# Patent

## Title
Inhibitors of the interaction between MDM2 and P53

## Abstract

The present invention provides compounds of formula (I), their use as an inhibitor of a p53-MDM2 interaction as well as pharmaceutical compositions comprising said compounds of formula (I) wherein n, m, p, s, t, R1, R2, A and Z have defined meanings.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the national stage of PCT Application No. PCT/EP2007/052579, filed Mar. 19, 2007, which claims priority from European Patent Application No. 06111531.7, filed Mar. 22, 2006, and U.S. Provisional Patent Application No. 60/784,780, filed Mar. 22, 2006, the entire disclosures of which are hereby incorporated in their entirely.


FIELD OF THE INVENTION
The present invention relates to compounds and compositions containing said compounds acting as inhibitors of the interaction between MDM2 and p53. Moreover, the present invention provides processes for the preparation of the disclosed inhibitors, compositions comprising them and methods of using them, for instance as a medicine.
p53 is a tumour suppressor protein which plays a pivotal role in the regulation of the balance between cell proliferation and cell growth arrest/apoptosis. Under normal conditions the half life of p53 is very short and consequently the level of p53 in cells is low. However, in response to cellular DNA damage or cellular stress (e.g. oncogene activation, telomere erosion, hypoxia), levels of p53 increase. This increase in p53 levels leads to the activation of the transcription of a number of genes which drives the cell into either growth arrest or into the processes of apoptosis. Thus, an important function of p53 is to prevent the uncontrolled proliferation of damaged cells and thus protect the organism from the development of cancer.
MDM2 is a key negative regulator of p53 function. It forms a negative autoregulatory loop by binding to the amino terminal transactivation domain of p53 and thus MDM2 both inhibits the ability of p53 to activate transcription and targets p53 for proteolytic degradation. Under normal conditions this regulatory loop is responsible for maintaining the low levels of p53. However, in tumours with wild-type p53, the equilibrium concentration of active p53 can be increased by antagonising the interaction between MDM2 and p53. This will result in restoration of the p53-mediated pro-apoptotic and anti-proliferative effects in such tumour cells.
MDM2 is a cellular proto-oncogene. Over-expression of MDM2 has been observed in a range of cancers. MDM2 is over-expressed in a variety of tumours due to gene amplification or increased transcription or translation. The mechanism by which MDM2 amplification promotes tumourigenesis is at least in part related to its interaction with p53. In cells over-expressing MDM2 the protective function of p53 is blocked and thus cells are unable to respond to DNA damage or cellular stress by increasing p53 levels, leading to cell growth arrest and/or apoptosis. Thus after DNA damage and/or cellular stress, cells over-expressing MDM2 are free to continue to proliferate and assume a tumorigenic phenotype. Under these conditions disruption of the interaction of p53 and MDM2 would release the p53 and thus allow normal signals of growth arrest and/or apoptosis to function.
MDM2 may also have separate 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. A domain of E2F1 shows striking similarity to the MDM2-binding domain of p53. Since the interactions of MDM2 with both p53 and E2F1 locate to the same binding site on MDM2, it can be expected that MDM2/p53 antagonists will not only activate cellular p53 but also modulate E2F1 activities, which are commonly deregulated in tumour cells.
Also the therapeutic effectiveness of DNA damaging agents currently used (chemotherapy and radiotherapy), may be limited through the negative regulation of p53 by MDM2. Thus if the MDM2 feed-back inhibition of p53 is interrupted, an increase in functional p53 levels will increase the therapeutic effectiveness of such agents by restoring the wild-type p53 function that leads to apoptosis and/or reversing of p53-associated drug resistance. It was demonstrated that combining MDM2 inhibition and DNA-damaging treatments in vivo led to synergistic anti-tumour effects (Vousden K. H., Cell, Vol. 103, 691-694, 2000).
Thus disruption of the interaction of MDM2 and p53 offers an approach for therapeutic intervention in tumours with wild-type p53, might even exhibit anti-proliferative effects in tumour cells that are devoid of functional p53 and furthermore can sensitise tumorigenic cells for chemotherapy and radiotherapy.
BACKGROUND OF THE INVENTION
JP 11130750, published on 18 May 1999, describes amongst others, substituted phenylaminocarbonylindolyl derivatives as 5-HT receptor antagonists. EP1129074, published on 18 May 2000, describes anthranilic acid amides as inhibitors of vascular endothelial growth factor receptors (VEGFR) and useful in the treatment of angiogenic disorders.
EP1317443, published on 21 Mar. 2002, discloses tricyclic tert-amine derivatives, useful as chemokine receptor CXCR4 or CCR5 modulators for treating human immunodeficiency virus and feline immunodeficiency virus.
EP1379239, published on 10 Oct. 2002, discloses N-(2-arylethyl)benzylamines as antagonists of the 5-HT6 receptor.
WO00/15357, published on 23 Mar. 2000, provides piperazine-4-phenyl derivatives as inhibitors of the interaction between MDM2 and p53. EP1137418, published on 8 Jun. 2000, provides tricyclic compounds for restoring conformational stability of a protein of the p53 family.
EP1443937, published on 22 May 2003, describes substituted 1,4-benzodiazepines and the uses thereof as inhibitors of the MDM2-p53 interactions.
EP1458380, published on 26 Jun. 2003, provides cis-2,4,5-triphenyl-imidazolones that inhibit the interaction of MDM2 protein with p53-like peptides and have antiproliferative activity.
EP1519932, published on 15 Jan. 2004, discloses bisarylsulfonamide compounds that bind to MDM2 and can be used in cancer therapy.
There continues to be a need for effective and potent small molecules that inhibit the interactions between MDM2 and p53.
The compounds of the present invention differs from the prior art in structure, in their pharmacological activity and/or in pharmacological potency.


DESCRIPTION OF THE INVENTION
The present invention provides compounds, compositions for, and methods of, inhibiting the interactions between MDM2 and p53 for treating cancer. Furthermore the compounds and compositions of the present invention are useful in enhancing the effectiveness of chemotherapy and radiotherapy.
This invention concerns compounds of formula (I)




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

    m is 0, 1, or 2 and when m is 0 then a direct bond is intended;
    n is 0, 1, 2, 3 or 4 and when n is 0 then a direct bond is intended;
    p is 0, or 1 and when p is 0 then a direct bond is intended;
    s is 0, or 1 and when s is 0 then a direct bond is intended;
    t is 0 or 1 and when t is 0 then a direct bond is intended;
    R1 and R2 are each independently hydrogen, halo, C1-6alkyl, C1-6alkyloxy, arylC1-6alkyloxy, heteroarylC1-6alkyloxy, phenylthio, hydroxyC1-6alkylcaibonyl, C1-6alkyl substituted with a substituent selected from amino, aryl and heteroaryl; or C3-7cycloalkyl substituted with a substituent selected from amino, aryl and heteroaryl;
    A is a radical selected from















wherein

    R4 and R5 are each independently selected from hydrogen, halo, C1-6alkyl, polyhaloC1-6alkyl, cyano, cyanoC1-6alkyl, hydroxyC1-6alkyl, hydroxy, amino, C1-6alkyloxy,
    
        C1-6alkylcarbonyl, methylsulfonylamino, aryl or heteroaryl;
    
    
    Z is a radical selected from






    
    
        wherein
        R6 or R7 are each independently selected from hydrogen, halo, hydroxy, amino, C1-6alkyl, nitro, polyhaloC1-6alkyl, cyano, cyanoC1-6alkyl, tetrazoloC1-6alkyl, aryl, heteroaryl, arylC1-6alkyl, heteroarylC1-6alkyl, aryl(hydroxy)C1-6alkyl, heteroaryl(hydroxy)C1-6alkyl, arylcarbonyl, heteroarylcarbonyl, C1-6alkylcarbonyl, arylC1-6alkylcarbonyl, heteroarylC1-6alkylcarbonyl, C1-6alkyloxy, C3-7cycloalkylcarbonyl, C3-7cycloalkyl(hydroxy)C1-6alkyl, arylC1-6alkyloxyC1-6alkyl, C1-6alkyloxyC1-6alkyloxyC1-6alkyl, C1-6alkylcarbonyloxyC1-6alkyl, C1-6alkyloxycarbonylC1-6alkyloxyC1-6alkyl, hydroxyC1-6alkyloxyC1-6alkyl, C1-6alkyloxycarbonylC2-6alkenyl C1-6alkyloxyC1-6alkyl, C1-6alkyloxycarbonyl, C1-6alkylcarbonyloxy, aminocarbonyl, hydroxyC1-6alkyl, aminoC1-6alkyl, hydroxycarbonyl, hydroxycarbonylC1-6alkyl and —(CH2)v—(C(═O)r)—(CHR10)u—NR8R9; wherein
        
            v is 0, 1, 2, 3, 4, 5, or 6 and when v is 0 then a direct bond is intended;
            r is 0, or 1 and when r is 0 then a direct bond is intended;
            u is 0, 1, 2, 3, 4, 5, or 6 and when u is 0 then a direct bond is intended;
            R10 is hydrogen or C1-6alkyl;
            R8 and R9 are each independently selected from hydrogen, C1-12alkyl, C1-6alkylcarbonyl, C1-6alkylsulfonyl, arylC1-6alkylcarbonyl,
            
                C3-7cycloalkyl, C3-7cycloalkylcarbonyl, —(CH2)k—NR11R12, C1-12alkyl substituted with a substituent selected from hydroxy, hydroxycarbonyl, cyano,
                C1-6alkyloxycarbonyl, C1-6alkyloxy, aryl or heteroaryl; or
                C3-7cycloalkyl substituted with a substituent selected from hydroxy,
                C1-6alkyloxy, aryl, amino, arylC1-6alkyl, heteroaryl or heteroarylC1-6alkyl; or
            
