CZ110599A3 - Medicament exhibiting MDM2 antagonistic activity, derivatives of phenoxyacetic acid and phenoxymethyltetrazol and pharmaceutical composition - Google Patents

Abstract

The use of phenoxyacetic acid derivatives a of phenoxymethyltetrazole of formula I wherein -O-C (R1) (R2HCH2) p-A it can be in ortho, meta or para position; A is -COOH, COO- (C 1-4) alkyl, -CN or tetrazolyl, optionally substituted (C i.4) alkyl; or A- (CH 2) p -C (R 1) (R 2) - is phenyl, benzyl or (indolyl) methyl, optionally substituted with R 4 groups; p is 0,1 or 2; R1 and R2 are each hydrogen or (C μ 4) alkyl or together with the carbon atom to which it is attached a (C 3-7) cycloalkyl group is attached; 0 to 2 substituents independently selected from chloro, bromo, iodine, fluorine, (C 1-4) alkyl-, hydroxy-, (C 1-4) alkoxy-, or (C μ 4) acyl groups; or a benzene ring to which it is attached R 4, is optionally substituted naphthyl, n is an integer from 1 to 4; m is 0 or 1; B is C 1-10 alkyl, -COC (R 3) = CFI-R, -CH = C (R 3) -CO-Ar, -CO-CH (R 3) -CH 2 -R 10 or -CH 2 -R 11; -CO-C (R 3) -CH 2 -NR 5 R 6, when m = nebo, or is -CH = C (R 3) -COAr, if m = 1; -R is hydrogen, -Ar, or -CO-Ar; -R 3 is hydrogen or (C 1-8) alkyl; -R 5 and R 6 are independently (C 1-4) alkyl or together with the nitrogen to which they are attached are attached, piperidine, piperazine, (C μ8) alkylpiperazine, morpholino or thiomorpholine a group; -Ar is optionally substituted phenyl; stereoisomers or salts with pharmaceutically acceptable salts acids or bases for the preparation of a drug having MDM2 antagonistic activity. New Compounds of Formula I a pharmaceutical compositions comprising at least one of these compounds.

Description

Medicament with MDM2 antagonistic activity, phenoxyacetic acid derivatives and phenoxymethyltetrazole and pharmaceutical composition

Technical field

The invention relates to the use of phenoxyacetic acid and phenoxymethyltetrazole derivatives for the manufacture of a medicament having MDM2 antagonist activity, certain phenoxyacetic acid derivatives and phenoxymethyltetrazole, and pharmaceutical compositions based thereon. These compounds exhibit anti-tumor properties and, in particular, act as antagonists of the interaction of MDM2 with p53, resulting in an intracellular increase in active p53, thus allowing the p53 protein to promote apoptosis in tumor cells.

BACKGROUND OF THE INVENTION

The proto-oncoprotein MDM2 (double minute 2 mouse, human equivalent protein is sometimes referred to as HDM2) is differentially expressed in a number of human tumors (Oliner et al. 1992, Nature 358, 80-83; Leach et al. 1993, Cancer Res. 54 Ebert et al 1994, Int J.Oncol.5, 1279-1284, Bueso-Ramos et al 1993, Blood 82, 2617-2623, Chilosi et al 1994, Blood 84, 4295-4300; et al.

1995, Diagn. Mol. Pathol. 4, 93-97; McCann et al. 1995, Brit. J. Cancer. 71, 981985; Cordon-Cardo et al. 1994, Cancer Res. 54, 794-799). MDM2 forms a negative anti-regulatory loop with p53 binding to its N-terminal domain (Kussie et al.

1996, Science 274, 948-953), thereby inhibiting p53 function (Momand et al 1992, Cell 69, 1237-1245; Oliner et al 1993, Nature 362, 857-860; Barak et al 1992, EMBO J. 11 Finlay 1993, Mol Cell Biol 13, 301-306, Chen et al 1996, Mol Cell Biol 16, 2445-2452, Haupt et al 1996, EMBO J. 15, 1596- 1606) and promotes proteolithic degradation of p53 (Kubbutat et al 1997, Nature 387, 299-302; Haupt et al 1997, Nature 387, 296-299; Midgley et al 1997, Oncogene 15, 1179-1189). Therefore, interference with this autoregulatory loop between MDM2 and p53 may be 9 99 99 9 9 9 9 999 999

9

9999 9 999 999 99 99 used to increase the concentration of active p53 in mammalian cells. Tumor cells in many cases have depleted p53, resulting in a defective cell cycle and regulation of apoptosis. However, only a portion of the tumor cells carry mutation defects in p53. For the remainder of the wild-type p53 tumor cells, the steady-state concentration of active p53 can be increased by preventing the interaction of MDM2 and p53. A peptide model antagonist of the MDM2-p53 interaction has been used to restore p53 activity both in vitro (Böttger et al. 1997, J. Mol. Biol. 269, 744-756) and in mammalian cells (Böttger et al. 1997, Current Biology 7 , 860-869). Inhibition of MDM2 expression

Synthetic nucleic acids have been shown to increase the amount of active p53 and thereby increase the sensitivity of cell lines to cytostatics (Chen et al. 1998). Proc, Natl Acad Sci USA 95, 195-200). Lack of functional p53 is the most common molecular defect correlating with tumor resistance to chemo- and radiotherapy. Control failure

MDM2 was considered to be the cause of this phenomenon (Kondo et al. 1995, Oncogene 10, 2001-2005, Blaydes et al. 1997, Oncogene 14, 1859-1868). Therefore, agents that increase the intracellular concentration of active p53 in tumor cells by preventing the interaction of MDM2 with p53 have therapeutic use in sensitizing tumor cells to chemo- and radiotherapy. In tumor types particularly sensitive to elevated functional p53 (Hansen et al 1995, Oncogene 11, 2535-2545), agents of this type alone are sufficient to induce apoptosis.

In addition to the effects mediated by increasing the intracellular concentration of active p53, MDM2 antagonists are able to influence cell cycle regulation of non-p53 mammalian cells. Transcription from E2F-dependent promoters is activated by mdm2, which interacts with E2F at the same binding site with a homologous epitope as p53 (Piette et al. 1997, Oncogene 15, 1001-1010, Brown et al. 1993, Mol. Cell. Biol. 13, 6849-6857). MDM2 therefore promotes general proliferation by increasing the passage of the S-phase (Leveillard and Wasylyk 1997, J. Biol. Chem. 272, 3065120 60661), increasing the expression of angiogenic mitogens (Kondo et al 1996, Oncogene 13,

1773-1779) and inhibits differentiation (Fiddler et al. 1996, Mol. Cell. Bilol. 16, 50485057). MDM2 inhibitors will therefore be therapeutically useful in MDM2 disregulated tumors independent of their p53 status.

It has now been found that derivatives of O-substituted phenol which carry an acidic group attached through an alkylene chain to phenol oxygen are effective antagonists of MDM2 activity, in particular antagonists of MDM2-p53 interaction.

