term "pharmaceutically acceptable salts" means pharmaceutically acceptable addition salts with an acid or base. It is intended that these pharmaceutically acceptable acid or base addition salts comprise therapeutically active addition salt forms with a non-toxic acid and a non-toxic base that the compounds of formula (I) are capable of forming. Compounds of formula (I) that have base properties can be converted to their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Suitable acids contain, for example, inorganic acids, such as hydrohalic acids, for example hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like 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, benzene sulfonic, -toluenesulfonic, cyclic, salicylic, p-aminosalicylic, pamic and the like acids.

Compounds of formula (I) which have acidic properties can be converted into their pharmaceutically acceptable base addition salts by treating said acid form with an appropriate organic or inorganic base. Suitable salt forms with a base include, for example, ammonium salts, alkali and alkaline earth metal salts, for example lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, for example salts of benzathine, N-methyl-D-glucamine, hydrabamine and salts with amino acids such as, for example, arginine, lysine and the like.

The terms acid or base addition salts also contain hydrates and solvent addition forms which the compounds of formula (I) are capable of forming. Examples of such forms are, for example, 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 salts. Examples of these organic or inorganic salts include halides, nitrates, sulfates, phosphates, acetates, trifluoroacetates, trichloroacetates, propionates, tartrates, sulfonates, for example methyl sulfonates, 4-methylphenyl sulfonates, salicylates, benzoates and the like, metals of the second main group of the periodic system, for example magnesium salt or calcium, the third or fourth main group, for example aluminum, tin, lead, as well as the first to eighth transition group of the periodic system, such as, for example, chromium, manganese, iron, cob alt, nickel, copper, zinc and the like.

As used herein, the term “stereochemically isomeric forms of the compounds of formula (I)” means all possible compounds derived from the same atoms linked by the same sequence of bonds, but having different three-dimensional structures that are not interchangeable, which the compounds of formula (I) may possess. Unless otherwise mentioned and indicated, the chemical designation of the compound encompasses a mixture of all possible stereochemically isomeric forms that the compound may possess. The specified mixture may contain all diastereomers and / or enantiomers of the main molecular structure of the specified compounds. All stereochemically isomeric forms of the compounds of formula (I), whether in pure form or in admixture with each other, are considered to be included within the scope of the claims of this invention.

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

The pure stereoisomeric forms of the compounds and intermediates referred to herein are characterized as isomers substantially free 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., a minimum of 90% of one isomer and a maximum of 10% of other possible isomers) up to a stereoisomeric excess of 100% (i.e. e. 100% of one isomer in the absence of others), more specifically, compounds or intermediates having a stereoisomeric excess of 90% up to 100%, more specifically, having a stereoisomeric excess of 94% up to 100% and, most specifically, having a stereoisomeric excess 97 % up to 1 00% off. The terms “enantiomerically pure” and “diastereomerically pure” should be understood in a similar manner, but as related to the enantiomeric excess, respectively, to the diastereomeric excess in the mixture in question.

The tautomeric forms of the compounds of formula (I) relate to the compounds of formula (I), where, for example, the enol group is converted to a keto group (keto-enol tautomerism).

The N-oxide forms of the compounds of formula (I) relate to the compounds of formula (I), where one or more nitrogen atoms are oxidized to the so-called N-oxide, especially those N-oxides, where one or more nitrogen atoms of piperidine, piperazine or pyridazinyl are N -oxidized.

The compounds of formula (I) can be converted into the corresponding N-oxide forms by the following procedures known in the art for the conversion of trivalent nitrogen to its N-oxide forms. Said N-oxidation reaction, as a rule, can be carried out by reacting the starting compound of formula (I) with the corresponding organic or inorganic peroxide. Suitable inorganic peroxides include, for example, hydrogen peroxide, alkali metal or alkaline earth metal peroxides, sodium peroxide, potassium peroxide; the corresponding organic peroxides may contain peroxyacids, such as, for example, benzenecarboxylic acid or halogen-substituted benzenecarboxylic acid, for example 3-chlorobenzenecarboxylic acid, peroxoalkanoic acids, for example peroxoacetic acid, alkyl hydroperoxides, for example tert-butyl. Suitable solvents are, for example, water, lower alcohols, for example ethanol and the like, hydrocarbons, for example toluene, ketones, for example 2-butanone, halogenated hydrocarbons, for example dichloromethane, and mixtures of such solvents.

This invention also relates to any isotopes 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.

Wherever used hereinafter, the term "compounds of formula (I)" also includes N-oxide forms, pharmaceutically acceptable addition salts with acids or bases, and all stereoisomeric forms.

The first group of compounds of interest consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

a) m is 0;

b) n is 2;

c) p is 1;

d) t is 0;

означает -CH=C<;e)

means -CH = C <;

f) R ¹ and R ² each independently represent hydrogen;

g) R ³ and R ⁴ each independently represent hydrogen;

h) R ⁵ represents hydrogen;

i) R ⁶ and R ⁷ each independently represent hydrogen or C _1-6 alkyl;

j) Z represents a radical selected from (a-1), (a-2) or (a-4); or

k) R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy, C _1-6 alkyloxycarbonyl or hydroxy C _1-6 alkyl.

The second group of compounds of interest consists of those compounds of formula (I) and those compounds of the first group of compounds of interest where one or more of the following restrictions are used:

a) m is 0;

b) n is 2;

c) p is 1;

d) t is 0;

означает -CH=C<;e)

means -CH = C <;

f) R ¹ and R ² each independently represent hydrogen;

g) R ³ and R ⁴ each independently represent hydrogen;

h) R ⁵ represents hydrogen;

i) R ⁶ and R ⁷ each independently represent hydrogen,

j) Z represents a radical selected from (a-2) or (a-4); or

k) R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy or hydroxy C _1-6 alkyl.

A group of preferred compounds consists of those compounds of formula (I) or any subgroup thereof, wherein m is 0; n is 0; p is 1; t is 0; R ¹ and R ² each independently represent hydrogen; R ³ and R ⁴ each independently represent hydrogen; R ⁵ represents hydrogen; R ⁶ and R ⁷ each independently represent hydrogen or C _1-6 alkyl; Z represents a radical selected from (a-1), (a-2) or (a-4); or R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy, C _1-6 alkyloxycarbonyl or hydroxy C _1-6 alkyl.

A group of more preferred compounds consists of such compounds of formula (I) or any subgroup thereof, where m is 0; n is 0; p is 1; t is 0; R ¹ and R ² each independently represent hydrogen; R ³ and R ⁴ each independently represent hydrogen; R ⁵ represents hydrogen; R ⁶ and R ⁷ each independently represent hydrogen; Z represents a radical selected from (a-2) or (a-4); or R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy or hydroxy C _1-6 alkyl.

Most preferred compounds are compound No. 1, connection No. 4 and connection No. 5.

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

Some such production methods will be described hereinafter in more detail. Other methods for preparing the final compounds of formula (I) are described in the examples.

Compounds of formula (I) can be prepared by reacting an intermediate of formula (II) with an intermediate of formula (III), where W represents an appropriate leaving group, such as, for example, halogen, for example fluorine, chlorine, bromine or iodine, or sulfonyloxy a radical such as methylsulfonyloxy, 4-methylphenylsulfonyloxy and the like. The reaction can be carried out in a reaction inert solvent, such as, for example, an alcohol, for example methanol, ethanol, 2-methoxyethanol, propanol, butanol and the like; ether, for example 4,4-dioxane, 1,1'-hydroxybispropane and the like; ketone, for example 4-methyl-2-pentanone; or N, N-dimethylformamide, nitrobenzene, acetonitrile, acetic acid and the like. The addition of an appropriate base, such as, for example, an alkali metal or alkaline earth metal carbonate or hydrogen carbonate, for example triethylamine or sodium carbonate, can be used to trap the acid that is released during the reaction. A small amount of the corresponding metal iodide, for example sodium or potassium iodide, may be added to promote the reaction. Stirring can improve the reaction rate. The reaction can easily be carried out at a temperature between room temperature and the boiling point to return the reflux of the reaction mixture and, if desired, the reaction can be carried out at elevated pressure.

Compounds of formula (I), where p is 1, referred to herein as compounds of formula (I-a), can be prepared by conversion of intermediates of formula (IV) using mixed lithium aluminum hydride in a suitable solvent such as tetrahydrofuran.

Compounds of formula (Ia) can also be prepared by reacting the corresponding carboxaldehyde of formula (VI) with an intermediate of formula (V) in the presence of an appropriate reagent such as sodium borohydride, for example sodium tetrahydroborate or cyanotrihydroborate on a polymer carrier, in a suitable solvent, such as alcohol e.g. methanol.

Similarly, compounds of formula (I), where t is 1, referred to herein as compounds of formula (I-b), can be prepared by reacting an intermediate of formula (II) with the corresponding carboxaldehyde of formula (VII).