            
            R8 and R9 together with the nitrogen to which they are attached can optionally form a morpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, or piperazinyl substituted with a substituent selected from C1-6alkyl, arylC1-6alkyl, arylC1-6alkyloxycarbonyl, heteroarylC1-6alkyl, C3-7cycloalkyl and C3-7cycloalkylC1-6alkyl; wherein
            
                k is 0, 1, 2, 3, 4, 5, or 6 and when k is 0 then a direct bond is intended;
                
                    R11 and R12 are each independently selected from hydrogen, C1-6alkyl, arylC1-6alkyloxycarbonyl, C3-7cycloalkyl, C1-12alkyl substituted with a substituent selected from hydroxy, C1-6alkyloxy, aryl, and heteroaryl; and C3-7cycloalkyl substituted with a substituent selected from hydroxy, C1-6alkyloxy, aryl, arylC1-6alkyl, heteroaryl, and heteroarylC1-6alkyl; or
                    R11 and R12 together with the nitrogen to which they are attached can optionally form a morpholinyl, a piperazinyl or a piperazinyl substituted with C1-6alkyloxycarbonyl;

aryl is phenyl or naphthalenyl;

                
                
            
            
        
        
    
    
    each phenyl or naphthalenyl can optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, hydroxyC1-6alkyl, C1-6alkyl, amino, polyhaloC1-6alkyl and C1-6alkyloxy; and
    each phenyl or naphthalenyl can optionally be substituted with a bivalent radical selected from methylenedioxy and ethylenedioxy;
    heteroaryl is pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl or tetrahydrofuranyl;
    each pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl, or tetrahydrofuranyl can optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, C1-6alkyl, amino, polyhaloC1-6alkyl, aryl, arylC1-6alkyl or C1-6alkyloxy; and
    each pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, benzofuranyl, or tetrahydrofuranyl can optionally be substituted with a bivalent radical selected from methylenedioxy or ethylenedioxy.


The compounds of formula (I) may also exist in their tautomeric forms. Such forms although not explicitly indicated in the above formula are intended to be included within the scope of the present invention.
A number of terms used in the foregoing definitions and hereinafter are explained hereunder. These terms are sometimes used as such or in composite terms.
As used in the foregoing definitions and hereinafter, halo is generic to fluoro, chloro, bromo and iodo; C1-6alkyl defines straight and branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms such as, e.g. methyl, ethyl, propyl, butyl, pentyl, hexyl, 1-methylethyl, 2-methylpropyl, 2-methyl-butyl, 2-methylpentyl and the like; hydroxyC1-6alkyl defines a hydroxy substituent on straight and branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms; trihalomethyl defines methyl containing three identical or different halo substituents for example trifluoromethyl; C3-7cycloalkyl includes cyclic hydrocarbon groups having from 3 to 10 carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and the like.
The term “addition salt” comprises the salts which the compounds of formula (I) are able to form with organic or inorganic bases such as amines, alkali metal bases and earth alkaline metal bases, or quaternary ammonium bases, or with organic or inorganic acids, such as mineral acids, sulfonic acids, carboxylic acids or phosphorus containing acids.
The term “addition salt” further comprises pharmaceutically acceptable salts, metal complexes and solvates and the salts thereof, that the compounds of formula (I) are able to form.
The term “pharmaceutically acceptable salts” means pharmaceutically acceptable acid or base addition salts. The pharmaceutically acceptable acid or base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and non-toxic base addition salt forms which the compounds of formula (I) are able to form. The compounds of formula (I) which have basic properties can be converted in their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. 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, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.
The compounds of formula (I) which have acidic properties may be converted in their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.
The terms acid or base addition salt also comprise the hydrates and the solvent addition forms which the compounds of formula (I) are able to form. Examples of such forms are e.g. hydrates, alcoholates and the like.
The term “metal complexes” means a complex formed between a compound of formula (I) and one or more organic or inorganic metal salt or salts. Examples of said organic or inorganic salts comprise the halogenides, nitrates, sulfates, phosphates, acetates, trifluoroacetates, trichloroacetates, propionates, tartrates, sulfonates, e.g. methylsulfonates, 4-methylphenylsulfonates, salicylates, benzoates and the like of the metals of the second main group of the periodical system, e.g. the magnesium or calcium salts, of the third or fourth main group, e.g. aluminium, tin, lead as well as the first to the eighth transition groups of the periodical system such as, for example, chromium, manganese, iron, cobalt, nickel, copper, zinc and the like.
The term “stereochemically isomeric forms of compounds of formula (I)”, as used hereinbefore, defines all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which are not interchangeable, which the compounds of formula (I) may possess. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of formula (I) both in pure form or in admixture with each other are intended to be embraced within the scope of the present invention.
Of special interest are those compounds of formula (I) which are stereochemically pure.
Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as 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” concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric excess of 90% up to 100%, even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%. The terms “enantiomerically pure” and “diastereomerically pure” should be understood in a similar way, but then having regard to the enantiomeric excess, respectively the diastereomeric excess of the mixture in question.
The tautomeric forms of the compounds of formula (I) are meant to comprise those compounds of formula (I) wherein e.g. an enol group is converted into a keto group (keto-enol tautomerism).
The N-oxide forms of the compounds of formula (I) are meant to comprise those compounds of formula (I) wherein one or several nitrogen atoms are oxidized to the so-called N-oxide, particularly those N-oxides wherein one or more of the piperidine-, piperazine or pyridazinyl-nitrogens are N-oxidized.
The compounds of formula (I) may be converted to the corresponding
N-oxide forms following art-known procedures for converting a trivalent nitrogen into its N-oxide form. Said N-oxidation reaction may generally be carried out by reacting the starting material of formula (I) with an appropriate organic or inorganic peroxide. Appropriate inorganic peroxides comprise, for example, hydrogen peroxide, alkali metal or earth alkaline metal peroxides, e.g. sodium peroxide, potassium peroxide; appropriate organic peroxides may comprise peroxy acids such as, for example, benzenecarboperoxoic acid or halo substituted benzenecarboperoxoic acid, e.g. 3-chlorobenzenecarboperoxoic acid, peroxoalkanoic acids, e.g. peroxoacetic acid, alkylhydroperoxides, e.g. t-butyl hydro-peroxide. Suitable solvents are, for example, water, lower alcohols, e.g. ethanol and the like, hydrocarbons, e.g. toluene, ketones, e.g. 2-butanone, halogenated hydrocarbons, e.g. dichloromethane, and mixtures of such solvents.
The present invention is also intended to include any isotopes of atoms present in the compounds of the invention. For example, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include C-13 and C-14.
Whenever used hereinafter, the term “compounds of formula (I)” is meant to include also the N-oxide forms, the pharmaceutically acceptable acid or base addition salts and all stereoisomeric forms.
A first group of interesting compounds consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

    a) m is 0;
    b) n is 0 or 2;
    c) p is 1;
    d) s is 0;
    e) t is 0;
    f) R1 and R2 are each independently hydrogen;
    g) A is a radical selected from (a-15), (a-21), (a-30), (a-39) or (a-40);
    h) R4 and R5 are each independently selected from hydrogen or C1-6alkyloxy;
    i) Z is the radical (b-2); or
    j) R6 and R7 are each independently selected from hydrogen.


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

    a) m is 0;
    b) n is 2;
    c) p is 1;
    d) s is 0;
    e) t is 0;
    f) R1 and R2 are each independently hydrogen;
    g) A is a radical selected from (a-21), (a-39) or (a-40);
    h) R4 and R5 are each independently selected from hydrogen or C1-6alkyloxy;
    i) Z is the radical (b-2); or
    j) R6 and R7 are each independently selected from hydrogen.


A group of preferred compounds consists of those compounds of formula (I) or any subgroup thereof, wherein m is 0; n is 0 or 2; p is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-15), (a-21), (a-30), (a-39) or (a-40); R4 and R5 are each independently selected from hydrogen or C1-6alkyloxy; Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen.
A group of more preferred compounds consists of those compounds of formula (I) or any subgroup thereof wherein m is 0; n is 2; p is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-21), (a-39) or (a-40); R4 and R5 are each independently selected from hydrogen or C1-6alkyloxy; Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen.
The most preferred compounds are compound No. 2, compound No. 3 or compound No. 5.




The compounds of formula (I), their pharmaceutically acceptable salts and N-oxides and stereochemically isomeric forms thereof may be prepared in conventional manner. The starting materials and some of the intermediates are known compounds and are commercially available or may be prepared according to conventional reaction procedures as generally known in the art.
A number of such preparation methods will be described hereinafter in more detail. Other methods for obtaining final compounds of formula (I) are described in the examples.
The compounds of formula (I) can be prepared by reacting an intermediate of formula (II) with an intermediate of formula (III) wherein W is an appropriate leaving group such as, for example, halo, e.g. fluoro, chloro, bromo or iodo, or a sulfonyloxy radical such as methylsulfonyloxy, 4-methylphenylsulfonyloxy and the like. The reaction can be performed in a reaction-inert solvent such as, for example, an alcohol, e.g. methanol, ethanol, 2-methoxy-ethanol, propanol, butanol and the like; an ether, e.g. 4,4-dioxane, 1,1′-oxybispropane and the like; a ketone, e.g. 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 or earth alkaline metal carbonate or hydrogen carbonate, e.g. triethylamine or sodium carbonate, may be utilized to pick up the acid which is liberated during the course of the reaction. A small amount of an appropriate metal iodide, e.g., sodium or potassium iodide may be added to promote the reaction. Stirring may enhance the rate of the reaction. The reaction may conveniently be carried out at a temperature ranging between room temperature and the reflux temperature of the reaction mixture and, if desired, the reaction may be carried out at an increased pressure.