Chalcone derivatives with a carboxymethoxy substituent on one of the two phenyl rings have anti-ulcer effects according to literature (Pol. J. Chem, vol 65 (2-3),

-2 · · · · · · •

369-75, 1991; DE 3537207, Biorex; Chem. Pharm. Bull. Vol. 27 (12), 2943-53 (1997); FR 2383157, Biorex; JP 54019948, Taisho), bactericidal activity (Pharmacie, vol. 44 (3), 190-1, 1989; Hacettepe Univ. Eczacilik Fak. Derg. Vol. 11 (1), 1-11, 1991), hypolipaemic activity ( FR 2639043, US 3,994,955, Searle, Taiwan Yao Hsueh Tsa Chih, vol 27 (1-2), 12-16, 1975), diuretic activity (Eur. J. Med. Chem.-Chim. Ther., Vol. 16 (6), 551-5 and 556-62, 1981; and BE 639727), pesticidal activity (CA 904291, Dow Chem.), Or reduce blood fibrinogen levels (DE 4327365, Boehrnger Mannheim). To date, no anti-tumor activity has been reported.

The chalcone derivatives with antitumor activity described in the prior art do not carry a carboxymethoxy or tetrazolylmethoxy group on the phenyl ring and are known to act as antitubulinics (US 4,904,697, Merrell Dow).

SUMMARY OF THE INVENTION

The present invention provides the use of compounds of formula (I):

where:

the -OC (R 1) (R 2) - (CH 2) _p -A group may be in the ortho, meta or para position;

- A is selected from COOH, -COO- (C 1 -C 4) alkyl, -CN or a group of the formula

R ’

NN ································· Wherein R 'is hydrogen or (C 1 -C ₄ ) alkyl;

or the group A- (CH ₂ ) _p -C (R 1) (R 2) - is selected from phenyl, benzyl or (indolyl) methyl group, which may be substituted with R 4 groups;

-p is 0, 1 or 2;

R 1 and R 2 are independently selected from hydrogen or (C 1 -C 8) alkyl, or may form together with the carbon atom to which they are attached a (C 3 -C 7) cycloalkyl group;

R 4 are from 0 to 2 substituents independently selected from chloro, bromo, iodo, fluoro, linear or branched (C 1 -C ₈ ) alkyl, hydroxy, (C 1 -C ₄ ) alkoxy, (C 1 -C ₄ ) acyl groups;

- or group

in formula (I) is a naphthyl group which may be substituted with R 4 groups;

- n is an integer from 1 to 4,

- m is 0 or 1

- B is selected from linear or branched C 1 -C 10 alkyl, -CO-C (R 3) = CH-R, CH = C (R 3) -CO-Ar, -CO-CH (R 3) -CH ₂ -R, or

-CO-C (R 3) -CH ₂ -NR 5 R 6 when m = 0, or -CH = CH (R 3) -CO-Ar when m = 1;

-4 · · · · · · · · · · · · · · · · · · · · · φ ·

R is selected from hydrogen, Ar or -CO-Ar;

- R 3 is hydrogen or (C 1 -C 8) alkyl;

- R 5 and R 6 are independently a (C 1 -C ₄ ) alkyl group or form, together with the nitrogen atom to which they are attached, a piperidine, piperazine, (C 1 -C 8) alkylpiperazine, morpholine or thiomorpholine group,

-Ar is a phenyl group which may be unsubstituted or substituted with from 1 to 3 groups independently selected from chlorine, bromine, iodine, fluorine, linear or branched (C 1 -C 8) alkyl, hydroxy, (C 1 -C ₄ ) alkoxy, ( C 1 -C ₄ ) acyl groups, stereoisomers of these groups, or salts thereof with pharmaceutically acceptable acids or bases, for the preparation of medicaments having MDM2 antagonist activity, preferably for the treatment of MDM2 disregulated tumors, for example sarcoma.

Another object of the present invention are novel compounds of formula (I) as defined above, with the proviso that when m - 0, A is selected from -COO- (C 1 -C ₄ ) alkyl, -CN or a group of the formula

FV

NN wherein R 'is hydrogen or (C 1 -C ₄ ) alkyl; and when m = 0 and A is -COO- (C 1 -C ₄ ) alkyl, then B can only be a group having the structure -CO-C (R 3) -CH ₂ -NR 5 R 6.

Preferred compounds of formula (I) are those wherein the -OC (R 1) (R 2) - (CH ₂ ) _p -A group is in the para position.

- 5 ·· · 9 · 9

9 9 9 9 9

Particularly preferred are compounds of formula (I) wherein R4 is formed from 1 to 2 chlorine atoms or wherein Ar is phenyl substituted with 1 to 2 chlorine atoms.

Even more preferred are compounds of formula (I) wherein m is 0, R is hydrogen and A is 5 tetrazole.

The most preferred compounds of formula (I) are:

-6 ··········

9 9 9 9 9 9 9 9 9 999

9 9 9 9 9

PREPARATION OF COMPOUNDS OF THE INVENTION

Compounds of formula (I) wherein m is 0 and B is a linear or branched C 1 -C ₁₀ alkyl, COCH ₃ , -CO-C (R 3) = CH-R or -CH = C (R 3) -CO-Ar group, may be prepared from intermediates of formula (II):

wherein R 4 is as defined above and B 'is a linear or branched C 1 -C 10 alkyl, COCH 3, -CO-C (R 3) = CH-R or -CH = C (R 3) -CO-Ar group, by reaction with an ester of formula ( III):

Hal-C (R 1) (R 2) - (CH ₂ ) _p -COO- (C 1 -C 4) alkyl (III) or with a nitrile of formula (IIP):

Hal-C (R 1) (R 2) - (CH ₂ ) _p -CN (IIP) wherein R 1, R 2 and _p are as defined above and Hal is a chlorine, bromine or iodine atom, or with a group of formula (III):

Aryl-Hal

-7 • 999 · • 9

• MM

99

9 9 9 9 • 9 9 9 • 999 · Μ

Wherein Hal is a chlorine, bromine or iodine atom and the aryl is selected from benzyl, (indolyl) methyl and phenyl activated by a group that can be simply removed after the reaction, such as a chromium (O) -tricarbonyl group.

The reaction of the intermediates of formula (II) with the intermediates of formula (III) or (III ') may be carried out in a solvent, preferably an aprotic dipolar solvent such as dimethylformamide or dimethylsulfoxide, and in the presence of a base such as an alkali or alkaline earth metal carbonate. The reaction temperature is preferably from room temperature to 100 ° C.

The product of the reaction of intermediates of formula (II) wherein B 'is not COCH ₃ , with intermediates of formula (III) is already a compound of formula (I) and can be converted to another compound of formula (I) with A = - COOOH by hydrolysis of the ester group. Such hydrolysis may preferably be carried out in the presence of a base such as an alkali metal or alkaline earth metal carbonate or hydroxide in a solvent such as an alcohol.

The reaction product of intermediates (II) wherein B ¹ is not COCH ₃ , with intermediates (III ¹ ) is converted to the compound of formula (I) in which A is the tetrazole group shown above, by reaction with sodium azide in a solvent such as dimethylformamide a nitrogen atom at the 1 or 2 position of the tetrazole ring using a suitable alkylating agent such as dimethyl sulfate in the presence of a base.