The compounds of formula (I) can also be converted into each other by methods known in the art for reactions or transformations of functional groups. Some of these conversions are described above. Other examples are hydrolysis of carboxylic acid esters to the corresponding carboxylic acid or alcohol; hydrolysis of amides to the corresponding carboxylic acids or amines; hydrolysis of nitriles to the corresponding amides; the amino groups on imidazole or phenyl may be substituted with hydrogen by diazotization reactions known in the art and subsequent substitution of the diazo groups with hydrogen; alcohols can be converted into esters and ethers; primary amines can be converted to secondary or tertiary amines; double bonds can be hydrogenated to the corresponding single bond; the iodine radical on the phenyl group can be converted to the ester group by the introduction of carbon monoxide in the presence of a suitable palladium catalyst.

Intermediates of formula (V), where m is 0, referred to herein as intermediates of formula (V-a), can be prepared by converting an intermediate of formula (VIII) with hydrazine hydrate in a suitable solvent such as methanol.

Intermediates of formula (Va) can also be prepared by reducing a nitro group to an amine starting from an intermediate of formula (XVI) in the presence of a metal catalyst, such as Raney nickel, and an appropriate reducing agent, such as hydrogen, in a suitable solvent, such as methanol or ethanol.

Intermediates of formula (X) can 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 can be carried out in the presence of a base, such as triethylamine, in a suitable solvent, such as a mixture of dichloromethane and tetrahydrofuran.

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

Intermediates of formula (VIII) (the same for intermediates of formula (XVI)), where t is 0, referred to herein as intermediates of formula (VIII-a), can be prepared by reacting an intermediate of formula (IX) with an intermediate of formula (XIV), where L represents an appropriate leaving group, such as, for example, halogen, for example fluorine, chlorine, bromine or iodine, or

C _1-6 alkyloxy, for example methyloxy, in the presence of a solution of hydrochloride in 2-propanol, in a reaction inert solvent, such as N, N-dimethylformamide.

Intermediates of formula (IX), wherein R ⁶ and R ⁷ are both hydrogen, referred to herein as intermediates of formula (IX-a), can be prepared by converting an intermediate of formula (XV) in the presence of sodium cyanoborohydride. The reaction can be carried out in a reaction inert solvent, such as, for example, acetic acid.

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 the configuration of R or S.

Some of the compounds of formula (I) and some of the intermediates of this invention may contain an asymmetric carbon atom. Stereochemically pure isomeric forms of these compounds and these intermediates can be obtained using procedures known in the art. For example, diastereoisomers can be separated by physical methods, such as selective crystallization or chromatographic techniques, for example, countercurrent distribution, liquid chromatography, and the like. Enantiomers can be obtained from racemic mixtures by first converting said racemic mixtures with appropriate resolving agents, such as, for example, chiral acids, into a mixture of diastereomeric salts or compounds; then the physical separation of these mixtures of diastereomeric salts or compounds by, for example, selective crystallization, chromatography with supercritical fluid or chromatographic techniques, for example, liquid chromatography, and the like methods; and finally, the conversion of these individual diastereomeric salts or compounds into the corresponding enantiomers. Pure stereochemically isomeric forms can also be obtained from pure stereochemically isomeric forms of the corresponding intermediates and starting materials, provided that the transition reactions occur stereochemically.

The compounds of formula (I), their pharmaceutically acceptable acid addition salts and stereoisomeric forms have useful pharmacological properties since they inhibit the interaction between p53 and MDM2.

As used herein, the term “MDM2” means a protein resulting from expression of the mdm2 gene. Within the meaning of this term, MDM2 encompasses all the proteins encoded by the mdm2 gene, their mutants, their alternative parts of proteins, and their phosphorylated proteins. Additionally, the term “MDM2” as used herein includes analogs of MDM2, for example MDMX, also known as MDM4, and homologs and analogs of MDM2 of other animals, for example, the human HDM2 homolog or human HDMX analogue.

The term "interaction inhibition" or "interaction inhibitor" is used here to prevent or reduce the direct or other association of one or more molecules, peptides, proteins, enzymes or receptors, or to 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 Section C.1. This enhancement may be caused, but not limited to, by one or more of the following mechanisms of action:

- inhibition of the interaction of p53 with MDM2,

- direct association with either the MDM2 protein or p53 protein,

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

- passivation or transportation of MDM2 and p53 to various cell compartments,

- modulation of proteins associated with MDM2, for example (but without limitation) p73, E2F-1, Rb, p21waf1 or cip1,

- down-regulation or interference by expression of MDM2 and / or activity of MDM2, for example (but not limited to) effects on its cellular 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 mis-folding,

- improving the expression of p53 or expression of members of the p53 family, for example p63 and p73,

- enhancing p53 activity, for example (but without limitation) by improving its transcriptional activity, and / or

- enhancing the expression of genes and proteins of the p53 signaling pathway, for example (but without limitation) p21waf1, cip1, MIC-1 (GDF-15), PIG-3 and ATF-3.

Therefore, this invention relates to compounds of formula (I) for use as a medicine.

In addition, the invention also relates to the use of a compound for the manufacture of a medicament for the treatment of a disorder mediated by p53-MDM2 interaction, wherein said compound is a compound of formula (I).

As used herein, the term “treatment method” or “treatment” encompasses any action against a disease and / or condition of an animal, in particular a human, and includes: (i) preventing the occurrence of a disease and / or condition in a subject that may be predisposed to the disease and / or a condition that has not yet been diagnosed that has such a disease and / or condition, (ii) inhibiting the disease and / or condition, i.e. cessation of its development, (iii) relieving the disease and / or condition, i.e. causing reverse development of the disease and / or condition.

The term "disorder mediated through the interaction of p53-MDM2" means any undesirable or detrimental condition that results from or results from the inhibition of the interaction between the MDM2 protein and p53 or other cellular proteins that cause apoptosis, cause cell death or regulate the cell cycle.

The invention also relates to a method for treating a disorder mediated through the interaction of p53-MDM2 by administering an effective amount of a compound of this invention to a subject, for example a mammal (and more specifically a human), if necessary, such treatment.

The compounds of the invention can exert antiproliferative effects in tumor cells, even if such cells lack functional p53. More specifically, the compounds of the invention may have antiproliferative effects in wild-type p53 tumors and / or in tumors overexpressing MDM2.

Thus, the present invention relates to a method for inhibiting tumor growth by administering an effective amount of a compound of this invention to a subject, such as a mammal (and more specifically a human), if such treatment is necessary.

Examples of tumors that can be inhibited are, but are not limited to, lung cancer (e.g., adenocarcinoma, including non-small cell lung cancer), pancreatic malignancies (e.g., pancreatic carcinoma, such as, for example, exocrine pancreatic carcinoma) malignant diseases of the colon (for example, colorectal carcinomas, such as, for example, adenocarcinoma of the colon and adenoma of the colon), esophageal cancer, squamous carcinoma of the oral sex sti, carcinoma of the tongue, gastric carcinoma, nasopharyngeal cancer, hematopoietic neoplasms of lymphoid origin (e.g. acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), non-Hodgkin's lymphoma, Hodgkin's disease, myeloid leukemia (e.g., acute myelogenous leukemia) thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g., fibrosarcoma and rhabdomyosarcoma), melanoma, teratocarcinoma, neuroblastoma, brain tumor, glioma, benign op skin khol (e.g., keratoacanthoma), breast carcinoma (e.g., advanced breast cancer), renal carcinoma, ovarian carcinoma, cervical carcinoma, endometrial carcinoma, bladder carcinoma, prostate cancer, including progressive disease, testicular neoplasms, osteomas head and neck cancer and epidermal carcinoma.

The compounds of this invention can also be used to treat and prevent inflammatory conditions.

Therefore, this invention relates to a method for treating and preventing inflammatory conditions by administering an effective amount of a compound of this invention to a subject, such as a mammal (and more specifically a human), if necessary, such treatment.

The compounds of this invention can also be used to treat and prevent autoimmune diseases and conditions. The term "autoimmune disease" refers to any disease in which the animal's immune system adversely reacts to its own antigen. The term "intrinsic antigen" means any antigen that is normally found in the body of an animal. Typical autoimmune diseases include, but are not limited to: Hashimoto's thyroiditis, Graves' disease, multiple sclerosis, pernicious anemia, Addison’s disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, systemic congestive seborrhea (SLE or lupus erythematosus), dermatomyositis, Cromer granulomas granulomas, granomegle granulomas, disease, antiphospholipid syndrome, herpetiform dermatitis, allergic encephalomyelitis, glomerulonephritis, membranous glomerulonephritis, Goodpasture syndrome, myasthenic syndrome Lambert-Eaton, severe myasthenia gravis, bullous pemphigoid, polyendocrinopathy, Reiter’s disease and stubborn syndrome.

Therefore, this invention also relates to a method for treating autoimmune diseases and conditions and treating diseases associated with conformational unstable or improperly folded proteins, administering an effective amount of a compound of this invention to a subject, such as a mammal (and more specifically a human), if such treatment is necessary.

The compounds of this invention can also be used to treat diseases associated with conformational unstable or improperly folded proteins.