The compounds of formula (I), wherein p is 1, herein referred to as compounds of formula (I-a) can be prepared by converting intermediates of formula (IV) with lithium aluminium hydride in a suitable solvent such as tetrahydrofuran.




The compounds of formula (I-a) can also be prepared by reacting an appropriate carboxaldehyde of formula (VI), with an intermediate of formula (V), in the presence of an appropriate reagent, such as a sodium borohydride e.g. sodium tetrahydroborate or polymer supported cyanotrihydroborate, in a suitable solvent, such as an alcohol e.g. methanol.




In an identical way the compounds of formula (I), wherein t is 1, herein referred to as compounds of formula (I-b), can be prepared by reacting an intermediate of formula (II) with an appropriate carboxaldehyde of formula (VII).




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




The compounds of formula (I) or their intermediates may also be converted into each other via art-known reactions or functional group transformations. A number of such transformations are already described hereinabove. Other examples are hydrolysis of carboxylic esters to the corresponding carboxylic acid or alcohol; hydrolysis of amides to the corresponding carboxylic acids or amines; hydrolysis of nitrites to the corresponding amides; amino groups on imidazole or phenyl may be replaced by a hydrogen by art-known diazotation reactions and subsequent replacement of the diazo-group by hydrogen; alcohols may be converted into esters and ethers; primary amines may be converted into secondary or tertiary amines; double bonds may be hydrogenated to the corresponding single bond; an iodo radical on a phenyl group may be converted in to an ester group by carbon monoxide insertion in the presence of a suitable palladium catalyst.
Intermediates of formula (II), wherein m is 0 and s is 0, herein referred to as intermediates of formula (II-a), can be prepared by a nitro to amine reduction reaction starting with an intermediate of formula (IX), in the presence of a metal catalyst such as Raney Nickel and an appropriate reductant such as hydrogen, in a suitable solvent such as methanol or ethanol.




Intermediates of formula (X), wherein s is 0, 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 may be performed in the presence of a base such as triethylamine, in a suitable solvent, such as, a mixture of dichloromethane and tetrahydrofuran.




The intermediates of formula (VI) can be prepared by reacting intermediates of formula (XIII) with lithium aluminium hydride in a suitable solvent such as tetrahydrofuran.




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




The intermediates of formula (IX), wherein p is 1, herein referred to as intermediates of formula (IX-a), can be prepared by treating an intermediate of formula (XVI) with an intermediate of formula (XVID), wherein Q is an appropriate leaving group such as, for example, halo, e.g. fluoro, chloro, bromo or iodo, or
C1-6alkyloxy, e.g. methyloxy, in diisopropylethyl amine.




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




The intermediates of formula (XVIII), wherein m is 0, herein referred to as intermediates of formula (XVIII-a), can be prepared by reacting an intermediate of formula (XVI), with an intermediate of formula (XIX), wherein Q is as defined above, in a suitable solvent such as diisopropylethyl amine.




The intermediates of formula (V) wherein m, n and s are 0, herein referred to as intermediates of formula (V-a), can be prepared by converting an intermediate of formula (XX) with hydrochloride solution.




The intermediates of formula (XX) can be prepared by reacting an intermediate of formula (XXI) with an intermediate of formula (III) wherein W is an appropriate leaving group such as, for example, halo. The reaction can be performed 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 centre in their structure. Such stereogenic centre may be present in an R or an S configuration.
Some of the compounds of formula (I) and some of the intermediates in the present invention may contain an asymmetric carbon atom. Pure stereochemically isomeric forms of said compounds and said intermediates can be obtained by the application of art-known procedures. For example, diastereoisomers can be separated by physical methods such as selective crystallization or chromatographic techniques, e.g. counter current distribution, liquid chromatography and the like methods. Enantiomers can be obtained from racemic mixtures by first converting said racemic mixtures with suitable resolving agents such as, for example, chiral acids, to mixtures of diastereomeric salts or compounds; then physically separating said mixtures of diastereomeric salts or compounds by, for example, selective crystallization, supercritical fluid chromatography or chromatographic techniques, e.g. liquid chromatography and the like methods; and finally converting said separated diastereomeric salts or compounds into the corresponding enantiomers. Pure stereochemically isomeric forms may also be obtained from the pure stereochemically isomeric forms of the appropriate intermediates and starting materials, provided that the intervening reactions occur stereospecifically.
The compounds of formula (I), the pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof have valuable pharmacological properties in that they inhibit the interaction between p53 and MDM2.
The term “MDM2” is used herein to mean a protein obtained as a result of expression of the mdm2 gene. Within the meaning of this term, MDM2 encompass all proteins encoded by mdm2, mutants thereof, alternative slice proteins thereof, and phosphorylated proteins thereof. Additionally, as used herein, the term “MDM2” includes MDM2 analogues, e.g. MDMX, also known as MDM4, and MDM2 homologues and analogues of other animals, e.g. the human homologue HDM2 or the human analogue HDMX.
The term “inhibiting the interaction” or “inhibitor of the interaction” is used herein to mean preventing or reducing the direct of indirect association of one or more molecules, peptides, proteins, enzymes or receptors; or preventing or reducing the normal activity of one or more molecules, peptides, proteins, enzymes, or receptors.
The term “inhibitor of the interaction of p53 with MDM2” or “p53-MDM2 inhibitor” is used herein to describe an agent which increases the expression of p53 in the assay described in C.1. This increase may be caused by, but is not limited to, one or more of the following mechanisms of action:

    
    
        inhibiting the interaction between p53 and MDM2,
        direct association with either the MDM2 or the p53 protein,
        interactions with upstream or downstream targets, e.g. kinases, or enzyme activities involved in ubiquitination or SUMO modification,
        sequestering or transportation of MDM2 and p53 into different cellular compartments,
        modulation of proteins associating with MDM2, for example (but not limited to), p73, E2F-1, Rb, p21waf1 or cip1,
        downregulating or interference with MDM2 expression and/or NMM2 activity, for example (but not limited to), impacting on its cellular localisation, post-translational modification, nuclear export or ubiquitin ligase activity,
        direct or indirect stabilization of the p53 protein, e.g. by keeping it in its functional structural form, or by preventing misfolding,
        enhancing p53 expression or expression of p53 family members, e.g. p63 and p73.
        increasing p53 activity, for example by (but not limited to), enhancing its transcriptional activity and/or
        increasing expression of genes and proteins of the p53-signalling pathway, for example (but not limited to) p21waf1, cip1, MIC-1 (GDF-15), PIG-3 and ATF-3.
    
    