Alternatively, the compound of formula (I) wherein A is a tetrazole group as defined above, m is 0 and B is -CO-C (R 3) = CH-R, where R 3 is H, can be obtained in a three-step process involving a) reaction the intermediates (II) mentioned above, wherein B 'is COCH ₃ with the intermediates of formula (IIP); b) reacting the compound obtained in step a) with sodium azide in a solvent such as dimethylformamide and optimally alkylating the nitrogen atom at the 1 or 2 position of the tetrazole ring with a suitable alkylating agent such as dimethyl sulfate in the presence of a base; and c) reacting the compound obtained in step b) with the aromatic aldehyde R-CHO in an inert solvent and in the presence of a base such as

-8111 · 11 11 1111 · 1 11

111 11 1 1112

110 19 1 1119

111 1 111 111 111

1119 9 111 9 1 alkali metal or alkaline earth metal hydroxide, at temperatures ranging from room temperature to reflux.

Alternatively, compounds of formula (I) wherein A is a tetrazole group, m is 0 and B is -CO-C (R 3) -CH ₂ , i.e. compounds wherein R is hydrogen, can be obtained from compounds of formula (ΙΓ):

wherein A is the tetrazole group shown above and R1, R2, R3, R4 and p are as defined above, by reaction with paraformaldehyde. Reaction of structure (ΙΓ) with paraformaldehyde and an amine salt hydrochloride of formula HNR5R6, wherein R5 and R6 are as defined above under Mannich conditions, and subsequent treatment with a weak base such as an alkali metal or alkaline earth metal bicarbonate, yields a compound of formula ( I) where

B '= -CO-C (R 3) -CH ₂ -NR 5 R 6, as the hydrochloride.

A compound of formula (II) wherein B 'is a group of formula -CO-C (R 3) = CH-R with R = Ar or R = -CO-Ar may be obtained from a compound of formula (IV):

(IV) reaction with an aldehyde of formula Ar-CHO or Ar-CO-CHO in an inert solvent and in the presence of a base such as an alkali metal or alkaline earth metal hydroxide at temperatures ranging from room temperature to reflux.

-999 99

999 99 9,999

999 99 9,999

999 99 99 999 999

9999 9999 9 9 • 999

9 «9 9 9 9

Analogously, a compound of formula (II) in which B 'is a group of formula Ar-CO-C (R 3) = CH, can be obtained from a compound of formula Ar-CO-CH ₂ -R ₃ with an aldehyde of formula (IV):

(IV ’)

Compounds of formula (IV), (IV '), II with B' C _C-I0 alkyl, Ar-CHO, Ar-CO-CHO and Ar-CO-CH ₂ -R3 are known compounds which may be prepared according to methods well known to the skilled chemist or are even commercially available.

Compounds of formula (II ') wherein A is a tetrazole group can be prepared from compounds of formula (IV) by reaction with compounds of formula (IIP) followed by sodium azide, as shown above.

Compounds of formula (I) wherein m = O and B is -CO-C (R 3) -CH ₂ -R may be prepared from compounds of formula (II) wherein B 'is -CO-C (R 3) = CHR , catalytic hydrogenation of the double bond and subsequent reaction with intermediates of formula (III) or (ΙΙΓ) as shown above. The compound of formula (I) obtained by reaction with intermediate (IIP) can be optimally converted to another compound of formula I, wherein A is a tetrazole group, by reaction with sodium azide as mentioned above.

Compounds of formula (I) wherein m is 1 and B is -CH = C (R 3) CO-Ar may be obtained from a compound of formula (V):

(CH ₂ ) n NH ₂ (V)

-10 ···· «0 (· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Where ρ, η, R1, R2 and R4 have the above meanings and A 'has the meaning of A except for COOH, by reaction with an intermediate of formula (VI):

Ar-CO-C (R 3) = CH-COOH (VI)

Compounds wherein B is -CO-C (R 3) = CH-CR or -CO-C (R 3) -CH 2 -R can be prepared in an analogous manner. Such a reaction is accomplished by activating the carboxyl group of compounds (VI), for example, through a mixed anhydride or acyl chloride, or by the presence of a suitable condensing agent such as dicyclohexyl carbodiimide. The compounds thus obtained are converted to other compounds of formula (I) by hydrolysis of the ester group under basic conditions when A '= -COO- (C 1 -C ₄ ) alkyl, or by reaction with an azide when A' = -CN, according to the procedures described above .

Compounds of formula (V) may be prepared by acid-catalyzed esterification of the corresponding acids (where A 1 = -COOH), which may be prepared again by the method described in Synthesis (1997), 778-782, which is incorporated herein by reference. Suitable esterification conditions may be methanol in the presence of sulfuric acid in an amount sufficient to protonate the amino groups and to catalyze the esterification reaction. Subsequent treatment with a weak base may release the amino groups.

Compounds of formula (VI) may be prepared by the method described in Am. Soc. 70, 3359 (1948), which is incorporated herein by reference.

BIOLOGICAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION

The compounds of the invention interact with the MDM2 protein, in particular the human MDM2 protein, and inhibit the interaction of MDM2 with other proteins, in particular the interaction of MDM2 with p53. MDM2 has different functions, the main one being the control of p53 activity during the cell cycle (processed by Piett et al. 1997, Oncogene 15, 1001-1010).

-11 ·

4444 44 44 4444 44 44

444 44 4444 4444 44 44 4444

444 44 444 444

4444 4444

MDM2 proteins form a hydrophobic pocket in their amino-terminal domain, occupied by the peptide epitope present at the amino terminus of p53 (Kussie et al. 1996, Science 274, 948-953). This interaction between the N-terminus of p53 and the N-terminal domain of MDM2 is a key prerequisite for enabling control of p53 by MDM2. Compounds binding to the hydrophobic pocket of the N-terminal domain of MDM2 therefore act as antagonists of MDM2 mediated inhibition and degradation of p53. By this mechanism, the level of active p53 can be increased, causing, in particular, in p53-sensitive tumor cells, mediated induction of apoptosis and cell cycle arrest Only peptides and proteins capable of this mode of intervention have been available to date (Böttger et al 1997, J. Mol. Biol. 269 744-756; Current Biology 7, 860869). The compounds of the invention now provide the first low molecular weight chemical entities capable of disrupting the MDM2-p53 interaction.

In addition, the compounds of the invention are capable of inhibiting the interaction of MDM2 by interacting its N-terminal domain with other proteins having a homologous interaction site such as E2F-1. The compounds of the present invention are therefore capable of exerting antiproliferative or sensitizing effects on tumor cells, regardless of the p53 tumor cell status (Example 15).