Examples of diseases associated with conformational unstable or improperly folded proteins include, but are not limited to: cystic fibrosis (CFTR), Marfan syndrome (fibrillin), amyotrophic lateral sclerosis (superoxide dismutase), scurvy (collagen), a disease in which urine similar to maple syrup (alpha-ketoacid dehydrogenase complex), poor osteogenesis (type 1 procollagen pro-alpha), Creutzfeldt-Jakob disease (prion), Alzheimer's disease (amyloid beta), familial amyloidosis (lysozyme), like arakty (crystallins), familial hypercholesterolemia (LDL receptor), αI-antitrypsin deficiency, Tay-Sachs disease (beta-Hexosaminidase), retinitis pigmentosa (rhodopsin), and leprechaunism (insulin receptor).

Therefore, this invention also relates to a method for treating diseases associated with conformational unstable or improperly folded proteins, by administering an effective amount of a compound of this invention to a subject, such as a mammal (and more specifically a human), if necessary, such treatment.

In view of their beneficial pharmacological properties, these compounds may be formulated in 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 an addition salt with a base or an acid as an active ingredient is combined when thoroughly mixed with a pharmaceutically acceptable carrier, this carrier may take various forms depending on the form of the preparation desired for administration. Such pharmaceutical compositions are desirable in unit dosage form, suitable, preferably, for administration by oral, rectal, subcutaneous or parenteral injection. For example, in the preparation of compositions in a dosage form for oral administration, any conventional pharmaceutical medium may be used, such as, for example, water, glycols, oils, alcohols and the like, in the case of liquid preparations for oral administration, such as suspensions, syrups , elixirs and solutions, or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrants and the like in the case of powders, pills, capsules and tablets.

Based on the ease of 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 usually contain sterile water, at least in large part, although other ingredients, for example, to improve solubility, may be included. Injection solutions, for example, can be prepared with a carrier that contains saline, glucose solution, or a mixture of saline and glucose solution. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. In compositions intended for subcutaneous administration, the carrier optionally contains a penetration enhancing agent and / or a suitable wetting agent, optionally combined with appropriate additives of any nature in small relative amounts, these additives should not have a noticeable harmful effect on the skin. These additives may facilitate the administration to the skin and / or may be useful in preparing the desired compositions. These compositions can be administered in various ways, for example using a transdermal patch, applied in the form of a spot, as an ointment. It is especially advantageous to prepare said pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. As used in the description and claims, the term "unit dosage form" refers to physically discrete units applicable as unit doses, each unit containing a predetermined amount of the active ingredient, calculated so as to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including notched or coated tablets), capsules, pills, powder sachets, cachets, injectable solutions or suspensions, forms measured with a teaspoon, tablespoon and the like.

It is especially advantageous to prepare said pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. As used in the description and claims, the term "unit dosage form" refers to physically discrete units applicable as unit doses, each unit containing a predetermined amount of the active ingredient, calculated so as to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including notched or coated tablets), capsules, pills, powder sachets, cachets, injectable solutions or suspensions, forms measured with a teaspoon, tablespoon and the like.

The compound of the invention is administered in an amount sufficient to inhibit the interaction between MDM2 and p53 or other cellular proteins that cause apoptosis, cause cell death, or regulate the cell cycle.

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

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

One skilled in the art can easily determine the effective amount based on the test results presented hereinafter. It is generally contemplated 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 the required dose as one, two, three, four or more subdivided doses at appropriate intervals throughout the day. Said subdivisions may be presented as unit dosage forms, for example, containing from 0.5 to 500 mg, and in particular from 10 mg to 500 mg of the active ingredient per unit dosage form.

In another aspect of the present invention, a combination of a p53-MDM2 inhibitor with another anti-cancer agent is considered, especially for use as a medicine, more particularly in the treatment of cancer or related diseases.

For the treatment of these conditions, the compounds of the invention can be advantageously used in combination with one or more other drugs, more specifically with other anti-cancer agents. Examples of anti-cancer agents include, but are not limited to, the following:

- coordination compounds of platinum, for example cisplatin, carboplatin or oxaliplatin;

- taxane compounds, for example paclitaxel or docetaxel;

- topoisomerase I inhibitors, such as camptothecin compounds, for example irinotecan or topotecan;

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

- anti-tumor vinca alkaloids, for example vinblastine, vincristine or vinorelbine;

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

- alkylating agents, such as nitrogen bitterness or nitrosourea, for example cyclophosphamide, chlorambucil, carmustine, thiotepa, mefalan or lomustine;

- antitumor anthracycline derivatives, for example daunorubicin, doxorubicin, doxil, idarubicin or mitoxantrone;

- molecules that target the IGF-1 receptor, for example picropodophilin;

- tetracarcinol derivatives, for example tetrocarcinol A;

- glucocorticolide, for example prednisone;

- antibodies, for example trastuzumab (HER2 antibody), rituximab (CD20 antibody), gemtuzamab, cetuximab, pertuzumab or bevacizumab;

- estrogen receptor antagonists or selective estrogen receptor modulators, for example tamoxifen, fulvestrant, toremifene, droloxifene, faslodex or raloxifene;

- aromatase inhibitors such as exemestane, anastrozole, letrazole and Vorozol;

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

DNA methyl transferase inhibitors, for example, azacitidine or decitabine;

- antifolates, for example, premetrexed disodium;

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

- antimetabolites, for example, chlorofarabin, aminopterin, cytosine arabinoside or methotrexate;

apoptosis inducing agents and anti-angiogenic 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, colchicinins or nocodazole;

kinase inhibitors, for example flavoperidol, imatinib mesylate, erlotinib or gefitinib;

- farnesyl transferase inhibitors, for example tipifarnib;

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

- inhibitors of the ubiquitin proteasome pathway, for example, PS-341, MLN.41 or bortezomib;

- yondelis;

- telomerase inhibitors, for example telomestatin;

- matrix metalloproteinase inhibitors, for example, batimastat, marimastat, prinostat or metastat.

As stated above, the compounds of this invention also have therapeutic applications in sensitizing tumor cells to chemotherapy and radiotherapy.

Therefore, the compounds of this invention can be used as a “radiosensitizer” and / or “chemosensitizer” or can be given in combination with another “radiosensitizer” and / or “chemosensitizer”.

As used herein, the term “radiosensitizer” 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 ionizing radiation and / or to promote the treatment of diseases that are treatable by ionizing radiation.

As used herein, the term “chemosensitizer” 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 to promote the treatment of diseases that can be treated with chemotherapeutic agents.

Various mechanisms of the action of radiosensitizers have been suggested as suggestions in the literature, including: radiosensitizers of hypoxic cells (for example, 2-nitroimidazole compounds and benzotriazine dioxide compounds) that mimic oxygen or exhibit alternative behavior similar to bioreductive agents for hypoxia; non-hypoxic cell radiosensitizers (e.g., halogenated pyrimidines) can be analogues of DNA bases and are mainly incorporated into the DNA of cancer cells and thereby promote radiation-induced destruction of DNA molecules and / or interfere with normal DNA replenishment mechanisms and various other potential mechanisms of action of radiosensitizers in the treatment of diseases have been proposed as hypotheses.

Many cancer treatment protocols currently use radiosensitizers in combination with x-ray irradiation. Examples of X-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, ethanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromide iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin and their therapeutically effective analogues and derivatives.

Photodynamic therapy (PDT) of malignant diseases uses visible light as a radiation activator of a sensitizing agent. Examples of photodynamic radiosensitizers include, but are not limited to, the following: hematoporphyrin derivatives, photofrin, benzoporphyrin derivatives, tin etioporphyrin, pheoborbid-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine and their therapeutically effective analogues.

Radiosensitizers can be administered in combination 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 regulate the flow of therapeutic agents, nutrients and / or oxygen into target cells; chemotherapeutic agents that act on the tumor, with or without additional radiation, or other therapeutically effective compounds for treating cancer or another disease.

Chemosensitizers can be administered in combination with a therapeutically effective amount of one or more other compounds, including, but not limited to, compounds that promote the incorporation of chemosensitizers into target cells; compounds that regulate the flow of therapeutic agents, nutrients and / or oxygen into target cells; chemotherapeutic agents that act on the tumor, or other therapeutically effective compounds for treating cancer or another disease. A calcium antagonist, for example verapamil, has been found to be applicable in combination with anti-neoplastic agents to impart chemosensitivity to tumor cells resistant to recognized chemotherapeutic agents and to enhance the efficacy of these compounds in drug-sensitive malignancies.

In view of their beneficial pharmacological properties, the components of the combinations of this invention, i.e. another drug and p53-MDM2 inhibitor may be included in various pharmaceutical forms for administration purposes. The components may be separately included in separate pharmaceutical compositions or included in a single pharmaceutical composition containing both components.

This invention therefore also relates to a pharmaceutical composition comprising another drug and a p53-MDM2 inhibitor together with one or more pharmaceutical carriers.

The present invention also relates to the use of the combination of this invention to obtain a pharmaceutical composition for inhibiting the growth of tumor cells.