Hence, the present invention discloses the compounds of formula (I) for use as a medicine.
Furthermore, the invention also concerns the use of a compound for the manufacture of a medicament for the treatment of a disorder mediated through a p53-MDM2 interaction, wherein said compound is a compound of formula (I)
The term “treating” or “treatment” as used herein covers any treatment of a disease and/or condition in an animal, particularly a human, and includes: (i) preventing a disease and/or condition from occurring in a subject which may be predisposed to the disease and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease and/or condition, i.e., arresting its development; (iii) relieving the disease and/or condition, i.e., causing regression of the disease and/or condition.
With the term “a disorder mediated through a p53-MDM2 interaction” is meant any undesired or detrimental condition that results in or from the inhibition of the interaction between the MDM2 protein and p53 or other cellular proteins that induce apoptosis, induce cellular death, or regulate the cell cycle.
This invention also provides a method for treating a disorder mediated through a p53-MDM2 interaction by administering an effective amount of a compound of the present invention, to a subject, e.g. a mammal (and more particularly a human) in need of such treatment.
The compounds of the invention can have antiproliferative effects in tumour cells, even if such cells are devoid of functional p53. More in particular, the compounds of the invention can have antiproliferative effects in tumours with wild-type p53 and/or in tumours overexpressing MDM2.
Thus, this invention also provides a method for inhibiting tumour growth by administering an effective amount of a compound of the present invention, to a subject, e.g. a mammal (and more particularly a human) in need of such treatment.
Examples of tumours which may be inhibited, but are not limited to, lung cancer (e.g. adenocarcinoma and including non-small cell lung cancer), pancreatic cancers (e.g. pancreatic carcinoma such as, for example exocrine pancreatic carcinoma), colon cancers (e.g. colorectal carcinomas, such as, for example, colon adenocarcinoma and colon adenoma), oesophageal cancer, oral squamous carcinoma, tongue carcinoma, gastric carcinoma, nasopharyngeal cancer, hematopoietic tumours of lymphoid lineage (e.g. acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), non-Hodgkin's lymphoma, Hodgkin's disease, myeloid leukemias (for example, acute myelogenous leukemia (AML)), thyroid follicular cancer, myelodysplastic syndrome (MD S), tumours of mesenchymal origin (e.g. fibrosarcomas and rhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas, brain tumors, gliomas, benign tumour of the skin (e.g. keratoacanthomas), breast carcinoma (e.g. advanced breast cancer), kidney carcinoma, ovary carcinoma, cervical carcinoma, endometrial carcinoma, bladder carcinoma, prostate cancer including the advanced disease, testicular cancers, osteosarcoma, head and neck cancer and epidermal carcinoma.
The compounds of the present invention can also be used for the treatment and prevention of inflammatory conditions.
Thus, this invention also provides a method for the treatment and prevention of inflammatory conditions by administering an effective amount of a compound of the present invention, to a subject, e.g. a mammal (and more particularly a human) in need of such treatment.
The compounds of the present invention can also be used for the treatment of autoimmune diseases and conditions. With the term “autoimmune diseases” is meant any disease in which an animal's immune system reacts adversely to a self-antigen. With the term “self-antigen” is meant any antigen that is normally found in the animal's body. Representative autoimmune diseases include but are not limited to: Hashimoto's thyroiditis, Grave's disease, multiple sclerosis, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), dermatomyositis, Crohn's disease, Wegener's granulomatosis, Anti Glomerular Basement Membrane Disease, Antiphospholipid Syndrome, 25 Dermatitis Herpetiformis, Allergic Encephalomyelitis, Glomerulonephritis, Membranous Glomerulonephritis, Goodpasture Syndrome, Lambert-Eaton, Myasthenic Syndrome, Myasthenia Gravis, Bullous Pemphigoid, Polyendocrinopathies, Reiter's Disease, and Stiff-Man Syndrome.
Thus, this invention also provides a method for the treatment of autoimmune diseases and conditions and the treatment of diseases associated with conformational unstable or misfolded proteins by administering an effective amount of a compound of the present invention, to a subject, e.g. a mammal (and more particularly a human) in need of such treatment.
The compounds of the present invention can also be useful for the treatment of diseases associated with conformational unstable or misfolded proteins.
Examples of diseases associated with conformational unstable or misfolded proteins include but are not limited to: cystic fibrosis (CFTR), Marfan syndrome (fibrillin), Amyotrophic lateral sclerosis (superoxide dismutase), scurvy (collagen), maple syrup urine disease (alpha-ketoacid dehydrogenase complex), osteogenesis imperfecta (typel procollagen pro-alpha), Creutzfeldt-Jakob disease (prion), Alzheimer's disease (beta-amyloid), familial amyloidosis (lysozyme), cataracts (crystallins), familial hypercholesterolemia (LDL receptor), α I-antitrypsin deficiency, Tay-Sachs disease (beta-hexosaminidase), retinitis pigmentosa (rhodopsin), and loprechaunism (insulin receptor).
Thus, this invention also provides a method for the treatment of diseases associated with conformational unstable or misfolded proteins by administering an effective amount of a compound of the present invention, to a subject, e.g. a mammal (and more particularly a human) in need of such treatment.
In view of their useful pharmacological properties, the subject compounds may be formulated into various pharmaceutical forms for administration purposes.
To prepare the pharmaceutical compositions of this invention, an effective amount of a particular compound, in base or acid addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for administration orally, rectally, percutaneously, or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets.
Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, to aid solubility for example, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, 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 the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause a significant deleterious effect to the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on, as an ointment. It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient, calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
The compound of the invention is administered in an amount sufficient to inhibit the interaction between MDM2 and p53 or other cellular proteins that induce apoptosis, induce cellular death, or regulate the cell cycle.
The oncogenic potential of MDM2 is not only determined by its ability to suppress p53, but also by its ability to regulate other tumour suppressor proteins, e.g. the retinoblastoma protein pRb and the closely associated E2F1 transcription factor.
Thus, the compound of the invention is administered in an amount sufficient to modulate the interaction between MDM2 and the E2F transcription factors.
Those skilled in the art could easily determine the effective amount from the test results presented hereinafter. In general it is 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 single, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 0.5 to 500 mg, and in particular 10 mg to 500 mg of active ingredient per unit dosage form.
As another aspect of the present invention, a combination of a p53-MDM2 inhibitor with another anticancer agent is envisaged, especially for use as a medicine, more specifically in the treatment of cancer or related diseases.
For the treatment of the above conditions, the compounds of the invention may be advantageously employed in combination with one or more other medicinal agents, more particularly, with other anti-cancer agents. Examples of anti-cancer agents include but are not limited to:

    
    
        platinum coordination compounds for example cisplatin, carboplatin or oxalyplatin;
        taxane compounds for example paclitaxel or docetaxel;
        topoisomerase I inhibitors such as camptothecin compounds for example irinotecan or topotecan;
        topoisomerase II inhibitors such as anti-tumour epipodophyllotoxins or podophyllotoxin derivatives for example etoposide or teniposide;
        anti-tumour vinca alkaloids for example vinblastine, vincristine or vinorelbine;
        anti-tumour nucleoside derivatives for example 5-fluorouracil, leucovorin, gemcitabine or capecitabine;
        alkylating agents such as nitrogen mustard or nitrosourea for example cyclophosphamide, chlorambucil, carmustine, thiotepa, mephalan or lomustine;
        anti-tumour anthracycline derivatives for example daunorubicin, doxorubicin, doxil, idarubicin or mitoxantrone;
        molecules that target the IGF-1 receptor for example picropodophilin;
        tetracarein derivatives for example tetrocarcin A;
        glucocorticoiden 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 vorozole;
        differentiating 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 azacytidine or decitabine;
        antifolates for example premetrexed disodium;
        antibiotics for example antinomycin D, bleomycin, mitomycin C, dactinomycin, caminomycin or daunomycin;
        antimetabolites for example chlofarabine, aminopterin, cytosine arabinoside or methotrexate;
        apoptosis inducing agents and antiangiogenic agents such as Bcl-2 inhibitors for example YC 137, BH 312, ABT 737, gossypol HA 14-1, TW 37 or decanoic acid;
        tubuline-binding agents for example combrestatin, colchicines or nocodazole;
        kinase inhibitors for example flavoperidol, imatinib mesylate, erlotinib or gefitinib;
        farnesyltransferase inhibitors for example tipifarnib;
        histone deacetylase (HDAC) inhibitors for example sodium butyrate, suberoylanilide hydroxamide acid (SAHA), depsipeptide (FR 901228), NVP-LAQ824, R306465, JNJ-26481585 or trichostatin A;
        Inhibitors of the ubiquitin-proteasome pathway for example PS-341, MLN 0.41 or bortezomib;
        Yondelis;
        Telomerase inhibitors for example telomestatin;
        Matrix metalloproteinase inhibitors for example batimastat, marimastat, prinostat or metastat.
    
    