Furthermore, the compounds of the present invention are particularly specific for the interaction with MDM2. Within mammalian cells, there are several proteins with hydrophobic pockets with the ability to host compounds or hydrophobic residues. An example of such a protein is glutathione S-transferase (GST) (Reinemer et al 1992, J. Mol. Biol. 227, 214-226, Cameron et al., Structure 3, 717-727; McTígue et al 1995, J. Mol. Biol 246, 2125 27). In the present invention, it has been observed that certain compounds are capable of interacting with both MDM2 and GST. An example of such a substance is ethacrynic acid, which was previously described as a GST inhibitor that binds to the hydrophobic pocket of this protein (Oakley et al. 1997, Biochemistry 36, 576-585; Ploemen et al 1993, Xenobiotics 23, 913-923). Surprisingly, an interaction of ethacrylic acid with MDM2 has been observed in the present invention. The present invention therefore provides assays

-12 ·· φ »» φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ MD aktivit MD MD MD ST ST ST ST The present invention further provides technology to identify compounds with a high binding affinity for MDM2 and a low or preferably absent affinity for GST. Compounds with a high MDM2-p53 interaction inhibitory activity and a comparatively low or no GST inhibitory activity are particularly preferred agents for inducing a therapeutic anti-tumor effect based on MDM2 inhibition because many tumors during the chemotherapy cycle produce resistant tumor cell populations, which in many examples have elevated levels of GST (Chen and Waxman 1994, Biochem. Pharmacol. 47, 1079-1087; Pidkett and Lu 1989,

Annu. Roar. Biochem. 58, 743-764). Therefore, the interaction of compounds with both MDM2 and GST results in the exhaustion of a compound inhibiting MDM2 due to competition. A compound of this type is, for example, LSM 83177 (the compound in Example 12, also called substance E), for which it has been discovered in the present invention to be a potent sensitizing agent for tumor cells regardless of their GST status (Example 15).

The chemical structure of LSM 83177 is:

In addition, low or no GST inhibitory activity is a desired property of a therapeutically useful MDM2 antagonist since side effects such as diuresis, hyperglycemia and hypercalcemia may be associated with GST inhibition (O'Dwyer et al. 1991, Cancer

Res. 51, 6059-6065; Oakley et al. 1997, Biochemistry 36, 576-585).

The following Examples 12-15 illustrate how the biological activity of the compounds of the present invention can be determined.

-13 «Α Μ <· ·» 9 ·· • · 9

9 9 • · ·

9 9 • 9 99

9 9

9 ·

9 9 9

9 9 9

9 9 9

9 * 99

9 9 9 • 9 A Α 99 999

9

99

The compounds of the present invention may be administered in doses of from 0.01 mg to 0.4 g per kilogram of body weight per day. A preferred dosage regimen for best results is one that provides the use of about 1 mg to 50 mg per kilogram of body weight per day, using the same dosages to administer from about 70 mg to 3.5 g of the active compound to patients in 24 hours. with an approximate weight of 70 kg. The dosage regimen can be adjusted to achieve the best therapeutic effects. For example, dosages may be administered taking into account the therapeutic situation of the patient. The active compound may be administered orally, intravenously, intramuscularly or subcutaneously.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of at least one compound in admixture with pharmaceutically compatible carriers.

Oral compositions generally comprise an inert diluent and an edible carrier. They may be placed in gelatin capsules or compressed into tablets. Other oral formulations are capsules, pills, elixirs, suspensions, or syrups.

Tablets, pills, capsules and the like may contain the following ingredients (in addition to the active ingredient); a binder such as microcrystalline cellulose, tragacanth or gelatin; carriers such as starch or lactose; disintegrating agents such as alginic acid, primogel, corn starch and the like; a lubricant such as magnesium stearate; a plasticizer such as silica; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as mint essence, methyl salicylate, or orange essence. If the selected composition is in the form of capsules, it may contain an additional liquid carrier such as a fatty oil. Other compositions may contain various materials that alter their physical form, for example, coating agents (for tablets or pills) such as sugar or shellac. The material used to prepare the composition should be pharmaceutically pure and non-toxic in the dosages used.

-14> ··· · <

0 0

0 · 0

0 0 0

0 0 ·

00 * 0 0000

0 0

0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0

000 000

0> 0 00

For the preparation of pharmaceutical compositions for parenteral administration, the active ingredient should be contained in a solution or suspension, which may additionally comprise the following components; a sterile diluent such as water for injection, saline, oil, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents;

antibacterial agents such as benzyl alcohol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminotetraacetic acid; buffers such as acetates, citrates or phosphates for adjusting the tone of the solution, such as sodium chloride or dextrose. The parenteral preparation may be contained in ampoules, disposable syringes, glass or plastic vials.

It is a further object of the present invention to provide pharmaceutical compositions comprising at least one compound of the present invention in admixture with pharmaceutical carriers.

CHEMICAL EXPERIMENTAL PART

The present invention is further illustrated by the following preparations and examples.

The numbering of the positions on the chalkon circle is as follows:

Preparation 1: 4'-hydroxy-3-chlorochalcone 30

• ·

A solution of 1.36 g of 4-hydroxyacetophenone in 14 ml of ethanol is added at room temperature to 0.64 g of lithium hydroxide monohydrate, followed by the addition of 6 ml of ethanol to facilitate stirring. 1.17 ml of 3-chlorobenzaldehyde is added and the reaction mixture is refluxed for 7 hours. The solvent is evaporated under vacuum and the residue is dissolved in 15 ml of water. The solution is cooled to 0 ° C and 15 ml of 1 N hydrochloric acid is added. A yellow solid precipitated, which was stirred for 1 hour, filtered and dried in vacuo overnight. The solid is crystallized from 2.5 mL of 96% ethanol to give 0.41 g of product, m.p. Mp 163-165 ° C.

1 H-NMR in d 6 -DMSO: 6.90 ppm (d, 2H); 7.4 ppm (m, H); 7.6 ppm (d, 1H); 7.8 ppm (m, 1H); 8 ppm (d, 1H); 8.05 ppm (m, 1H); 8.1 ppm (d, 2H); 10.45 ppm (s, 1H).

Preparation 2: 3 ', 4'-dichloro-4-hydroxychalcone

A solution of 4-hydroxybenzaldehyde (1.22 g) in 20 ml of ethanol is added at room temperature to 0.63 g of lithium hydroxide monohydrate and 1.89 g of 3,4-dichloroacetophenone, refluxed for 6 hours (after 4 hours a deep red solid separates and stirred at room temperature overnight. The solid is filtered off and the mother liquors are concentrated to dryness, the residue is dissolved in a mixture of ethyl acetate and 1 N hydrochloric acid. The organic phase is separated, washed with brine, dried over sodium sulfate and concentrated to dryness. The residue (1.4 g) is dissolved in 30 ml of water, ethyl acetate and 1 N hydrochloric acid to acidic pH. After stirring for 30 minutes, the organic phase is separated, washed twice with brine, dried over sodium sulfate and concentrated to dryness. The residue is crystallized from ethyl acetate (30 mL) at reflux to give 0.946 g of the product as a yellow solid, m.p. Mp 198-199 ° C.

1 H-NMR in d 6 -DMSO: 6.85 ppm (d, 2H); 7.7-7.9 ppm (m, 5H); 8.1 ppm (dd, 1H); 8.4 ppm (d, 1H); 10.15 ppm (s, 1H).

Preparation 3: 3. 4-Dichloro-4'-hydroxychalcone

-16 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · ·

A solution of 4-hydroxyacetophenone (5.45 g) in 60 ml of ethanol is added to 3.36 g of lithium hydroxide monohydrate and 7 g of 3,4-dichlorobenzaldehyde and refluxed for 2 hours. An additional 3 g of 3,4-dichlorobenzaldehyde are added and the reaction mixture is refluxed for a further 2 hours and left at room temperature overnight. The solid obtained by filtration is dissolved in 50 ml of water and 50 ml of 1N hydrochloric acid. The resulting yellow solid is separated and stirred for 1 hour, collected by filtration and dried under vacuum at 40 ° C for several hours to give 7.3 g of product which is crystallized from a mixture of ethyl acetate (90 ml) and isopropanol (5 ml). 1.3 g of product are obtained. An additional 2.4 g is obtained by purification to dry concentrated concentrated solutions by silica gel column chromatography, m.p. Mp = 190-192 ° C.