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

The other drug and p53-MDM2 inhibitor can be administered simultaneously (for example, in separate or single compositions) or sequentially in any order. In the latter case, two compounds should be administered during such a period and in such an amount and in such a way that they are sufficient to ensure that a beneficial or synergistic effect is achieved. It should be borne in mind that the preferred method and order of administration and the corresponding dosage amounts and modes for each component of the combination will depend on the particular other drug and p53-MDM2 inhibitor that is administered, their route of administration, the particular tumor being treated, and the particular subject being treated. The optimal method and order of administration and dosage amounts and regimen can easily be determined by specialists using traditional methods and taking into account the information provided here.

The platinum coordination compound is preferably administered at a dose of from 1 to 500 mg per square meter (mg / m ² ) of body surface area, for example from 50 to 400 mg / m ² , in particular for cisplatin at a dose of about 75 mg / m ² and for carboplatin about 300 mg / m ² per course of treatment.

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

The camptothecin compound is preferably administered at a dose of from 0.1 to 400 mg per square meter (mg / m ² ) of body surface area, for example from 1 to 300 mg / m ² , in particular for irinotecan at a dose of from about 100 to 350 mg / m ² and for topotecan from about 1 to 2 mg / m ² per course of treatment.

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

The anticancer vinca-alkaloid is preferably administered at a dose of 2 to 30 mg per square meter (mg / m ² ) of body surface area, in particular for vinblastine at a dose of about 3 to 12 mg / m ² , for vincristine at a dose of about 1 to 2 mg / m ² and for vinorelbine at a dose of about 10 to 30 mg / m ² per course of treatment.

The antitumor derivative of the nucleoside is preferably administered at a dose of 200 to 2500 mg per square meter (mg / m ² ) of body surface area, for example, 700 to 1500 mg / m ² , in particular for 5-FU, at a dose of 200 to 500 mg / m ² , for a dose of gemcitabine from about 800 to 1200 mg / m ² and for capecitabine from about 1000 to 2500 mg / m ² per course of treatment.

Alkylating agents, such as nitrogen bitterness or nitrosourea, are preferably administered at a dose of 100 to 500 mg per square meter (mg / m ² ) of body surface area, for example 120 to 200 mg / m ² , in particular for a cyclophosphamide in a dose of about 100 to 500 mg / m ² , for a dose of about 0.1 to 0.2 mg / m ² for chlorambucil, for a dose of about 150 to 200 mg / m ² for carmustine, and about 100 to 150 mg for lomustine / m ² per course of treatment.

The antitumor derivative of anthracycline is preferably administered at a dose of from 10 to 75 mg per square meter (mg / m ² ) of body surface area, for example from 15 to 60 mg / m ² , in particular for doxorubicin at a dose of from about 40 to 75 mg / m ² for daunorubicin at a dose of about 25 to 45 mg / m ² and for idarubicin at a dose of about 10 to 15 mg / m ² per course of treatment.

The antiestrogen agent is preferably administered at a dose of about 1 to 100 mg per day, depending on the particular agent and the condition being treated. Tamoxifen is preferably administered orally at a dose of from 5 to 50 mg, preferably from 10 to 20 mg, twice a day, continuing treatment for a sufficient time to achieve and maintain a therapeutic effect. Toremifen is preferably administered orally in a dose of about 60 mg once a day, continuing treatment for a sufficient time to achieve and maintain a therapeutic effect. Anastrozole is preferably administered orally at a dose of about 1 mg once a day. Droloxifene is preferably administered orally at a dose of about 20-100 mg once a day. Raloxifene is preferably administered orally at a dose of about 60 mg once a day. Exemestane is preferably administered orally at a dose of about 25 mg once a day.

Antibodies are preferably administered at a dose of about 1 to 5 mg per square meter (mg / m ² ) of body surface area, or as is customary in medicine, if different. Trastuzumab is preferably administered at a dose of 1 to 5 mg per square meter (mg / m ² ) of body surface area, in particular 2 to 4 mg / m ² per treatment course.

The indicated doses can be administered, for example, once, twice or more times per treatment course, which can be repeated, for example, every 7, 14, 21 or 28 days.

The compounds of formula (I), their pharmaceutically acceptable acid addition salts and stereoisomeric forms can have valuable diagnostic properties that can be used to detect or identify p53-MDM2 interactions in a biological sample, including detecting or measuring complex formation between the labeled compound and / or p53 and / or MDM2 and / or other molecules, peptides, proteins, enzymes or receptors.

Compounds that are labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances and the like can be used in detection or identification methods. Examples of radioisotopes include ¹²⁵ I, ¹³¹ I, ³ H and ¹⁴ C. Enzymes are usually made detectable by combining with an appropriate substrate, which in turn catalyzes the detection reaction. Examples thereof include, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably horseradish peroxidase. Luminescent substances include, for example, luminol, derivatives of luminol, luciferin, equorin 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 invention.

experimental part

Hereinafter, “DMF” means N, N-dimethylformamide, “DCM” means dichloromethane, “EtOAc” means ethyl acetate, “EtOH” means ethanol, “MeOH” means methanol, and “THF” means tetrahydrofuran.

Melting points

For some compounds, melting points were determined using a Kofler thermostat containing a heated plate with a linear temperature gradient, a differential index, and a temperature scale in degrees Celsius.

LCMS

Method a

The HPLC gradient was provided by the Alliance HT 2795 (Waters) system, which contained a four-stage pump with a degasser, an automatic sampling device, and a diode detector (DAD). The flow from the column was diverted to the MS detector. The MS detector was shaped with an electrospray ionization source. The capillary pointer voltage was 3 kV, and the source temperature was maintained at 100 ° C. on an LCT (Flight-Z-spray mass spectrometer time from Waters). Nitrogen was used as spray gas. Data acquisition was carried out by the Waters-Micromass MassLynx-Openlynx data system information system. Reverse phase HPLC was performed on an n Xterra-RP C18 column (5 μm, 3.9 × 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; used to create a gradient condition from 85% A, 15% B (retention for 3 minutes) to 20% A, 80% B for 5 minutes, holding at 20% A and 80% B for 6 minutes and restoring equilibrium with the initial conditions for 3 minutes.The injection volume was 20 μl, the cone voltage was 20 V for positive ionization mode. scanning from 100 to 900 in 8 seconds, using a delay (InterScan delay) of 0.08 seconds.

Method B

The LC gradient was provided by an Acquity UPLC system (Waters) containing a binary pump, a sampling device, a column heater (set to 55 ° C) and a diode detector (DAD). The flow from the column was diverted to the MS detector. The MS detector was shaped with an electrospray ionization source. The mass spectrum was obtained by scanning from 100 to 1000 in 0.18 seconds using a delay of 0.02 seconds. The voltage of the capillary pointer was 3.5 kV, and the temperature of the source was maintained at 140 ° C. Nitrogen was used as spray gas. Data acquisition was carried out by the Waters-Micromass MassLynx-Openlynx data system information system.

Reverse phase super performance liquid chromatography (UPLC) was performed on a bridged ethyl siloxane / silica (BEH) C18 column (1.7 μm, 2.1 × 50 mm) with a flow rate of 0.8 ml / min. Two mobile phases (mobile phase A: 0.1% formic acid in a mixture of H ₂ O / methanol 95/5; mobile phase B: methanol) were used to create a gradient condition from 95% A to 5% A, 95% B for 1 3 minutes and hold for 0.2 minutes. An injection volume of 0.5 μl was used.

The cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.

A. Preparation of Intermediates

Example A1

a) Preparation of Intermediate 1

To a solution of 5-nitroindoline (10.0 g, 0.061 mol) and 4-chloropyridine hydrochloride (11.0 g, 0.073 mol) in DMF (60 ml) in an argon atmosphere at 0 ° C are added portions of potassium tert-butoxide (17, 0 g, 0.15 mol). The mixture is heated up to 100 ° C for 16 hours. The mixture was poured onto ice and extracted twice with EtOAc. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified twice by silica gel column chromatography (40-63 μm) (eluent: cyclohexane / EtOAc / MeOH 50/50/0 to 0/80/20). Pure fractions were collected and the solvent was evaporated, yielding 2.49 g (17%) of intermediate 1 as a brownish-orange solid.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.47 (dd, 2H, J = 6.4, J = 1.6), 8.06 (m, 2H), 7.41 (d, 1H , J = 9.6), 7.28 (dd, 2H, J = 6.4, J = 1.6), 4.16 (t, 2H, J = 8.6), 3.22 (t, 2H, J = 8.6).