As stated above, the compounds of the present invention also have therapeutic applications in sensitising tumour cells for chemotherapy and radiotherapy.
Hence the compounds of the present invention can be used as “radiosensitizer” and/or “chemosensitizer” or can be given in combination with another “radiosensitizer” and/or “chemosensitizer”.
The term “radiosensitizer”, as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of the cells to ionizing radiation and/or to promote the treatment of diseases which are treatable with ionizing radiation.
The term “chemosensitizer”, as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of cells to chemotherapy and/or promote the treatment of diseases which are treatable with chemotherapeutics.
Several mechanisms for the mode of action of radiosensitizers have been suggested in the literature including: hypoxic cell radiosensitizers (e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds) mimicking oxygen or alternatively behave like bioreductive agents under hypoxia; non-hypoxic cell radiosensitizers (e.g., halogenated pyrimidines) can be analogoues of DNA bases and preferentially incorporate into the DNA of cancer cells and thereby promote the radiation-induced breaking of DNA molecules and/or prevent the normal DNA repair mechanisms; and various other potential mechanisms of action have been hypothesized for radiosensitizers in the treatment of disease.
Many cancer treatment protocols currently employ radiosensitizers in conjunction with radiation of x-rays. Examples of x-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO 9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.
Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives, tin etioporphyrin, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.
Radiosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds which promote the incorporation of radiosensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and/or oxygen to the target cells; chemotherapeutic agents which act on the tumour with or without additional radiation; or other therapeutically effective compounds for treating cancer or other disease.
Chemosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds which promote the incorporation of chemosensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and/or oxygen to the target cells; chemotherapeutic agents which act on the tumour or other therapeutically effective compounds for treating cancer or other disease. Calcium antagonist, for example verapamil, are found useful in combination with antineoplastic agents to establish chemosensitivity in tumor cells resistant to accepted chemotherapeutic agents and to potentiate the efficacy of such compounds in drug-sensitive malignancies.
In view of their useful pharmacological properties, the components of the combinations according to the invention, i.e. the other medicinal agent and the p53-MDM inhibitor may be formulated into various pharmaceutical forms for administration purposes. The components may be formulated separately in individual pharmaceutical compositions or in a unitary pharmaceutical composition containing both components.
The present invention therefore also relates to a pharmaceutical composition comprising the other medicinal agent and the p53-MDM inhibitor together with one or more pharmaceutical carriers.
The present invention further relates to the use of a combination according to the invention in the manufacture of a pharmaceutical composition for inhibiting the growth of tumour cells.
The present invention further relates to a product containing as first active ingredient a p53-MDM2 inhibitor according to the invention and as second active ingredient an anticancer agents as a combined preparation for simultaneous, separate or sequential use in the treatment of patients suffering from cancer.
The other medicinal agent and p53-MDM2 inhibitor may be administered simultaneously (e.g. in separate or unitary compositions) or sequentially in either order. In the latter case, the two compounds will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved. It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimes for each component of the combination will depend on the particular other medicinal agent and p53-MDM2 inhibitor being administered, their route of administration, the particular tumour being treated and the particular host being treated. The optimum method and order of administration and the dosage amounts and regime can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
The platinum coordination compound is advantageously administered in a dosage of 1 to 500 mg per square meter (mg/m2) of body surface area, for example 50 to 400 mg/m2, particularly for cisplatin in a dosage of about 75 mg/m2 and for carboplatin in about 300 mg/m2 per course of treatment.
The taxane compound is advantageously administered in a dosage of 50 to 400 mg per square meter (mg/m2) of body surface area, for example 75 to 250 mg/m2, particularly for paclitaxel in a dosage of about 175 to 250 mg/m2 and for docetaxel in about 75 to 150 mg/m2 per course of treatment.
The camptothecin compound is advantageously administered in a dosage of 0.1 to 400 mg per square meter (mg/m2) of body surface area, for example 1 to 300 mg/m2, particularly for irinotecan in a dosage of about 100 to 350 mg/m2 and for topotecan in about 1 to 2 mg/m2 per course of treatment.
The anti-tumour podophyllotoxin derivative is advantageously administered in a dosage of 30 to 300 mg per square meter (mg/m2) of body surface area, for example 50 to 250 mg/m2, particularly for etoposide in a dosage of about 35 to 100 mg/m2 and for teniposide in about 50 to 250 mg/m2 per course of treatment.
The anti-tumour vinca alkaloid is advantageously administered in a dosage of 2 to 30 mg per square meter (mg/m2) of body surface area, particularly for vinblastine in a dosage of about 3 to 12 mg/m2, for vincristine in a dosage of about 1 to 2 mg/m2, and for vinorelbine in dosage of about 10 to 30 mg/m2 per course of treatment.
The anti-tumour nucleoside derivative is advantageously administered in a dosage of 200 to 2500 mg per square meter (mg/m2) of body surface area, for example 700 to 1500 mg/m2, particularly for 5-FU in a dosage of 200 to 500 mg/m2, for gemcitabine in a dosage of about 800 to 1200 mg/m2 and for capecitabine in about 1000 to 2500 mg/m2 per course of treatment.
The alkylating agents such as nitrogen mustard or nitrosourea is advantageously administered in a dosage of 100 to 500 mg per square meter (mg/m2) of body surface area, for example 120 to 200 mg/m2, particularly for cyclophosphamide in a dosage of about 100 to 500 mg/m2, for chlorambucil in a dosage of about 0.1 to 0.2 mg/kg, for carrnustine in a dosage of about 150 to 200 mg/m2, and for lomustine in a dosage of about 100 to 150 mg/m2 per course of treatment.
The anti-tumour anthracycline derivative is advantageously administered in a dosage of 10 to 75 mg per square meter (mg/m2) of body surface area, for example 15 to 60 mg/m2, particularly for doxorubicin in a dosage of about 40 to 75 mg/m2, for daunorubicin in a dosage of about 25 to 45 mg/m2, and for idarubicin in a dosage of about 10 to 15 mg/m2 per course of treatment.
The antiestrogen agent is advantageously administered in a dosage of about 1 to 100 mg daily depending on the particular agent and the condition being treated. Tamoxifen is advantageously administered orally in a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect. Toremifene is advantageously administered orally in a dosage of about 60 mg once a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect. Anastrozole is advantageously administered orally in a dosage of about 1 mg once a day. Droloxifene is advantageously administered orally in a dosage of about 20-100 mg once a day. Raloxifene is advantageously administered orally in a dosage of about 60 mg once a day. Exemestane is advantageously administered orally in a dosage of about 25 mg once a day.
Antibodies are advantageously administered in a dosage of about 1 to 5 mg per square meter (mg/m2) of body surface area, or as known in the art, if different. Trastuzumab is advantageously administered in a dosage of 1 to 5 mg per square meter (mg/m2) of body surface area, particularly 2 to 4 mg/m2 per course of treatment.
These dosages may be administered for example once, twice or more per course of treatment, which may be repeated for example every 7, 14, 21 or 28 days.
The compounds of formula (I), the pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof can have valuable diagnostic properties in that they can be used for detecting or identifying an p53-MDM2 interaction in a biological sample comprising detecting or measuring the formation of a complex between a labelled compound and/or p53 and/or MDM2 and or other molecules, peptides, proteins, enzymes or receptors.
The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances, etc. Examples of the radioisotopes include 125I, 131I, 3H and 14C. Enzymes are usually made detectable by conjugation of an appropriate substrate which, in turn catalyses a detectable reaction. Examples thereof include, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably horseradish peroxidase. The luminous substances include, for example, luminol, luminol derivatives, luciferin, aequorin and luciferase.
Biological samples can be defined as body tissue or body fluids. Examples of body fluids are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva and the like.
The following examples illustrate the present invention.
EXPERIMENTAL PART
Hereinafter, “DCM” is defined as dichloromethane, “DIPE” is defined as diisopropyl ether, “EtOAc” is defined as ethyl acetate, “EtOH” is defined as ethanol, “MeOH” is defined as methanol and “THF” is defined as tetrahydrofuran.
Melting Points
For a number of compounds, indicated by (Kofler), melting points were obtained with a Kofler hot bench, consisting of a heated plate with linear temperature gradient, a sliding pointer and a temperature scale in degrees Celsius.
LCMS
General Procedure
The HPLC gradient was supplied by an Alliance HT 2795 (Waters) system comprising a quaternary pump with degasser, an autosampler and a diode-array detector (DAD). Flow from the column was split to a MS detector. The MS detector was configured with an clectrospray ionization source. The capillary needle voltage was 3 kV and the source temperature was maintained at 100° C. on the LCT (Time of Flight-Z-spray mass spectrometer from Waters. Nitrogen was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data system.
Method A
In addition to the general procedure: Reversed phase HPLC was carried out on a 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; were employed to run a gradient condition from 85% A, 15% B (hold for 3 minutes) to 20% A, 80% B in 5 minutes, hold at 20% A and 80% B for 6 minutes and reequilibrated with initial conditions for 3 minutes. An injection volume of 20 μl was used. Cone voltage was 20 V for positive and negative ionization mode. Mass spectra were acquired by scanning from 100 to 900 in 0.8 seconds using an interscan delay of 0.08 seconds.
Method B
In addition to the general procedure: Reversed phase HPLC was carried out on a 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; were employed to run a gradient condition from 100% A (hold for 1 minutes) to 50% A, 50% B in 4 minutes, hold at 50% A and 50% B for 9 minutes and reequilibrated with initial conditions for 3 minutes. An injection volume of 20 μl was used. Cone voltage was 20 V for positive ionization mode and 20 V for negative ionization mode. Mass spectra were acquired by scanning from 100 to 900 in 0.8 seconds using an interscan delay of 0.08 seconds.
A. Preparation of the Intermediate Compounds
Example A1
Preparation of Intermediate 1




A mixture of benzo[b]furan-3-one (400 mg, 0.0029 mol) and N-methoxy-N-methyl(triphenylphosphoranylidene)acetamide (1.19 g, 0.0032 mol) in xylene (10 ml) was stirred at 135° C. for 35 hours. The reaction was quenched with water and basified with a saturated solution of sodium hydrogen-ocarbonate. The mixture was extracted with EtOAc, the organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: EtOAc/cyclohexane 50/50). The pure fractions were collected and the solvent was evaporated, yielding 210 mg (32%) of intermediate 1 as a brown oil.
1H NMR (300 MHz, CDCl3) δ 7.63 (m, 2H), 7.46 (d, 1H, J=7.1), 7.25 (m, 2H), 3.84 (s, 2H), 3.70 (s, 3H), 3.22 (s, 3H).
MS (ES+) m/z 220 (M+1).
Example A2
a) Preparation of Intermediate 2




A mixture of 1H-pyrrolo[2,3-b]pyridine-3-ethanamine (0.014 mol), 1-fluoro-4-nitro-benzene (0.015 mol) and DIPE (0.048 mol) was stirred at 210° C. for 30 minutes. DIPE was evaporated. The precipitate was dissolved in DCM/MeOH. The organic layer was washed with potassium carbonate 10%, dried (MgSO4), filtered and the solvent was evaporated. The residue (2 g) was purified by column chromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH4OH 97/3/0.3). The pure fractions were collected and the solvent was evaporated, yielding 0.152 g (28,%) of intermediate 2, melting point 201° C. (Kofler).
b) Preparation of Intermediate 3




A mixture of intermediate 2 (0.004 mol) and Raney Nickel (1 g) in MeOH (20 ml) was hydrogenated at room temperature for 2 hours under a 3 bar pressure, then filtered over celite. Celite was washed with MeOH. The filtrate was evaporated, yielding 0.92 g (100%) of intermediate 3.
Example A3
a) Preparation of Intermediate 4




A mixture of imidazo[1,2-a]pyridine-3-acetonitrile (0.028 mol) and Rh/Al2O3 5% (4.5 g) in EtOH (45 ml) and MeOH/NH3 (12.5 ml) was hydrogenated at room temperature for 72 hours under a 3 bar pressure, then filtered over celite. Celite was washed with DCM/EtOH. The filtrate was evaporated. The residue was dissolved in DCM. The organic layer was washed with potassium carbonate 10%, dried (MgSO4), filtered and the solvent was evaporated, yielding: 3.4 g (73%) of intermediate 4.
b) Preparation of Intermediate 5




A mixture of intermediate 4 (0.006 mol), 1-fluoro-4-nitro-benzene (0.007 mol) and DIPE (0.021 mol) was heated at 210° C. for 30 minutes. DIPE was evaporated. The crude oil was diluted in DCM and EtOH (90/10). The organic layer was washed with potassium carbonate 10%, dried (MgSO4), filtered and the solvent was evaporated. The residue (1.3 g) was purified by column chromatography over silica gel (20-45 μm) (eluent: DCM/MeOH 98/2). The pure fractions were collected and the solvent was evaporated. The residue (0.36 g) was crystallized from acetonitrile. The precipitate was filtered off and dried, yielding 0.199 g of intermediate 5, melting point 171° C. (Kofler).
c) Preparation of Intermediate 6