1 H-NMR in d 6 -DMSO: 6.9 ppm (d, 2H); 7.65 ppm (d, 5H); 7.7 ppm (d, 1H); 7.8 ppm (d, 1H); 8.05 ppm (s, 1 H); 8.1 ppm (d, 2H); 8.3 ppm (s, 1H); 10.4 ppm (s, 1H).

Preparation 4: 3,4-dichloro-4'-hydroxy-dihydrochalcone

A solution of 0.1 g of 3,4-dichloro-4'-hydroxychalcone in 10 ml of ethanol and 3 ml of dioxane is added to 0.14 g of 10% palladium on charcoal and hydrogenated for 1 hour 15 minutes.

The catalyst is filtered off through celite and the reaction mixture is concentrated to dryness. The residue was crystallized from diethyl ether to give 0.124 g of product, m.p. Mp 128-130 ° C. An additional 0.221 g of product is obtained by purifying the mother liquors concentrated to dryness by silica gel chromatography (eluent petroleum ether / ethyl acetate 8: 2).

Preparation 5: 2,3-dichloro-4-butyrylphenol

144 g of 2,3-dichloroanisole are dissolved in 288 ml of carbon disulfide and added to 92.3 g of butyryl chloride. While stirring and cooling with ice, 115 g of aluminum chloride are added in portions, keeping the temperature below 25 ° C. The reaction mixture is allowed to stand at room temperature for 1 hour, then heated at 45 ° C for 45 min. After addition of 280 ml of n-heptane a

- 17 • · · · φ f

* 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 * 9 e 9 · 9 »9» The reaction mixture is allowed to react through 115 g of aluminum chloride. overnight, the carbon disulfide is then distilled off and 200 ml of n-heptane is added dropwise. The separated solid is heated with stirring at 80-90 ° C for 3 hours and left at room temperature overnight. The solid is collected by filtration, treated with 86 mL concentrated hydrochloric acid and 1 L water. The mixture is extracted 3 times with diethyl ether and the organic extracts are combined and washed with water and 750 mL of 5% sodium hydroxide. The extracts are then treated with concentrated hydrochloric acid and the separated oil crystallizes on cooling. 114.7 g of product are obtained.

Preparation 6: (2,3-dichloro-4-butyrylphenoxy) acetonitrile g of 2,3-dichloro-4-butyrylphenol is mixed with 26.7 g of potassium carbonate and 16 g of chloroacetonitrile in 190 ml of dimethylsulfoxide and the mixture is heated with stirring at 85 ° C 2 hours 30 minutes. The reaction mixture is then quenched with 490 g of ice. The separated oil is extracted 4 times with diethyl ether and the organic phase is treated with 400 ml of 5% sodium hydroxide and washed with water. The organic phase is then dried over magnesium sulfate and concentrated to dryness to give 45.2 g of an oil which, after distillation at 188 ° C and 0.8 mm Hg, yields 40 g of product.

Preparation 7: 5- ((2,3-dichloro-4-butyrylphenoxy) methyltetrazole g of (2,3-dichloro-4-butyrylphenoxy) acetonitrile is mixed with 11.5 g of sodium azide and 9.5 g of ammonium chloride in 294 The reaction mixture is heated with stirring at 120-130 ° C for 30 min, the solvent is then evaporated under reduced pressure (80 ° C) and the residue is treated with stirring with 1.3 L of water. 44 g of crude product is obtained, which is crystallized from a mixture of 400 ml of methanol and 200 ml of water to give 34.8 g of the product, mp 135-137 ° C.

Elementary analysis. (% calculated / found): C 45.73 / 45.43; H 3.84 / 3.73; N

17.78 / 17.32; Cl 22.50 / 22.56.

-18 · · 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 »»

Preparation 8: 5 - ((((2,3-dichloro-4-butyryl) phenoxy) methyl) -1-methyltetrazole and 5 - (((2,3-dichloro-4-butyryl) phenoxy) methyl) -2-methyltetrazole

8.53 g of 5 - [((2,3-dichloro-4-butyryl) phenoxy) methyl] tetrazole is dissolved in 120 ml of water and 480 ml of acetone. 31.8 g of sodium carbonate is added, then 52.8 g of dimethyl sulfate are added dropwise and the reaction mixture is allowed to react for 20 hours. The mixture is then poured into water and the organic solvent is evaporated under reduced pressure at 90 ° C. An oil is separated from the aqueous phase which crystallizes upon cooling. The solid is filtered and dried to give 7.93 g of a 1: 1 mixture of the above tetrazoles. The solid is recrystallized from 1: 1 benzene / cyclohexane to give 2.55 g of 5 - (((2,3-dichloro-4-butyryl) phenoxy) methyl] -1-methyltetrazole. The filtrate is concentrated to dryness and the residue is crystallized from cyclohexane to give 3.5 g of 5 - (((2,3-dichloro-4-butyryl) phenoxy) methyl] 2-methyltetrazole.

Preparation 9: 4- (cyanomethoxy) acetophenone

To a solution of 4-hydroxyacetophenone (0.272 g) in DMF (8 mL, dried over molecular sieves) was added 2-chloroacetonitrile (0.164 mL) followed by potassium carbonate (0.636 g) and the resulting mixture was heated at 60 ° C for 1 hour. After cooling to room temperature and diluting with water (40 mL), the solution was acidified with HCl to pH 4 and extracted with ethyl acetate. The organic layer was washed once with brine, dried over sodium sulfate and evaporated to dryness to give

4- (cyanomethoxy) acetophenone as a dark solid (0.354 g). This material was used for the next step.

Preparation 10: 4- (5-Tetrazolylmethoxy) acetophenone

Sodium azide (0.165 g) and aluminum chloride (0.107 g) were added to a solution of 430 (cyanmethoxy) acetophenone (0.349 g) in DMF (5 mL, dried over molecular sieves) and

-19 The mixture obtained was heated to 70 ° C. 2.5 hours. The mixture was cooled to 0 ° C and treated with 1N HCl (50 mL). After stirring at 0 ° C for 0.5 hour, the yellow precipitate was filtered off and dried under vacuum at 40 ° C overnight to give 4- (5-tetrazolylmethoxy) acetophenone (0.32 g) as a brownish solid. This material was used for the next step.

1 H-NMR in d 6 -DMSO: 2.45 ppm (s, overlaps with DMSO signal); 5.6 ppm (s, 2H); 7.15 ppm (d, 2 H); 8.0 ppm (d, 2 H); 16.9 ppm (brs, 1H).