MS (ES + C m / z 242 (M + 1).

b) Preparation of Intermediate 2

A mixture of intermediate 1 (2.4 g, 0.010 mol) and Raney nickel (5 ml, 50% suspension in water) in EtOH (45 ml) and THF (45 ml) was stirred at room temperature at 30 psi (psi). inch) of hydrogen for 3 hours. After filtering through a celite pad, the solvent was evaporated, yielding 2.01 g (96%) of intermediate 2 as a brown solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.32 (dd, 2H, J = 6.4, J = 1.5), 7.15 (d, 1H, J = 8.5), 6.92 (dd, 2H, J = 6.4, J = 1.5), 6.63 (d, 1H, J = 2.2), 6.50 (dd, 1H, J = 8.3, J = 2 4), 3.92 (t, 2H, J = 8.3), 3.44 (br s, 2H), 3.08 (t, 2H, J = 8.3).

c) Preparation of Intermediate 3

To a solution of intermediate 2 (1.9 g, 0.0089 mol) in DCM (20 ml) and THF (20 ml) in an argon atmosphere, indole-3-acetic acid (2.0 g, 0.012 mol) was successively added, hydrate 1-hydroxybenzotriazole (1.6 g, 0.012 mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2.2 g, 0.012 mol). The reaction mixture was stirred at room temperature for 40 hours. To complete the reaction, indole-3-acetic acid (1.6 g, 0.0089 mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.7 g, 0.0089 mol) are added and the mixture is stirred at room temperature for more than 16 hours. The solvents were evaporated and the residue was purified by silica gel column chromatography (40-63 μm) (eluent: DCM / MeOH / NH ₄ OH 95/5 / 0.1 to 80/20 / 0.1). The collected fractions were evaporated, the resulting solid was washed with MeOH and dried, yielding 1.95 g (60%) of intermediate 3.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.93 (br s, 1H), 10.08 (br s, 1H), 8.31 (d, 2H, J = 6.3), 7.88 (d, 1H, J = 8.1), 7.58 (m, 2H), 7.34 (m, 2H), 7.26 (d, 1H, J = 2.1), 7, 17 (d, 2H, J = 6.6), 7.07 (t, 1H, J = 6.9), 6.98 (t, 1H, J = 7.4), 3.99 (t, 2H , J = 8.3), 3.71 (s, 2H), 3.12 (t, 2H, J = 8.2).

MS (ES +) Cm / z 369 (M + 1).

Example A2

a) Preparation of Intermediate 4

To a solution of 5-nitroindoline (5.0 g, 0.030 mol) and 4-chloroquinoline (6.0 g, 0.037 mol) in DMF (30 ml) in an argon atmosphere, potassium tert-butoxide (8.4 g, 0.075 mol) is added portionwise. ) The mixture was stirred at 100 ° C for 16 hours, then at room temperature for 70 hours. The mixture was poured onto ice and extracted 3 times with EtOAc. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (40-63 μm) (eluent: EtOAc / cyclohexane 50/50 to 100/0). Pure fractions were collected and the solvent was evaporated, yielding 2.26 g (26%) of intermediate 4 as an orange solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.93 (d, 1H, J = 4.9), 8.11 (m, 2H), 7.92 (dd, 1H, J = 8.9, J = 2.5), 7.85 (d, 1H, J = 7.7), 7.82 (dt, 1H, J = 7.0, J = 1.4), 7.60 (dt, 1H, J = 6.8, J = 1.2), 7.54 (d, 1H, J = 4.9), 6.26 (d, 1H, J = 8.9), 4.30 (t, 2H , J = 8.4), 3.35 (t, 2H, J = 8.2).

MS (ES +) Cm / z 292 (M + 1).

b) Preparation of Intermediate 5

A mixture of intermediate 4 (2.0 g, 0.0069 mol) and Raney nickel (3 ml, 50% suspension in water) in EtOH (30 ml) and THF (30 ml) was stirred at room temperature under a hydrogen pressure of 30 psi per within 4.5 hours. After filtering through a celite pad, the solvent was evaporated, yielding 1.81 g (100%) of intermediate 5 as an orange solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.71 (d, 1H, J = 5.1), 8.07 (d, 1H, J = 8.5), 8.01 (d, 1H, J = 8.5), 7.67 (t, 1H, J = 7.0), 7.41 (t, 1H, J = 7.1), 7.09 (d, 1H, J = 5.1) 6.67 (s, 1H), 6.49 (d, 1H, J = 8.3), 6.38 (dd, 1H, J = 8.3, J = 1.9), 4.04 ( t, 2H, J = 7.9), 3.39 (br s, 2H), 3.12 (t, 2H, J = 7.8).

MS (ES +) Cm / z 262 (M + 1).

c) Preparation of Intermediate 6

To a solution of intermediate 5 (1.7 g, 0.0064 mol) in DCM (15 ml) and THF (15 ml) under argon atmosphere, indole-3-acetic acid (1.5 g, 0.0084 mol) was added sequentially. , 1-hydroxybenzotriazole hydrate (1.1 g, 0.0084 mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.6 g, 0.0084 mol). The mixture was stirred at room temperature for 16 hours. The solvents were evaporated and the residue was purified by silica gel column chromatography (40-63 μm) (eluent: DCM / MeOH / NH ₄ OH 90/10 / 0.1). Pure fractions were collected and the solvent was evaporated, yielding 1.15 g (43%) of intermediate 6 as a brown foam.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.74 (d, 1H, J = 5.1), 8.61 (br s, 1H), 8.09 (d, 1H, J = 8.1 ), 7.91 (d, 1H, J = 8.4), 7.70-7.62 (m, 2H), 7.41 (m, 4H), 7.22 (m, 3H), 7, 09 (d, 1H, J = 5.1), 6.74 (dd, 1H, J = 8.4, J = 1.8), 6.38 (d, 1H, J = 8.4), 4 04 (t, 2H, J = 8.0), 3.89 (s, 2H), 3.15 (t, 2H, J = 7.9).

MS (ES +) m / z 419 (M + 1).

Example A3

Preparation of Intermediate 7

A mixture of indole-3-acetic acid (2.0 g, 0.013 mol) and 1,1-carbonyldiimidazole (2.1 g, 0.013 mol added in portions) in DCM (28 ml) was stirred under argon at room temperature for 2 hours. N, O-dimethylhydroxylamine hydrochloride (1.3 g, 0.013 mol) was added, the mixture was stirred at room temperature for 16 hours and then poured into ice with water. pH adjusted to 10 4 N. sodium hydroxide solution and the aqueous layer was extracted twice with EtOAc. The organic layer is separated, washed with 3 N. hydrochloride solution, dried (MgSO ₄ ), filtered and the solvent was evaporated, yielding 2.37 g (89%) of intermediate 7 as a pink solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.09 (br s, 1H), 7.66 (d, 1H, J = 7.5), 7.36 (d, 1H, J = 7.5 ), 7.22-7.10 (m, 3H), 3.92 (s, 2H), 3.66 (s, 3H), 3.23 (s, 3H).

Example A4

a) Preparation of Intermediate 8

A mixture of 5-nitroindoline (4.0 g, 0.024 mol) and 4-chloro-2-pyridinecarboxylic acid methyl ester (5.0 g, 0.029 mol) in acetic acid (24 ml) is heated up to 120 ° C for 25 minutes in the Biotage Initiator microwave. The reaction was quenched with ice, the pH was adjusted to pH 9 by the addition of a saturated solution of potassium carbonate. The mixture was extracted 3 times with DCM. 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 μm) (eluent: DCM / MeOH 100/0 to 90/10). Pure fractions were collected and the solvent was evaporated, yielding 1.37 g (19%) of intermediate 8 as a yellow solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.65 (d, 1H, J = 5.6), 8.16 (dd, 1H, J = 8.9, J = 2.1), 8.11 (s, 1H), 7.98 (d, 1H, J = 2.5), 7.35 (d, 1H, J = 8.9), 7.30 (dd, 1H, J = 5.7, J = 2.5), 4.23 (t, 2H, J = 8.5), 4.05 (s, 3H), 3.32 (t, 2H, J = 8.4).

MS (ES +) m / z 300 (M + 1).

b) Preparation of Intermediate 9

A mixture of intermediate 8 (1.2 g, 0.0045 mol) and Raney nickel (4 ml, 50% suspension in water) in MeOH (20 ml) and THF (20 ml) was stirred at room temperature in a hydrogen atmosphere for 20 hours. After filtering through a celite pad, the solvent was evaporated, yielding 1.06 g (88%) of intermediate 9 as an orange solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.40 (d, 1H, J = 5.8), 7.77 (d, 1H, J = 2.5), 7.18 (d, 1H, J = 8.4), 7.05 (dd, 1H, J = 5.8, J = 2.6), 6.62 (d, 1H, J = 2.2), 6.52 (dd, 1H, J = 8.4, J = 2.4) , 4.00 (m, 5H), 3.09 (t, 2H, J = 8.3).

MS (ES +) m / z 270 (M + 1).

Example A5

a) Preparation of Intermediate 10

A mixture of 5-aminoindole (1.1 g, 0.0086 mol) and phthalic anhydride (2.6 g, 0.017 mol) in DMF (20 ml) was stirred at 100 ° C for 5 hours, then at room temperature for 64 hours. The reaction mixture was diluted in EtOAc, washed twice with a saturated solution of ammonium chloride, dried (MgSO ₄ ), filtered and the solvent was evaporated. The resulting oil was taken up in acetic acid (15 ml) and stirred at room temperature for 20 minutes, then heated to 80 ° C for 1.5 hours. Ice was added and the pH was adjusted to pH 4 with saturated sodium carbonate solution. The mixture was extracted twice 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 (40-63 μm) (eluent: EtOAc / cyclohexane 40/60). Pure fractions were collected and the solvent was evaporated, yielding 2.06 g (91%) of intermediate 10.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.32 (br s, 1H), 7.97 (m, 2H), 7.79 (m, 2H), 7.66 (d, 1H, J = 2.0), 7.49 (d, 1H, J = 8.6), 7.27 (dd, 1H, J = 5.7, J = 2.8), 7.17 (dd, 1H, J = 8.6, J = 2.0), 6.61 (t, 1H, J = 1.1).