A mixture of intermediate 5 (0.003 mol) and Raney Nickel (1 g) in MeOH (20 ml) was hydrogenated at room temperature for 3 hours under a 3 bar pressure, then filtered over celite. Celite was washed with MeOH. The filtrate was evaporated, yielding 1 g (>100%) of intermediate 6.
Example A4
a) Preparation of Intermediate 7




A mixture of 1-fluoro-4-nitro-benzene (0.0083 mol), 1H-indol-5-amine (0.0076 mol) and DIPE (0.0166 mol) was stirred at 210° C. for 18 hours, then taken up in DCM. The organic layer was washed with hydrochloric acid 3N, then with NaHCO3, dried (MgSO4), filtered, and the solvent was evaporated. The residue was taken up in EtOH. The precipitate was filtered, washed with diethyl ether and dried, yielding 1.08 g (65%) of intermediate 7, melting point 180° C.
b) Preparation of Intermediate 8




A mixture of intermediate 7 (0.0039 mol) and raney Nickel (1 g) in MeOH (20 ml) was hydrogenated at room temperature for 2 hours under a 3 bar pressure, then filtered over celite. Celite was washed with DCM/MeOH. The filtrate was evaporated, yielding 1 g (>100%) of intermediate 8.
Example A5
Preparation of Intermediate 9




1,1′-Carboxyldiimidazole (315 mg, 0.0019 mol) was added dropwise to a solution of (6-methoxy-benzofuran-3-yl)-acetic acid (400 mg, 0.0019 mol) in DCM (10 ml). The mixture was stirred at room temperature for 3 hours. O,N-Dimethyl-hydroxylamine hydrochloride (190 mg, 0.0019 mol) was added and the mixture was stirred at room temperature for 4 hours. The reaction was quenched with ice and basified with a 4N solution of sodium hydroxide. The mixture was extracted with DCM, the organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: EtOAc/cyclohexane 50/50). The pure fractions were collected and the solvent was evaporated, yielding 410 mg (85%) of intermediate 9 as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 7.54 (m, 2H), 6.99 (s, 1H), 6.88 (d, 1H, J=6.4), 3.83 (s, 3H), 3.79 (s, 2H), 3.69 (s, 3H), 3.21 (s, 3H).
Example A6
a) Preparation of Intermediate 10




A mixture of (3-iodo-2-thienyl)-carbamic acid, 1,1-dimethylethyl ester (0.0105 mol), 4-bromo-2-butenoic acid, ethyl ester (0.0158 mol) and potassium carbonate (0.021 mol) in N,N-dimethylformamide (100 ml) was stirred at room temperature for 16 hours. Triphenyl-phosphine (0.001 mol) and palladium acetate (0.0005 mol) were added. The mixture was stirred at 70° C. for 8 hours, then washed with water. The organic layer was separated with EtOAc, dried (MgSO4) and the solvent was evaporated. The residue was purified by column chromatography over silica gel (eluent: EtOAc/cyclohexane 10/90), yielding 2.3 g (70%) of intermediate 10.
b) Preparation of Intermediate 11




A solution of intermediate 10 (0.0016 mol) in THF (10 ml) was stirred at −78° C. under Argon. A solution of N,O-dimethylhydroxylamine HCl (0.0004 mol) in THF (40 ml) was stirred at −78° C. under Argon. Butyllithium (1.6M in hexane) (0.016 mol) was added dropwise at −78° C. to N,O-dimethylhydroxylamine HCl (0.0004 mol). The mixture was stirred at room temperature for 20 minutes, then cooled again to −78° C. Intermediate 10 was added dropwise. The mixture was stirred at −78° C. for 2 hours, poured out into saturated NH4Cl at −78° C. and extracted with EtOAc. The organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (eluent: EtOAc/cyclohexane 10/90 to 30/70). The pure fractions were collected and the solvent was evaporated, yielding 0.4 g (77%) of intermediate 11.
c) Preparation of Intermediate 12




Intermediate 11 (0.0012 mol) was dissolved in DCM and absorbed on silica gel. The mixture was stirred at 60° C. for 24 hours in vacuo. The residue was purified by column chromatography over silica gel (eluent: EtOAc/cyclohexane 80/20). The pure fractions were collected and the solvent was evaporated, yielding (62%) of intermediate 12.
d) Preparation of Intermediate 13




Aluminum lithium hydride (0.0008 mol) was added slowly at 0° C. to a solution of intermediate 12 (0.0008 mol) in THF (4 ml, dry). The mixture was stirred at 0° C. for 1 hour, poured out on ice, washed with KHSO4 5% and extracted with EtOAc. The organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated, yielding intermediate 13. This product was used directly in the next reaction step.
B. Preparation of the Final Compounds
Example B1
Preparation of Compound 1




A 1N lithium aluminium hydride solution in THF (0.65 ml) was added dropwise at 0° C. to a suspension of intermediate 1 (162 mg, 0.00065 mol) in THF (4 ml). The mixture was stirred for 1 hour at 0° C. The reaction was quenched at 0° C. by slow addition of a 5% potassium bisulfate solution in water and extracted twice with EtOAc. The organic layer was separated, dried (MgSO4), filtered, and the solvent was evaporated to give benzofuran-3-yl-acetaldehyde.
To a solution of N-4-pyridinyl-1,4-benzenediamine in MeOH (3 ml) and acetic acid (4 drops) were added sodium cyanoborohydride (57 mg, 0.00091 mol) and the previous aldehyde dissolved in MeOH (1 ml). The mixture was stirred at room temperature for 18 hours. The solvent was evaporated, the residual oil taken up in EtOAc and washed with a saturated solution of sodium hydrogen carbonate. The organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM/MeOH 95/5). The pure fractions were collected and the solvent was evaporated, yielding 41 mg (20%) of compound 1, melting point 152° C.-154° C.
1H NMR (300 MHz, MeOH-d4) δ 7.98 (d, 2H, J=6.6), 7.59 (m, 2H), 7.43 (d, 1H, J=7.4), 7.24 (m, 2H), 7.00 (dd, 2H, J=8.7, J=3.3), 6.68 (m, 4H), 3.45 (t, 2H, J=7.2), 2.98 (t, 2H, J=7.2).
MS (ES+) m/z 330 (M+1).
Example B2
Preparation of Compound 2




A mixture of intermediate 3 (0.004 mol) and 4-bromo-pyridine hydrochloride (0.004 mol) in acetic acid (5 ml) was stirred at 140° C. for 15 minutes, then evaporated. The residue was dissolved in DCM/MeOH. The organic layer was washed with potassium carbonate 10%, dried (MgSO4), filtered and the solvent was evaporated. The residue (1 g) was purified by column chromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH4OH 90/10/1). The pure fractions were collected and the solvent was evaporated. The residue (0.74 g, 63%) was crystallized from acetonitrile. The precipitate was filtered off and dried, yielding 0.541 g of compound 2, melting point 182° C. (Kofler).
1H NMR (DMSO-d6) δ 2.96 (2H, t, J=7.7 Hz), 3.30 (2H, t, J=7.7 Hz), 5.6 (1H, br, t, J=7.6 Hz), 6.58 (4H, m), 6.95 (2H, d, J=7.7 Hz), 7.03 (1H, m), 7.33 (1H, m), 7.96 (1H, d, J=7.7 Hz), 8.03 (2H, d, J=7.6 Hz), 8.17-8.21 (1H, m), 8.22 (1H, br, s), 11.38 (1H, br, s)
LCMS (ES+) m/z 330 (M+1), Rt=7.10, Method A.
Example B3
Preparation of Compound 3




4-Bromo-pyridine hydrochloride (0.004 mol) was added to a solution of intermediate 6 (0.004 mol) in acetic acid (10 ml). The mixture was heated at 140° C. for 15 minutes in a microwave oven. Acetic acid was evaporated. The crude oil was dissolved in DCM/EtOH (90/10). The organic layer was washed with potassium carbonate, dried (MgSO4), filtered and the solvent was evaporated. The residue (1 g) was purified by column chromatography over silica gel (15-40 cm) (eluent: DCM/MeOH/NH4OH 90/10/0.5 to 90/10/1). The pure fractions were collected and the solvent was evaporated, yielding 0.63 g (48%) of compound 3.
1H NMR (DMSO-d6) δ 1.75-1.93 (4H, m), 2.65-2.78 (4H, m), 3.32 (2H, s), 3.70 (2H, t, J=6.11 Hz), 5.62 (1H, br, t, J=7.6 Hz), 6.58-6.53 (5H, m), 6.95 (2H, d, J=7.7 Hz), 8.05 (2H, d, J=7.6 Hz), 8.24 (1H, s)
LCMS (ES+) m/z 334 (M+1), Rt 5.18, Method B.
melting point 256° C. (Kofler).
Example B4
Preparation of Compound 4




4-Bromo-pyridine hydrochloride (0.002 mol) was added to a solution of intermediate 8 (0.0022 mol) in acetic acid (2.8 ml). The mixture was heated at 110° C. for 30 minutes, poured out into ice water and basified with potassium carbonate 10%. The precipitate was filtered off and dried. The residue (0.739 g) was purified by column chromatography over kromasil (5 μm) (eluent: DCM/MeOH/NH4OH 96/4/0.4 to 88/12/1.2). The pure fractions were collected and the solvent was evaporated, yielding 0.018 g of compound 4.
1H NMR (DMSO-d6) δ 6.32 (1 H, br s), 6.68 (1 H, d, J=5.6 Hz), 6.9 (1 H, dd, J=10.2 Hz, J=2.5 Hz), 6.55 (2H, d, J=10.2 Hz), 7.0 (2 H, d, J=10.2 Hz), 7.27 (1 H, m), 7.32 (1 H, d, J=10.2 Hz), 7.7 (1 H, br s), 8.17 (1 H, dd, J=10.2 Hz, J=2.5 Hz), 8.37 (1H, br s), 10.9 (1 H, br s).
LCMS (ES+) m/z 301 (M+1), Rt 7.73, Method A.
Example B5
Preparation of Compound 5