Example 1: 4 '- (ethoxycarbonylmethoxy) -3-chlorochalcone

A solution of 4-hydroxy-3-chlorochalcone (1.3 g) in 24 ml of anhydrous DMF is added at room temperature and under a nitrogen atmosphere to 1.74 g of potassium carbonate and 0.84 ml of ethyl bromoacetate. The reaction mixture is heated at 60 ° C for 1 hour 30 minutes, diluted with 100 mL of water and extracted with ethyl acetate (3x20 mL). The organic extracts are combined and washed with brine, the organic phase is dried over sodium sulfate and concentrated to dryness to give a residue (1.83 g) which is purified by silica gel chromatography (eluent petroleum ether / ethyl acetate 7.5: 2.5). 0.66 g of product is obtained, m.p. 68-70 [deg.] C.

1 H-NMR in CDCl ₃ : 1.39 ppm (t, 3H); 4.3 ppm (q, 2 H); 4.75 ppm (s, 2 H); 7.0 ppm (d, 2 H); 7.43 ppm (s, 2 H); 7.5 ppm (m, 1 H); 7.55 ppm (d, 1 H); 7.6 ppm (m, 1 H); 7.7 ppm (d, 1 H); 8.0 ppm (d, 2 H).

Example 2: 4 '- (carboxymethoxy) -3-chlorochalcone

A suspension of 0.345 g of 4 '- (ethoxycarbonylmethoxy) -3-chlorchalcone in 4 ml of ethanol and 4 ml of water is added at room temperature to 0.212 g of sodium carbonate and kept at room temperature overnight. An additional 1.5 mL of ethanol and 1.5 mL of water are added and the reaction mixture is refluxed for 1 hour and cooled to room temperature with separation of a yellow solid. The solid is collected by filtration, dissolved in a mixture of water / ethyl acetate and

- 20 000 000 ·· 0 9 9 9

9 · 0 0 • 0 0 0 1 »hydrochloric acid is added to the acidic reaction. The organic phase is separated, washed with brine, dried over sodium sulfate and concentrated to dryness to give 0.29 g of a yellowish solid which crystallizes from ethyl acetate (20 ml) to give 0.12 g of product, m.p. M.p. 180-181 ° C.

1 H-NMR in d 6 -DMSO: 4.9 ppm (s, 2H); 7.05 ppm (d, 2 H); 7.5 ppm (m, 2 H); 7.7 ppm (d, 1 H); 7.85 ppm (m, 1 H); 8.05 ppm (d, 1 H); 8.1 ppm (d, 1 H); 8.2 ppm (d, 2 H); 13.1 ppm (s, 1 H).

Example 3: 3 ', 4'-dichloro-4- (ethoxycarbonylmethoxy) chalcone

A solution of 3 ', 4'-dichloro-4-hydroxychalcone (0.586 g) in 9 mL of anhydrous DMF is added at room temperature and under a nitrogen atmosphere to 0.69 potassium carbonate and 0.334 ethyl bromoacetate. The reaction mixture is heated at 60 ° C for 3 hours, cooled to room temperature and poured into a mixture of 40 mL of water and 20 mL of ethyl acetate. The mixture is kept under stirring until the solid has dissolved, the organic phase is separated, washed with ethyl acetate, dried over sodium sulfate and concentrated to dryness to give 0.737 g of the product as a yellow solid.

1 H-NMR in d 6 -DMSO: 1.2 ppm (t, 3H); 4.2 ppm (q, 2 H); 4.9 ppm (s, 2 H); 7.0 ppm (d, 2 H); 7.7-8.0 ppm (m, 5H); 8.15 ppm (dd, IH); 8.4 ppm (d, 1 H).

Example 4: 3 ', 4'-dichloro-4- (carboxymethoxy) chalcone

A suspension of 0.38 g of 3 ', 4'-dichloro-4- (ethoxycarbonylmethoxy) chalcone in 4 ml of ethanol and 4 ml of water is mixed with 0.212 g of sodium carbonate and refluxed for 3 hours and 30 minutes.

The reaction mixture is cooled to room temperature and the solid is collected by filtration, partitioned between water, 1N hydrochloric acid is added to the acidic pH and ethyl acetate. The organic phase is separated, washed with brine, dried over sulfate

-21 Sodium and concentrated to dryness. The residue is crystallized from ethyl acetate (10 mL) to give 0.158 g of product, m.p. Mp 203-206 ° C.

1 H-NMR in d 6 -DMSO: 4.8 ppm (s, 2H); 7.0 ppm (d, 2 H); 7.7 - 8.0 ppm (m, 5H); 8.1 ppm (dd, 1H); 8.4 ppm (d, 1H); 13.1 ppm (s, 1H).

Example 5: ^{3,4-Dichloro-4} '- (ethoxycarbonylmethoxy) dihydrochalcone

A solution of 3,4-dichloro-4'-hydroxychalcone (0.221 g) in three ml of anhydrous DMF is added to 0.125 ml of ethyl bromoacetate and 0.26 g of potassium carbonate and the mixture is heated at 60 ° C for 1 hour 45 minutes. The reaction mixture is cooled to room temperature and diluted with 20 mL of water and 20 mL of ethyl acetate. The mixture is acidified with 1N HCl to pH 3-4, the organic phase is separated, washed with brine, dried over sodium sulfate and concentrated to dryness. 0.268 g of product is obtained as a yellowish oil.

1 H-NMR in CDCl 3: 1.3 ppm (t, 3H); 3.0 ppm (t, 2 H); 3.2 ppm (t, 2 H); 4.25 ppm (q, 2 H); 4.6 ppm (s, 2H); 6.9 ppm (d, 2 H); 7.05 ppm (dd, 1H); 7.3 ppm (m, 2 H); 7.9 ppm (d, 2 H).

Example 6: 3,4-dichloro-4 '- (carboxymethoxy) dihydrochalcone

A solution of 3,4-dichloro-4 '- (ethoxycarbonylmethoxy) dihydrochalcone in 3 ml of ethanol is added to 3 ml of water, then 0.15 g of potassium carbonate is added and the mixture is heated at 70 ° C for 2 hours. The reaction mixture is concentrated to dryness, then 4 ml of water and 2 ml of 1N HCl are added to pH 2-3. After stirring for 30 min, the solid is collected by filtration, washed with water on the filter and dried under vacuum at 50 ° C overnight. 0.169 g of product is obtained as a white powder, m.p. 148-150 ° C.

1 H-NMR in d 6 -DMSO: 2.9 ppm (t, 2H); 3.3 ppm (t, 2 H); 4.7 ppm (s, 2H); 7.0 ppm (d,

2H); 7.25 ppm (dd, 1H); 7.6 ppm (m, 2 H); 7.9 ppm (d, 2 H); 13.1 ppm (s, 1H).