MS (ES + C m / z 263 (M + 1).

b) Preparation of Intermediate 11

A mixture of intermediate 10 (2.1 g, 0.0079 mol) and sodium cyanoborohydride (987 mg, 0.016 mol) in acetic acid (30 ml) was stirred at room temperature for 18 hours. Ice was added and the pH was adjusted to 6 with saturated sodium carbonate solution. The mixture was extracted twice with EtOAc. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (40-63 μm) (eluent: EtOAc / cyclohexane 30/70 to 70/30). Pure fractions were collected and the solvent was evaporated, yielding 726 mg (35%) of intermediate 11 as a yellow solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 7.93 (m, 2H), 7.77 (m, 2H), 7.09 (s, 1H), 7.00 (d, 1H, J = 8, 2), 6.70 (d, 1H, J = 8.2), 3.62 (t, 2H, J = 8.4), 3.09 (t, 2H, J = 8.4).

MS (ES + C m / z 265 (M + 1).

c) Preparation of Intermediate 12

A mixture of intermediate 11 (570 mg, 0.0022 mol), 4-bromo-6,7-dihydro-5H- [1] pyridin-7-ol (554 mg, 0.0026 mol) and 5 N. a solution of hydrochloride in 2-propanol (0.57 ml, 0.0029 mol) in DMF (11 ml) is heated up to 120 ° C for 1 hour in a Biotage Initiator microwave apparatus. The reaction was quenched with saturated sodium bicarbonate solution and extracted 3 times with DCM. The solvents are evaporated. The residue was purified by silica gel column chromatography (40-63 μm) (eluent: EtOAc / MeOH 100/0 to 80/20, then DCM / MeOH 95/5 to 90/10). Pure fractions were collected and the solvent was evaporated, yielding 651 mg (76%) of intermediate 12 as a yellow solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.30 (d, 1H, J = 5.6), 7.95 (m, 2H), 7.79 (m, 2H), 7.23-7, 12 (m, 3H), 6.94 (d, 1H, J = 8.4), 5.24 (t, 1H, J = 7.0), 4.18 (m, 1H), 4.08 ( m, 1H), 3.25 (m, 2H), 3.00 (m, 1H), 2.85 (m, 1H), 2.57 (m, 1H), 2.06 (m, 1H).

MS (ES + C m / z 398 (M + 1).

d) Preparation of Intermediate 13

At room temperature, hydrazine hydrate (137 μl, 0.0028 mol) was added to a suspension of intermediate 12 (557 mg, 0.0014 mol) in MeOH (5.5 ml), and the resulting mixture was heated up to 70 ° C for 40 minutes. The reaction was quenched with water and extracted 4 times with EtOAc. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated, yielding 392 mg (100%) of intermediate 13 as a brownish-orange solid.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.18 (d, 1H, J = 5.8), 7.07 (d, 1H, J = 5.8), 6.49 (d, 1H, J = 8.3), 6.63 (d, 1H, J = 2.1), 6.48 (dd, 1H, J = 8.3, J = 2.4), 5.21 (m, 1H) 4.19 (br s, 2H), 4.03 (m, 3H), 3.05 (m, 3H), 2.84 (m, 1H), 2.50 (m, 1H), 2, 03 (m, 1H).

Example A6

a) Preparation of Intermediate 14

A mixture of 2,3-dihydro-3,3-dimethyl-5-nitro-1H-indole (0.003 mol) and 4-bromo-pyridine (0.003 mol) in 1-butanol (5 ml) is heated in a sealed tube in a microwave oven 140 ° C for 30 minutes, then taken up in a 10% potassium carbonate solution and extracted with EtOAc. The organic layer was washed with water, then with NaCl and brine, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue (0.8 g) was purified by silica gel column chromatography (20-45 μm) (eluent: DCM / MeOH 100/0 to 98/20. The pure fractions were collected and the solvent was evaporated. The residue (0.45 g, 64%) was crystallized) from acetonitrile The precipitate was filtered off and dried, yielding 0.216 g (31%) of intermediate 14, melting point 217 ° C (Kofler).

¹ H NMR (DMSO-d ₆ ) δ 1.4 (6H, s), 3.92 (2H, s), 7.3 (2H, m), 7.45 (1H, m), 8.10 ( 2H, m), 8.47 (2H, m)

LCMS (ES +) m / z 270 (M + 1), R _t = 0.77, method B

b ) Preparation of Intermediate 15

A mixture of intermediate 14 (0.003 mol) and Raney nickel (0.9 g) in MeOH (20 ml) was hydrogenated at room temperature for 1 hour under a pressure of 3 bar, then filtered through celite. Celite washed with MeOH. The filtrate was evaporated, yielding 0.8 g (100%) of intermediate 15.

c) Preparation of Intermediate 16

Bromtris (pyrrolidino) phosphonium hexafluorophosphate (0.004 mol) is added portionwise at room temperature to a solution of intermediate 15 (0.003 mol), 1H - indole-3-acetic acid (0.004 mol), 1-hydroxybenzotriazole (0.04 mol) and diisopropyl simple ether (0.005 mol) in DCM (15 ml). The mixture was stirred at room temperature overnight. The organic layer was washed with 10% potassium carbonate solution, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue (3.2 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH ₄ OH 95/5 / 0.2). Pure fractions were collected and the solvent was evaporated. The residue (0.7 g, 52%) was crystallized from acetonitrile. The precipitate was filtered off and dried, yielding 0.55 g (41%) of intermediate 16, melting point 158 ° C (Kofler).

¹ H NMR (DMSO-d ₆ ) δ 1.28 (6H, s), 3.7 (2H, s), 3.73 (2H, s), 6.99 (1H, t, J = 7.7 Hz), 7.07-7.11 (3H, m), 2.25 (1H, d, J = 3.6 Hz), 7.30-7.39 (3H, m), 7.55 (1H , br.s.d, J = 3.6 Hz), 7.62 (1Н, d, J = 7.7 Hz), 8.3 (2H, d, J = 7.7 Hz), l0.03 (1Н , br.s), 10.92 (1H, br.s)

LCMS (ES +) m / z 397 (M + 1), R _t = 8.43, Method A

B. Preparation of final compounds

Example B1

Getting connection 1

Mixed lithium aluminum hydride (423 mg, 0.0011 mol) was added portionwise to a suspension of intermediate 3 (1.0 g, 0.0027 mol) in THF (60 ml) under argon. The mixture was stirred at room temperature for 24 hours, quenched with ice and a dilute solution of potassium tartrate-sodium, and extracted 3 times with DCM. 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 μm) (eluent: DCM / MeOH / NH ₄ OH 95/5 / 0.1). Pure fractions were collected and the solvent was evaporated, yielding 170 mg (18%) of compound 1 as a yellow foam.

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.36 (br s, 1H), 8.32 (d, 2H, J = 6.6), 7.63 (d, 1H, J = 7.8 ), 7.39 (d, 1H, J = 7.5), 7.24-7.11 (m, 3H), 7.07 (d, 1H, J = 1.8), 6.93 (d , 2H, J = 6.6), 6.58 (s, 1H), 6.45 (dd, 1H, J = 8.4, J = 2.1), 3.93 (t, 2H, J = 8.2), 3.62 (br s, 1H), 3.46 (t, 2H, J = 7.5), 3.09 (m, 4H).

LCMS (ES +) m / z 355 (M + 1), R _t = 8.30, method A

Example B2

Preparation of Compound 2

In an argon atmosphere at room temperature, lithium aluminum hydride (391 mg, 0.010 mol) was added portionwise to a solution of intermediate 6 (1.1 g, 0.0026 mol) in THF (55 ml). The mixture was stirred at room temperature for 18 hours. The reaction is quenched with MeOH at 0 ° C. Ice and a dilute solution of potassium sodium tartrate are added and the mixture is extracted 3 times with DCM. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (40-63 μm) (eluent: DCM / MeOH / NH ₄ OH 97/3 / 0.5). Pure fractions were collected and the solvent was evaporated, yielding 121 mg (12%) of compound 2 as an orange foam.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.70 (d, 1H, J = 5.1), 8.18 (br s, 1H), 8.07 (d, 1H, J = 8.7 ), 8.03 (d, 1H, J = 8.7), 7.66 (m, 2H), 7.41 (m, 2H), 7.22 (t, 1H, J = 7.7), 7.12 (m, 3H), 6.63 (s, 1H), 6.56 (d, 1H, J = 8.4), 6.34 (dd, 1H, J = 8.4, J = 2 , 1), 4.06 (t, 2H, J = 7.8), 3.46 (t, 2H, J = 6.8), 3.12 (m, 4H).

MS (ES + C m / z 405 (M + 1).