A 1N lithium aluminium hydride solution in THF (1.0 ml) was added dropwise at 0° C. to a suspension of intermediate 9 (249 mg, 0.0010 mol). The mixture was stirred for 1 hour at 0° C. The reaction was quenched at 0° C. by slow addition of a 5% potassium bisulfate solution in water and extracted twice with EtOAc. The organic layer was separated, dried (MgSO4), filtered, and the solvent was evaporated to give the (6-methoxy-benzofuran-3-yl)-acetaldehyde.
To a solution of N-4-pyridinyl-1,4-benzenediamine in MeOH (5 ml) and acetic acid (5 drops) were added sodium cyanoborohydride (90 mg, 0.0014 mol) and the previous aldehyde dissolved in MeOH (1 ml). The mixture was stirred at room temperature for 18 hours. The solvent was evaporated, the residual oil taken up in EtOAc and washed with a saturated solution of sodium hydrogen carbonate. The organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated. The residue was washed with MeOH and dried, yielding 125 mg (34%) of compound 5, melting point 184° C.-186° C.
1H NMR (300 MHz, DMSO-d6) δ 8.23 (s, 1H), 8.01 (dd, 2H, J=6.3, J=1.5), 7.71 (s, 1H), 7.51 (d, 1H, J=8.5), 7.14 (d, 1H, J=2.1), 6.92 (d, 2H, J=8.6), 6.85 (dd, 1H, J=8.5, J=2.2), 6.58 (m, 4H), 5.64 (t, 1H, J=5.6), 3.77 (s, 3H), 3.30 (t, 2H, J=7.0), 2.48 (t, 2H, J=7.0).
MS (ES+) m/z 360 (M+1).
Example B6
Preparation of Compound 6




Acetic acid (few drops), then intermediate 13 (0.0004 mol) were added dropwise to a solution of N-4-pyridinyl-1,4-benzenediamine (0.0008 mol) and sodiumcyanoborohydride (0.001 mol) in MeOH (4 ml). Intermediate 13 (0.0004 mol) was dissolved in MeOH (4 ml). The mixture was stirred at room temperature for 20 hours, poured out into water, washed with saturated NaHCO3 and extracted with EtOAc. The organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (eluent: DCM/MeOH 85/15). The pure fractions were collected and the solvent was evaporated, yielding 0.086 g (34%) of compound 6.
Table F-1 lists the compounds that were prepared in one of the above examples.







TABLE F-1













Co. No. 1; Ex. [B1]; mp. 152° C.-154° C.


 








Co. No. 2; Ex. [B2]; mp. 182° C.


 








Co. No. 3; Ex. [B3]; mp. 256° C.


 








Co. No. 4; Ex. [B4]


 








Co. No. 5; Ex. [B5]; mp. 184° C.-186° C.


 








Co. No. 6; Ex. [B6]









C. Pharmacological Example
The capacity of the compounds to preserve p53 in A2780 cells was measured with the p53 enzyme linked immunosorbent assay. The p53 assay is a “sandwich” enzyme immunoassay employing two polyclonal antibodies. A polyclonal antibody, specific for the p53 protein, has been immobilized onto the surface of the plastic wells. Any p53 present in the sample to be assayed will bind to the capture antibody. The biotinylated detector polyclonal antibody also recognizes p53 protein, and will bind to any p53, which has been retained by the capture antibody. The detector antibody, in turn, is bond by horseradish peroxidase-conjugated streptavidin. The horseradish peroxidase catalyses the conversion of the chromogenic substrate o-phenylene diamine, the intensity of which is proportional to the amount of p53 protein bond to the plate. The colored reaction product is quantified using a spectrophotometer. Quantitation is achieved by the construction of a standard curve using known concentrations of purified recombinant HIS tagged p53 protein (see example C.1.).
Cellular activity of the compounds of formula (I) was determined on U87MG tumour cells using a colorimetric assay for cell toxicity or survival (see example C.2).
U87MG cells are human glioblastoma cells with wild type p53. In this cell line MDM2 tightly controls p53 expression.
C.1. p53 ELISA
A2780 cells (ATCC) were cultivated in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and gentamycin at 37° C. in a humidified incubator with 5% CO2.
A2780 cells were seeded at 20.000 cells per well in a 96 well plate, cultured for 24 hours and treated with 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, low salt RIPA buffer (20 mM tris, pH7.0, 0.5 mM EDTA, 1% Nonidet P40, 0.5% DOC, 0.05% SDS, 1 mM PMSF, 1 μg/ml aprotinin and 0.5 μ/ml leupeptin) was added. Plates were placed on ice for 30 minutes to complete the lysis. p53 protein was detected in de lysates by using the sandwich ELISA, described below.
High binding polystyrene EIA/RIA 96 well plates (Costar 9018) were coated with the capture antibody pAb1801 (Abeam ab28-100) at a concentration of 1 μg/ml in coating buffer (0.1 M NaHCO3 pH8.2), 50 μl per well. The antibody was allowed to adhere overnight at 4° C. Coated plates were washed once with phosphate-buffered saline (PBS)/0.05% Tween 20 and 300 μl of blocking buffer (PBS, 1% bovine serum albumins (BSA)) was added, for an incubation period of 2 hours at room temperature. Dilutions of purified recombinant HIS tagged p53 protein, ranging from 3-200 ng/ml, were made 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, 20 μl of lysis buffer was added. The samples were added to the other wells at 20 μl lysate/well. After an overnight incubation at 4° C., plates were washed twice with PBS/0.05% Tween 20. Aliquots of 100 μl secondary polyclonal antibody p53(FL-393) (Tebubio, sc-6243) at a concentration of 1 μg/ml in blocking buffer were added to each well and allowed to adhere for 2 hours at room temperature. Plates were washed three times with PBS /0.05% Tween 20. Detection antibody anti-rabbit HRP (sc-2004, Tebubio) at 0.04 μg/ml in PBS/1% BSA was added and incubated for 1 hour at room temperature. Plates were washed three times with PBS/0.05% Tween 20 and 100 μl of substrate buffer was added (substrate buffer was prepared shortly before use by adding 1 tablet of 10 mg o-phenylene diamine (OPD) from Sigma and 125 μl 3% H2O2 to 25 ml OPD buffer: 35 mM citric acid, 66 mM Na2HPO4, pH5.6). After 5 to 10 minutes, colour reaction was stopped by adding 50 μl stop buffer (1 M H2SO4) per well. The absorbance at dual wavelengths of 490/655 nm was measured using a Biorad micro plate reader and the results were then analyzed.
For each experiment, controls (containing no drug) and a blank incubation (containing no cells or drugs) were run in parallel. The blank value was subtracted from all control and sample values. For each sample, the value of p53 (in absorbance units) was expressed as the percentage of the value for p53 present in the control. Percentage preservation higher than 140% was defined as significant. Herein the effects of test compounds are, expressed as the lowest dose giving at least 140% of the value for p53 present in the control (LAD) (see table F-2).
C.2. Proliferation Assay
All compounds tested were dissolved in DMSO and further dilutions were made in culture medium. Final DMSO concentrations never exceeded 0.1% (v/v) in cell proliferation assays. Controls contained U87MG cells and DMSO without compound and blanks contained DMSO but no cells.
U87MG cells were seeded in 96-well cell culture plates at 3000 cells/well/100 μl. 24 hours later, medium was changed and compound and/or solvent were added to a final volume of 200 μl. Following 4 days of incubation, medium was replaced by 200 μl fresh medium and cell growth was assessed using a MTT-based assay. Therefore, 25 μl of the MTT solution (0.5% MTT research grade from Serva in phosphate-buffered saline ) was added to each well and the 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 to each well 25 μl 0.1M glycin and 100 μl DMSO. The plates were shaken for another 10 min on a micro plate shaker before reading absorbance at 540 nm by a Biorad micro plate reader.
Within an experiment, the results for each experimental condition are the mean of 3 replicate wells. For initial screening purposes, compounds were tested at a single fixed concentration of 10−5 M. For active compounds, the experiments were repeated to establish full concentration-response curves. For each experiment, controls (containing no drug) and a blank incubation (containing no cells or drugs) were run in parallel. The blank value was subtracted from all control and sample values. For each sample, the mean value for cell growth (in absorbance units) was expressed as a percentage of the mean value for cell growth of the control. When appropriate, IC50-values (concentration of the drug, needed to reduce cell growth to 50% of the control) were computed using probit analysis for graded data (Finney, D. J., Probit Analyses, 2nd Ed. Chapter 10, Graded Responses, Cambridge University Press, Cambridge 1962). Herein the effects of test compounds are expressed as pIC50 (the negative log value of the IC50-value) (see table F-2).
In some of the experiments the proliferation assay was adapted for 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.














cell
cell




A2780
proliferation
proliferation



Co
p53-elisa
pIC50
pIC50



No
LAD
384 well
96 well
















1
1.0E−05

5.49



2
3.0E−06

5.36



3
3.0E−06

5.15



4
1.0E−06
5.85



5
1.0E−05

5.35



6
1.0E−05
<5










D. Composition Example
Film-Coated Tablets
Preparation of Tablet Core
A mixture of 100 g of a compound of formula (I), 570 g lactose and 200 g starch is mixed well and thereafter humidified with a solution of 5 g sodium dodecyl sulphate and 10 g polyvinyl-pyrrolidone in about 200 ml of water. The wet powder mixture is sieved, dried and sieved again. Then there is added 100 g microcrystalline cellulose and 15 g hydrogenated vegetable oil. The whole is mixed well and compressed into tablets, giving 10.000 tablets, each comprising 10 mg of a compound of formula (I).
Coating
To a solution of 10 g methyl cellulose in 75 ml of denaturated ethanol there is added a solution of 5 g of ethyl cellulose in 150 ml of dichloromethane. Then there are added 75 ml of dichloromethane and 2.5 ml 1,2,3-propanetriol 10 g of polyethylene glycol is molten and dissolved in 75 ml of dichloromethane. The latter solution is added to the former and then there are added 2.5 g of magnesium octadecanoate, 5 g of polyvinyl-pyrrolidone and 30 ml of concentrated colour suspension and the whole is homogenated. The tablet cores are coated with the thus obtained mixture in a coating apparatus.