-22 • · · ·

Example 7; 5 - [((2,3-dichloro-4- (2'-methylenebutyryl) phenoxy) methyl] tetrazole

39.2 g of 5 - [((2,3-dichloro-4-butyryl) phenoxy) methyl] tetrazole, 33 g of paraformaldehyde and 5 11.2 g of dimethylammonium chloride in 1 ml of acetic acid are heated at 80-90 ° C for 2 hours . After cooling to room temperature, the reaction mixture is partitioned between water and diethyl ether. The aqueous solution is treated with sodium bicarbonate and the resulting precipitate is collected by filtration. The solid (22 g) is treated with 200 mL of water, 200 mL of 2

The mixture is heated to complete dissolution and further 1 hour. The aqueous phase is acidified and extracted with diethyl ether. The organic extracts are combined, dried over magnesium sulfate and concentrated to dryness. The residue (7.66 g) is crystallized from 70 ml of benzene. 5.3 g of product are obtained.

Elementary analysis. (% calculated / found): C 47.72 / 47.01; H 3.70 / 3.52; N 15, 17.13 / 16.78; Cl, 21.97 / 21.43.

Example 8: 5 - [((2,3-dichloro-4- (2'-methylenebutyryl) phenoxy) methyl-1-methyltetrazole)

9.5 g of 5 - [((2,3-dichloro-4-butyryl) phenoxy) methyl] -1-methyltetrazole, 1.01 g of 20 paraformaldehyde and 2.53 g of dimethylammonium chloride in 5 drops of acetic acid are heated at 80 ° C. 90 ° C for 2 hours. The mixture is concentrated to dryness and poured into 100 ml of water. 200 ml of sodium bicarbonate solution are added and the mixture is kept under stirring for 4 hours until a solid separates. The solid is filtered off to give

6.3 g of crude material which is recrystallized from 130 ml of 1: 1 benzene / cyclohexane 25. 5.58 g of product are obtained, m.p. Mp 124-125 ° C.

Elementary analysis. (% calculated / found): C 49.28 / 49.69; H, 4.14 / 4.33; N, 16.42 / 16.41; Cl 20.79 / 20.53.

Example 9: 5 - (((2,3-dichloro-4- (2'-methylenebutyryl) phenoxy) methyl) -1-methyltetrazole

9 9 9 • 9 g of 5 - [((2,3-dichloro-4-butyryl) phenoxy) methyl] -2-methyltetrazole, 0.89 g of paraformaldehyde and 2.29 g of dimethylammonium chloride in 4 drops of acetic acid are heated at 80 ° C. -90 ° C for 2 hours. The mixture is concentrated to dryness and diluted with water.

The separated solid is filtered off. The filtrate is treated with sodium bicarbonate and heated on a water bath. The resulting solid is collected by filtration to give 7.76 g of product which is crystallized from 400 mL of cyclohexane. 4.16 g of product are obtained.

Elementary analysis. (% calculated / found): C 49.28 / 49.11; H, 4.14 / 4.55; N, 16.42 / 16.13; Cl 20.79 / 20.54.

Example 10: Ethyl 2- (4- (2- (3- (4-chlorobenzoyl) acrylylamino) ethyl) phenoxy) -2-methylpropionate (Compound D)

A solution of 10.3 g of P- (4-chlorobenzoyl) acrylic acid is dissolved in 100 ml of tetrahydrofuran, 7.02 ml of triethylamine is added and the mixture is cooled to -15 ° C. 5.25 ml of ethyl chloroformate is added dropwise and the reaction mixture is stirred for 15 min. A solution of 12.6 g of ethyl 2- (4- (2-aminoethyl) phenoxy) -2-methylpropionate in 20 ml of tetrahydrofuran is added dropwise to the reaction mixture, which is then held at 15 ° C for 30 minutes, at 0 ° C for 2 hours. and at room temperature overnight. The mixture is concentrated to dryness and dissolved in diethyl ether. The organic phase is washed with 2 N hydrochloric acid, 2 N NaOH and water, dried over sodium sulfate and concentrated to dryness. The residue after crystallization from diethyl ether gives 10 g of product,

m.p. Mp 76-77 ° C.

Elementary analysis. (% calculated / found): C 64.94 / 64.77; H 5.90 / 5.59; N, 3.15 / 3.14; Cl 7.98 / 8.12.

-24 · · · · φ · · · ·

Example 1: 2- (4- (2- (3- (4-chlorobenzoyl) acrylylamino) ethyl) phenoxy) -2-methylpropionic acid · · •φφφφφφφφφφφφφφφφ • • • Ethyl 2- (4- (2- (3- (4-chlorobenzoyl) acrylylamino) ethyl) phenoxy) -2-methylpropionate is dissolved in 70 ml ethanol and added to 100 ml 1 N KOH solution. Stirring is continued for 3 hours at 45 ° C. After cooling to room temperature, the mixture is concentrated to dryness, dissolved in diethyl ether and treated with hydrochloric acid. The organic phase is separated, dried over sodium sulfate and concentrated to dryness to give 8 g of product as a brown amorphous solid.

Example 12: 3,4-dichloro-4 '- (carboxymethoxy) chalcone (compound E or LSM83177)

A solution of 3,4-dichloro-4'-hydroxychalcone (0.586 g) (Preparation 3) in 9 mL of anhydrous DMF is added at room temperature and under nitrogen to 0.69 g of potassium carbonate and 0.334 g of ethyl bromoacetate. The reaction mixture is heated at 60 ° C for 3 hours, then cooled to room temperature and poured into a mixture of 40 mL of water and 20 mL of ethyl acetate. The mixture is stirred until the solid dissolves, then the organic phase is separated, washed with ethyl acetate, dried over sodium sulfate and concentrated to dryness to give 3,4-dichloro-4 '- (ethoxycarbonylmethoxy) chalcone. The 3,4-dichloro-4 '(ethoxycarbonylmethoxy) chalcone is saponified in analogy to Example 4 to 3,4-dichloro-4' - (carboxymethoxy) chalcone.

Example 12A: ^{3,4-Dichloro-4} '- (5-tetrazolylmethoxy) chalcone (součenina F)

To a stirred suspension of 4- (4-tetrazolylmethoxy) acetophenone (0.15 g) in ethanol (2 mL) under a nitrogen atmosphere is added lithium hydroxide hydrate (0.059 g) followed by 3,4-dichlorobenzaldehyde (0.123 g). The resulting mixture was refluxed for 2 hours. After cooling to room temperature, the precipitate was filtered off and suspended in water (2 ml). The suspension was acidified by treatment with 1 N HCl and stirred for 2 hours. Solids

-259 4

449 ··· · 4 · 9 · 4 9

4 was separated by filtration and recrystallized from MeOH to give 3,4-dichloro-4 '- (5-tetrazolylmethoxy) chalcone as a yellow powder (117 mg), m.p. Mp > 230 ° C.

BIOLOGICAL EXPERIMENTAL PART

Example 13

Preparation of MDM2: A DNA fragment encoding human MDM2 amino acids 1-118 was obtained by PCR and inserted into a modified plasmid vector (pQE40; QIAGEN Inc, Chatsworth, CAS, USA) under the control of a T5 promoter suitable for expression in E. coli in combination with pUBS 520 by the repressor (Brinkmann et al. 1989, Gene, 85, 109-114). BL21 cells (E. coli) were grown in LB medium at 37 ° C and were induced for expression by addition of 1 mM IPTG; MDM2 protein accumulation was more than>

15 hours. Cells were harvested, disrupted by French Press and insoluble MDM2 protein prepared according to standard protocols. MDM2 is further solubilized and subsequently back-converted to 100 mM Tris, pH 7; 1 mM EDTA; 10 mM DTT (according to Rudoph et al. 1997, Protein Function; A practical approach, 2 ^nd ed., IRL Press, 5799), mostly MDM2 having a purity of > 80% is obtained, suitable for analyzing the interaction of compounds. Further purification is performed by standard chromatographic techniques (hydrophobic interaction chromatography). The longer MDM2 fragment I-213aa was obtained and purified analogously to the described protocol.