Example B3

Preparation of Compound 3

At 0 ° C. under argon, lithium aluminum hydride (14 mg, 0.00036 mol) was added to a solution of intermediate 7 (74 mg, 0.00036 mol) in THF (1 ml). The mixture was stirred at 0 ° C for 1 hour, quenched with 5% potassium hydrogen sulfate solution and extracted twice with EtOAc. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated to give indol-3-yl acetaldehyde as an orange oil.

A mixture of intermediate 9 (200 mg, 0.00074 mol) and sodium cyanoborohydride (64 mg, 0.0010 mol) in MeOH (2.3 ml) and acetic acid (2 drops) was added dropwise to a solution of the indicated aldehyde (236 mg , 0.0015 mol) in MeOH (2 ml). The reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched with water, made basic with saturated sodium hydrogen carbonate solution and extracted 3 times with EtOAc. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (40-63 μm) (eluent: EtOAc / MeOH 100/0 to 90/10). Pure fractions were collected and the solvent was evaporated, yielding 190 mg (44%) of compound 3 as an orange foam.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.42 (d, 1H, J = 5.7), 8.08 (br s, 1H), 7.79 (d, 1H, J = 2.4 ), 7.63 (d, 1H, J = 7.8), 7.40 (d, 1H, J = 8.1), 7.25-7.07 (m, 5H), 6.59 (s , 1H), 6.48 (dd, 1H, J = 8.7, J = 2.1), 4.00 (m, 5H), 3.48 (t, 2H, J = 6.8), 3 12 (m, 4H).

MS (ES +) m / z 413 (M + 1).

Example B4

Preparation of Compound 4

Sodium borohydride (92 mg, 0.0024 mol) was slowly added to a solution of compound 3 (100 mg, 0.00024 mol) in MeOH (3 ml). The mixture was stirred at room temperature for 1 hour, then at 80 ° C for 4 hours and again at room temperature for 85 hours. The reaction was quenched with water and the mixture was extracted with EtOAc. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (40-63 μm) (eluent: DCM / MeOH 90/10 to 85/10). Pure fractions were collected and the solvent was evaporated, yielding 50 mg (54%) of compound 4 as a yellow foam.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.83 (br s, 1H), 8.14 (d, 1H, J = 5.7), 7.54 (d, 1H, J = 7 8), 7.35 (d, 1H, J = 7.8), 7.22 (m, 2H), 7.14 (d, 1H, J = 2.1), 7.70 (dt, 1H , J = 7.6, J = 1.2), 6.98 (dt, 1H, J = 7.5, J = 0.9), 6.86 (dd, 1H, J = 6.0, J = 2.4), 6.62 (d, 1H, J = 2.1), 6.45 (dd, 1H, J = 8.7, J = 2.2), 5.39 (br.s, 2H), 4.49 (d, 2H, J = 4.0), 3.92 (t, 2H, J = 8.2), 3.30 (m, 2H), 3.06 (t, 2H, J = 8.1), 2.96 (t, 2H, J = 7.5).

MS (ES +) m / z 385 (M + 1).

Example B5

Preparation of Compound 5

At 0 ° C. under argon, lithium aluminum hydride (14 mg, 0.00036 mol) was added to a solution of intermediate 7 (74 mg, 0.00036 mol) in THF (1 ml). The mixture was stirred at 0 ° C for 1 hour, quenched with 5% potassium hydrogen sulfate solution and extracted twice with EtOAc. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated to give indol-3-yl acetaldehyde as an orange oil.

To a mixture of intermediate 13 (100 mg, 0.00037 mol) and sodium cyanoborohydride (33 mg, 0.00052 mol) in MeOH (1.5 ml) and acetic acid (2 drops) was added dropwise to a solution of the indicated aldehyde (57 mg, 0.00036 mol) in MeOH (0.5 ml). The reaction mixture was stirred at room temperature for 18 hours. The reaction was quenched with saturated sodium bicarbonate and extracted twice with EtOAc. The organic layer was separated, washed with brine, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography (40-63 μm) (eluent: DCM / MeOH 100/0 to 90/10). Pure fractions were collected and the solvent was evaporated, yielding 79 mg (52%) of compound 5 as a yellow foam.

¹ H NMR (300 MHz, CMCl ₃ ) δ 8.37 (br s, 1H), 8.15 (d, 1H, J = 5.8), 7.63 (d, 1H, J = 7.8 ), 7.37 (d, 1H, J = 7.1), 7.21 (dt, 1H, J = 7.6, J = 1.2), 7.12 (dt, 1H, J = 7, 6, J = l, l), 7.07 (m, 2H), 6.83 (d, 1H, J = 8.5), 6.56 (d, 1H, J = 2.0), 6, 40 (dd, 1H, J = 8.5, J = 2.3), 5.22 (t, 1H, J = 6.7), 4.19 (br s, 1H), 4.04 (m , 3H), 3.44 (t, 2H, J = 6.7), 3.06 (m, 5H), 2.83 (m, 1H), 2.54 (m, 1H), 2.08 ( m, 1H).

MS (ES +) Cm / z 411 (M + 1).

Example B6

Preparation of Compound 6

Intermediate 16 (0.001 mol) was added portionwise to a solution of lithium aluminum tetrahydride (0.002 mol) in THF (10 ml) in a stream of N ₂ . The mixture was stirred and refluxed for 24 hours, poured into ice water and filtered through celite. Celite washed with EtOAc. The filtrate was extracted with EtOAc. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue (0.45 g) was purified by silica gel column chromatography (10 μm) (eluent: DCM / MeOH / NR ₄ OH 95/5 / 0.5). Pure fractions were collected and the solvent was evaporated, yielding 0.042 g of compound 6.

¹ H NMR (DMSO-d ₆ ) δ 1.28 (6H, s), 2.95 (2H, t, J = 7.7 Hz), 3.27-3.34 (2H, m), 3, 65 (2H, s), 5.38 (1H, br t, J = 6.4 Hz), 6.45 (1H, dd, J = 4 Hz, 7.7 Hz), 6.57 (1H, d, J = 4 Hz), 6.96-7.02 (3H, m), 7.07 (1H, t, J = 7.7 Hz), 7.15-7.5 (3H, m), 7.35 (1H, d, J = 7.7 Hz), 7.55 (1H, d, J = 7.7 Hz), 8.22 (1H, d, J = 7.7 Hz), 10, 83 (1H, broad s).

MS (ES + C m / z 383 (M + 1), R _t = 9.17, Method A

Table F-1 lists the compounds that were obtained in one of these examples.

C. Pharmacological example

The ability of compounds to retain p53 in A2780 cells was measured by enzyme-linked immunosorbent assay p53. The p53 assay is an enzyme immunoassay using an immunosandwich of two polyclonal antibodies. A polyclonal antibody specific for the p53 protein was immobilized on the surface of plastic wells. Any p53 present in the test sample will bind to the immobilized antibody. The biotinylated identifying polyclonal antibody also recognizes the p53 protein and will bind to any p53 that is retained by the immobilized antibody. The identifying antibody, in turn, binds to streptavidin conjugated to horseradish peroxidase. Horseradish peroxidase catalyzes the conversion of chromogenic substrate o-phenylenediamine, the intensity of which is proportional to the amount of p53 protein bound to the plate. The color reaction product is quantified using a spectrophotometer. Quantitative analysis is carried out by constructing a standard curve using known concentrations of purified recombinant HIS-labeled p53 protein (see Example C.1).

Cellular activity of the compounds of formula (I) was determined on U87MG tumor cells using a colorimetric analysis of cell toxicity or cell survival (see Example C.2).

U87MG cells are human glioblastoma cells with wild-type p53. In this cell line, MDM2 strictly controls p53 expression.

C.1 ELISA p53

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

A2780 cells were seeded at 20,000 cells per well on a 96-well plate, cultured for 24 hours and treated with the compound for 16 hours at 37 ° C in a humidified incubator. After incubation, the cells were washed once with phosphate-buffered saline and 30 μl per well of RIPA saline buffer (20 mM Tris, pH 7.0, 0.5 mM EDTA, 1% Nonidet P40, 0.5% DOC, 0.05%) was added. SDS, 1 mM PMSF, 1 μg / ml aprotinin and 0.5 μg / ml leipeptin). Plates were placed on ice for 30 minutes to complete lysis. The p53 protein was determined in lysates using an ELISA using an immunosandwich, which is described below.