## Claims

1. A compound of formula (I),



a N-oxide form, an addition salt or a stereochemically isomeric form thereof, wherein
m is 0, 1, or 2 and when m is 0 then a direct bond is intended;
n is 0, 1, 2, 3 or 4 and when n is 0 then a direct bond is intended;
p is 0, or 1 and when p is 0 then a direct bond is intended;
s is 0, or 1 and when s is 0 then a direct bond is intended;
t is 0 or 1 and when t is 0 then a direct bond is intended;
R1 and R2 are each independently hydrogen, halo, C1-6alkyl, C1-6alkyloxy, arylC1-6alkyloxy, heteroarylC1-6alkyloxy, phenylthio, hydroxyC1-6alkylcarbonyl, C1-6alkyl substituted with a substituent selected from amino, aryl and heteroaryl; or C3-7cycloalkyl substituted with a substituent selected from amino, aryl and heteroaryl;
A is a radical selected from













wherein
R4 and R5 are each independently selected from hydrogen, halo, C1-6alkyl, polyhaloC1-6alkyl, cyano, cyanoC1-6alkyl, hydroxyC1-6alkyl, hydroxy, amino, C1-6alkyloxy,
C1-6alkylcarbonyl, methylsulfonylamino, aryl or heteroaryl;

Z is a radical selected from





wherein
R6 or R7 are each independently selected from hydrogen, halo, hydroxy, amino, C1-6alkyl, nitro, polyhaloC1-6alkyl, cyano, cyanoC1-6alkyl, tetrazoloC1-6alkyl, aryl, heteroaryl, arylC1-6alkyl, heteroarylC1-6alkyl, aryl(hydroxy)C1-6alkyl, heteroaryl(hydroxy)C1-6alkyl, arylcarbonyl, heteroarylcarbonyl, C1-6alkylcarbonyl, arylC1-6alkylcarbonyl, heteroarylC1-6alkylcarbonyl, C1-6alkyloxy, C3-7cycloalkylcarbonyl, C3-7cycloalkyl(hydroxy)C1-6alkyl, arylC1-6alkyloxyC1-6alkyl, C1-6alkyloxyC1-6alkyloxyC1-6alkyl, C1-6alkylcarbonyloxyC1-6alkyl, C1-6alkyloxycarbonylC1-6alkyloxyC1-6alkyl, hydroxyC1-6alkyloxyC1-6alkyl, C1-6alkyloxycarbonylC2-6alkenyl C1-6alkyloxyC1-6alkyl, C1-6alkyloxycarbonyl, C1-6alkylcarbonyloxy, aminocarbonyl, hydroxyC1-6alkyl, aminoC1-6alkyl, hydroxycarbonyl, hydroxycarbonylC1-6alkyl and —(CH2)v—(C(═O)r)—(CHR10)u—NR8R9; wherein
v is 0, 1, 2, 3, 4, 5, or 6 and when v is 0 then a direct bond is intended;
r is 0, or 1 and when r is 0 then a direct bond is intended;
u is 0, 1, 2, 3, 4, 5, or 6 and when u is 0 then a direct bond is intended;
R10 is hydrogen or C1-6alkyl;
R8 and R9 are each independently selected from hydrogen, C1-12alkyl, C1-6alkylcarbonyl, C1-6alkylsulfonyl, arylC1-6alkylcarbonyl, C3-7cycloalkyl, C3-7cycloalkylcarbonyl, —(CH2)k—NR11R12, C1-2alkyl substituted with a substituent selected from hydroxy, hydroxycarbonyl, cyano, C1-6alkyloxycarbonyl, C1-6alkyloxy, aryl or heteroaryl; or
C3-7cycloalkyl substituted with a substituent selected from hydroxy, C1-6alkyloxy, aryl, amino, arylC1-6alkyl, heteroaryl or heteroarylC1-6alkyl; or

R8 and R9 together with the nitrogen to which they are attached can optionally form a morpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, or piperazinyl substituted with a substituent selected from C1-6alkyl, arylC1-6alkyl, arylC1-6alkyloxycarbonyl, heteroarylC1-6alkyl, C3-7cycloalkyl and C3-7cycloalkylC1-6alkyl; wherein
k is 0, 1, 2, 3, 4, 5, or 6 and when k is 0 then a direct bond is intended
 R11 and R12 are each independently selected from hydrogen, C1-6alkyl, arylC1-6alkyloxycarbonyl, C3-7cycloalkyl, C1-12alkyl substituted with a substituent selected from hydroxy, C1-6alkyloxy, aryl, and heteroaryl;
 and C3-7cycloalkyl substituted with a substituent selected from hydroxy, C1-6alkyloxy, aryl, arylC1-6alkyl, heteroaryl, and
 heteroarylC1-6alkyl; or
 R11 and R12 together with the nitrogen to which they are attached can optionally form a morpholinyl, a piperazinyl or a piperazinyl substituted with C1-6alkyloxycarbonyl;



aryl is phenyl or naphthalenyl;
each phenyl or naphthalenyl can optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, hydroxyC1-6alkyl, C1-6alkyl, amino, polyhaloC1-6alkyl and C1-6alkyloxy; and
each phenyl or naphthalenyl can optionally be substituted with a bivalent radical selected from methylenedioxy and ethylenedioxy;
heteroaryl is pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl or tetrahydrofuranyl;
each pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl, or tetrahydrofuranyl can optionally be substituted with one, two or three substituents each independently selected from halo, hydroxy, C1-6alkyl, amino, polyhaloC1-6alkyl, aryl, arylC1-6alkyl or C1-6alkyloxy; and
each pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, benzofuranyl, or tetrahydrofuranyl can optionally be substituted with a bivalent radical selected from methylenedioxy or ethylenedioxy.

2. A compound according to claim 1 wherein;
m is 0; n is 0 or 2; p is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-15), (a-21), (a-30), (a-39) or (a-40);
R4 and R5 are each independently selected from hydrogen or C1-6alkyloxy; Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen.

3. A compound according to claim 1 wherein
m is 0; n is 2; p is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-21), (a-39) or (a-40);
R4 and R5 are each independently selected from hydrogen or C1-6alkyloxy;
Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen.

4. A compound selected from the group consisting of:

5. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound as claimed in claim 1.

6. A process of preparing a pharmaceutical composition wherein the pharmaceutically acceptable carriers and a compound as claimed in claim 1 are intimately mixed.

7. A method of treating in a subject a disorder mediated by a p53-MDM2 interaction comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier, wherein the disorder is tumour growth.

8. A combination of an anti-cancer agent and a compound according to claim 1.

9. A process for preparing a compound as claimed in claim 1, said process comprising the steps of:
a) reacting an intermediate of formula (II) with an intermediate of formula (III) wherein W is an appropriate leaving group,




b) converting an intermediate of formula (IV) into compounds of formula (I), wherein p is 1, herein referred to as compounds of formula (I-a), in the presence of lithium aluminum hydride in a suitable solvent,




c) reacting an appropriate carboxaldehyde of formula (VI), with an intermediate of formula (V), in the presence of an appropriate reagent, in a suitable solvent,




d) reacting an intermediate of formula (II) with an appropriate carboxaldehyde of formula (VII) with the formation of a compound of formula (I), wherein t is 1, herein referred to as compounds of formula (I-b), or




e) reacting an intermediate of formula (VIII) with lithium aluminum hydride in a suitable solvent, with the formation of a compound of formula (I), wherein s is 1, herein referred to as compounds of formula (I-c).

10. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound as claimed in claim 4.

11. A process of preparing a pharmaceutical composition wherein the pharmaceutically acceptable carriers and a compound as claimed in claim 4 are intimately mixed.

12. A method of treating in a subject a disorder mediated by a p53-MDM2 interaction comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound of claim 4 and a pharmaceutically acceptable carrier, wherein the disorder is tumour growth.

13. A combination of an anti-cancer agent and a compound according to claim 4.

14. A compound according to claim 2 wherein
m is 0; n is 2; p is 1; s is 0; t is 0; R1 and R2 are each independently hydrogen; A is a radical selected from (a-21), (a-39) or (a-40);
R4 and R5 are each independently selected from hydrogen or C1-6alkyloxy;
Z is the radical (b-2); or R6 and R7 are each independently selected from hydrogen.

15. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound as claimed in claim 14.

16. A process of preparing a pharmaceutical composition wherein the pharmaceutically acceptable carriers and a compound as claimed in claim 14 are intimately mixed.

17. A method of treating in a subject a disorder mediated by a p53-MDM2 interaction comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound of claim 14 and a pharmaceutically acceptable carrier, wherein the disorder is tumour growth.