EIA / RIA highly binding polystyrene 96-well plates (Costar 9018) were coated with immobilized pAb 1801 antibody (Abcam ab28-100) at a concentration of 1 μg / ml in coating buffer (0.1 M NaHCO ₃ pH 8.2), 50 μl per well. The antibody was allowed to adhere overnight at 4 ° C. The coated plates were washed once with phosphate-buffered saline (PBS) / 0.05% Tween 20 and 300 μl blocking buffer (PBS, 1% bovine serum albumin (BSA)) was added, for an incubation period of 2 hours at room temperature. Dilutions of purified recombinant HIS-labeled p53 protein in the range of 3-200 ng / ml were performed 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 80 μl / well. To the standards was added 20 μl of lysis buffer. Samples were added to other wells at 20 μl of lysate / well. After overnight incubation at 4 ° C, the plates were washed twice with PBS / 0.05% Tween 20. Aliquots of 100 μl of secondary polyclonal antibody p53 (F1-393) (Tebubio, sc-6243) at a concentration of 1 μg / ml in blocking buffer added to each well and allowed to adhere for 2 hours at room temperature. The plates were washed three times with PBS / 0.05% Tween 20. An anti-rabbit HRP identification antibody (sc-2004, Tebubio) at 0.04 μg / ml in PBS / 1% BSA was added and incubated for 1 hour at room temperature. The plates were washed three times with PBS / 0.05% Tween 20 and 100 μl of substrate buffer was added (substrate buffer was prepared shortly before use by adding 1 tablet of 10 mg of o-phenylenediamine (OPD) from Sigma and 125 μl of 3% H ₂₀₂ to 25 ml of buffer OPD: 35 mM citric acid, 66 mM Na ₂ HPO ₄ , pH 5.6). After 5-10 minutes, the color reaction was stopped by adding 50 μl of stop buffer (1 MH ₂ SO ₄ ) per well. The optical density was measured at two wavelengths of 490/655 nm using a Biorad microplate reader, and the results were then analyzed.

For each experiment, controls (containing no drugs) and empty incubation (containing no cells or drugs) were performed in parallel. The blank value was subtracted from all control values and samples. For each sample, p53 (in units of optical density) was expressed as a percentage of the value for p53 present in the control. Relative safety higher than 140% was determined to be significant. Here, the effects of the test compounds are expressed as the smallest dose giving at least 140% of the value for p53 present in the control (LAD) (see table F-2).

C.2. Proliferation Analysis

All test compounds 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. The controls contained U87MG and DMSO cells without compound, and empty samples contained DMSO, but without cells.

U87MG cells were seeded in 96-well cell culture plates at 3000 cells / well / 100 μl. After 24 hours, the medium was replaced and the compound and / or solvent was added to a final volume of 200 μl. After 4 days of incubation, the medium was replaced with 200 μl of fresh medium and cell growth was evaluated using an MTT assay. Therefore, 25 μl of MTT solution (0.5% MTT grade for Serva assays in phosphate buffered saline) was added to each well and cells were further incubated for 2 hours at 37 ° C. The medium was then carefully aspirated and the blue MTT-formazan product was dissolved by adding 25 μl of 0.1 M glycine and 100 μl DMSO to each well. The plates were shaken for the next 10 minutes on a microplate shaker before reading the absorbance at a 540 nm Biorad microplate reader.

In each experiment, the results for each experimental condition are the average of 3 replicate wells. For the purpose of initial screening, compounds were tested at a single fixed concentration of 10 ^-5 M. For active compounds, experiments were repeated to establish complete concentration-response curves. For each experiment, controls (containing no drugs) and empty incubation (containing no cells or drugs) were performed in parallel. The blank value was subtracted from all control values and samples. For each sample, the average value of cell growth (in units of optical density) was expressed as a percentage of the average value for the growth of control cells. When appropriate, IC ₅₀ values (drug concentration necessary to reduce cell growth to 50% of control) were calculated using probit analysis of variable data (Finney, DJ, Probit Analyses, 2 ^nd Ed. Chapter 10, Graded Responses, Cambridge University Press, Cambridge 1962). Here, the effects of the test compounds are expressed as pIC ₅₀ (negative logarithm of IC ₅₀ ) (see table F-2).

In some experiments, proliferation analysis was adapted and used in 384 well culture plates (see table F-2).

Table F-2
Table F-2 lists the results of the compounds that were tested according to example C.1 and C.2

D. Compositional Example: Film-Coated Tablets

Getting the core of the pill

A mixture of 100 g of the compound of formula (I), 570 g of lactose and 200 g of starch is thoroughly mixed and then moistened with a solution of 5 g of sodium dodecyl sulfate and 10 g of polyvinylpyrrolidone in approximately 200 ml of water. The wet powder mixture is passed through a sieve, dried and again passed through a sieve. Then add 100 g of microcrystalline cellulose and 15 g of hydrogenated vegetable oil. All of this is thoroughly mixed and compressed into tablets, obtaining 10,000 tablets, each of which contains 10 mg of the compound of formula (I).

Coating

To a solution of 10 g methyl cellulose in 75 ml of denatured ethanol is added a solution of 5 g of ethyl cellulose in 150 ml of dichloromethane. Then add 75 ml of dichloromethane and 2.5 ml of 1,2,3-propanetriol, 10 g of polyethylene glycol is melted and dissolved in 75 ml of dichloromethane. The last solution is added to the previous one and then 2.5 g of magnesium octadecanoate, 5 g of polyvinylpyrrolidone and 30 ml of a concentrated color suspension are added and all this is homogenized. The tablet cores are coated with the mixture thus obtained in a coating apparatus.

Claims (14)

1. The compound of formula (I)

its N-oxide form, an addition salt or a stereochemically isomeric form, where
m is 0, 1 and when m is 0, then a direct relationship is implied;
n is 0, 1, 2, or 3, and when n is 0, then a direct relationship is implied;
p is 1;
t is 0 or 1, and when t is 0, then a direct relationship is implied;

means -CR ⁸ = C <, and then the dashed line represents the relationship, where
R ⁸ represents hydrogen;
R ¹ and R ² represents hydrogen;
R ³ and R ⁴ represents hydrogen;
R ⁵ represents hydrogen;
R ⁶ and R ⁷ are each independently selected from hydrogen or C _1-6 alkyl;
Z represents a radical selected from

where R ¹⁰ and R ¹¹ are each independently selected from values such as hydrogen, hydroxy, C _1-6 alkylcarbonyl, C _1-6 alkylcarbonyloxy C _1-6 alkyl, C _1-6 alkyloxycarbonyl, C _1-6 alkylcarbonyloxy, hydroxy C _1-6 alkyl. 2. The compound according to claim 1, where
n is 0; t is 0; R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy, C _1-6 alkyloxycarbonyl or hydroxy C _1-6 alkyl. 3. The compound according to claim 1 or 2, where
n is 0; t is 0; R ⁶ and R ⁷ are hydrogen; Z represents a radical selected from (a-2) or (a-4); R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy or hydroxy C _1-6 alkyl. 4. The compound according to claim 1 or 2, where the compound is compound No.1, compound No.4 or compound No.5.

compound No.1 compound No.4

compound No.5 5. The compound according to claim 1 or 2 for use in medicine for the treatment of cancer. 6. A pharmaceutical composition for treating cancer, comprising pharmaceutically acceptable carriers and, as an active ingredient, a therapeutically effective amount of a compound according to claims 1 to 4. 7. The method of obtaining the pharmaceutical composition according to claim 6, where the pharmaceutically acceptable carriers and the compound according to claims 1-4 are thoroughly mixed. 8. The use of compounds according to claim 1, 2, 3 or 4 for the manufacture of a medicament for the treatment of cancer. 9. The combination of anti-cancer agents and compounds according to claim 1, 2, 3 or 4. 10. The method of obtaining the compound according to claim 1, characterized by the interaction of an intermediate compound of formula (II) with an intermediate compound of formula (III), where W represents an appropriate leaving group, such as, for example, halogen,

where the variables have the meanings as defined in claim 1. 11. The method of obtaining the compound according to claim 1, characterized by the conversion of an intermediate compound of formula (IV) into a compound of formula (I), where p is 1, designated as a compound of formula (1-a), in the presence of lithium aluminum hydride in a suitable solvent

where the variables have the meanings as defined in claim 1. 12. The method of obtaining the compound according to claim 1, characterized by the interaction of the corresponding carboxaldehyde of the formula (VI) with an intermediate compound of the formula (V) in the presence of the corresponding reagent in a suitable solvent

where the variables have the meanings as defined in claim 1. 13. The method of obtaining the compound according to claim 1, characterized by the interaction of an intermediate compound of formula (II) with the corresponding carboxaldehyde of the formula (VII) with the formation of the compounds of formula (I), where t is 1, designated as the compound of formula (Ib)

where the variables have the meanings as defined in claim 1. 14. The use of claim 8, where the cancer is selected from the group consisting of: lung cancer (for example, adenocarcinoma and, including non-small cell lung cancer), pancreatic malignant diseases (for example, pancreatic carcinoma, such as, for example, exocrine pancreatic carcinoma ), malignant diseases of the colon (e.g. colorectal carcinomas, such as, for example, colon adenocarcinoma and colon adenoma), esophageal cancer, squamous oral carcinoma, tongue carcinoma, gastric carcinoma ohm, nasopharyngeal cancer, hematopoietic neoplasms of lymphoid origin (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 myeloid follicle syndrome (MDS), tumors of mesenchymal origin (e.g., fibrosarcoma and rhabdomyosarcoma), melanomas, teratocarcinomas, neuroblastomas, brain tumors, gliomas, benign skin tumors (e.g., keratoacanthomas), rtsinoma mammary gland (e.g., progressive breast cancer), kidney carcinoma, ovarian carcinoma, cervical carcinoma, endometrial carcinoma, bladder carcinoma, prostate cancer, including progressive disease, testicular tumors, osteosarcoma, head and neck cancer and epidermal carcinoma.