Example 14

BIACORE analysis of MDM2'a and p53 interaction: To quantify the effect of selected compounds on the binding properties of p53 to the MDM2 protein biosensor, measurements are performed using BIACORE 2000. BIACORE 2000 is a biosensor system from BIACORE AB. The technology of this biosensor is based on the optical phenomenon of surface plasmon resonance (SRP), which detects changes in the refractive index

-264 4 4 · 1

4

4 44 ► 4 4 «near the sensor chip surface. The refractive index correlates directly with the mass concentration in the layer and increases when the analyte binds to a surface immobilized ligand. The experiments are carried out under continuous flow conditions. The SPR response, expressed in resonance units (RU), is continuously recorded against time and the result is a sensorgram. When one interaction is complete, the surface can be regenerated with solutions that remove the bound analyte without affecting the activity of the bound ligand. The N-terminal biotinized peptide (5 μΜ in PBST buffer), which corresponds to amino acids 19-32 of wild-type human p53 obtained from Genosys Biotechnologies (Cambridge), was captured at a flow rate of 5 μΐ / min for 6 min on the SA-sensor chip ( the sensor chip was pre-immobilized with streptavidin, BIACORE AB). 40 μΐ MDM2 1-213 (100 nM in PBST) was mixed with 40 μΐ sample (40 μΜ in PBST with 2% DMSO). After 15 minutes incubation at 10 ° C, 30 µΐ of the mixture was injected onto the sensor chip at a flow rate of 10 µΐ / rnin. after an additional 4 min wash of the surface with PBST buffer, the signal was recorded.

By comparing the sample signals with the buffer signals, the inhibitory effects of the samples could be evaluated. The sensor surface was regenerated by washing with 100 mM HCl and 100 mM H ₃ PO ₄ . Figure 1 shows the inhibitory effects of selected compounds on the interaction of MDM2 with p53 derived peptides in relative units.

Example 15

Spectrophotometric analysis of GST activity: Human cell cultures, such as HT29 human colon carcinoma cell culture, are grown in monolayers at 37 ° C in 5% CO ₂ in DMEM supplemented with antibiotic and 10% fetal calf serum and are passaged twice a week. GST activity in the cytosol is determined by Habig et al. (Habig, WH, Pabst, MJ, Jakoby, WB, J. Biol. Chem. 249, 7130-7139, 1974) using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione. GST catalyzes the conjugation of CDNB with glutathione and results in a CDNB-glutathione product with strong molar absorption at 340 nm. The change in absorption is monitored for 5 min.

-27 99 · · «· 99 99 99 99 99 99 99 99 99 99. 99 ♦ 9

9 9 9 9 9

9 9 9 9 9 * x 10 ⁶ cells are collected and washed once in ice-cold phosphate buffer at 1000 rpm for 10 min at 4 ° C. The cell pellet is resuspended in 200 μΐ of ice-cold phosphate buffer and sonicated for 2 min on ice. The sonicate is then centrifuged at 11750 g at 4 ° C for 15 min in an Eppendorf centrifuge and the GST activity is determined in the supernatant. The intrinsic inhibitory effect of ethacrylic acid (EA) and selected MDM2 antagonists on the catalytic activity of HT29 cytosolic GST is investigated by the addition of drugs directly to the cell extracts immediately prior to the addition of glutathione.

Table 1 shows the inhibitory effect of EA and LSM 83177 on GST activity in HT29 cell extracts.

Example 16

Biological determination of radiosensitizing activity of MDM2 antagonist compounds:

Human tumor cell cultures containing wild type p53 and low levels of GST, such as MCF7 (breast cancer), or p53 mutant and high GST, such as MCF7 ADR (adriamycin-resistant breast cancer) are cultured in RPMI medium with 10% germline calf serum to semiconfluent state.

To determine the radiosensitizing activity of selected compounds on these cells, a minimum toxic dose of 2Gy (gray: J / kg) with a ¹³⁷ Cesium source at room temperature was determined. After irradiation of the monolayer of cells in the exponential phase of growth with a dose of 2Gy, the cells were incubated with different concentrations of selected compounds for 2-6 h.

Cells were trypsinized and plated in appropriate 6-well plates. Surviving cells were trypsinized after 12-13 days and the number of cells was determined by trypan blue staining (living / dead cells). The protocol was prepared according to Khil et al., 1996, Int. J. Rad. Oncol. Biol. Phys. 34, 375-380. Table 2 shows the differential radiosensitizing activity obtained with selected compounds on low and high GST content MCF-7 cell culture.

-28 • 999 99 ·

9999

98 • 9 9 9

9 9 9

999 999

9

99

Radiosensitization of both ethacrylic acid and LSM 83177 was observed in MCF-7 cells; drug irradiation enhancement has a factor of 1.5 when the drug is incubated for 2 h on cells and a factor of 2 when incubated for 6 h.

GST-independent radiosensitizing activity in the target cell was observed only with MDM2-specific compounds such as LSM 83177: specifically targeted MDM2 results in radiosensitization in MCF-7 ADR cells due to the specific interaction of MDM-2 with E2F and Rb, regardless of the fact that the p53 mutant cannot be activated in this cell culture. Ethacrynic acid does not mediate any radiosensitization between MCF-7 ADR cells.

-29 • 4 * 44 44 * 4444 44 44

444 44 4 4 444

4 44 · · 4444

444 4 4 · 444 444 • 444 4444

Table 1:

Inhibition of GST activity in HT29 cells by potential inhibitors

Concentration EA LSM 83177 [mg / ml] [% GST activity] 10 20 0 0 10 4 26 8 17 46 5 36 73 2.5 48 95 15 2 57 100 ALIGN! 1.3 64 100 ALIGN! 1 84 100 ALIGN! 0.4 91 100 ALIGN! 0.2 99 100 ALIGN! 20 0,04 100 ALIGN! 100 ALIGN! IC / 50 [mg / ml] 2.2 : 7,7

-30 •• 44

4 «44 * 4

44

4 4

4 4

445 44 4

4

4 «4 4

Table 2

Compounds were incubated at a final concentration of 20 µg / ml with the corresponding cells.

A-MCF-7 cells

Incubation drug duration after irradiation % of live cells per control without drug effect ethacrynic acid 2 GY + ethacrynic acid LSM 83177 2 Gy + LSM 83177 0 hrs 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! 2 hrs 52.3 42.7 64.1 41.8 6 hours 43.1 20.5 36.4 17

B-MCF-7ADR

incubation drug duration after irradiation % of live cells per control without drug effect ethacrynic acid 2GY + ethacrynic acid LSM 83177 2 Gy + LSM 83177 0 hrs 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! 2 hrs 88.9 82.5 87.2 72.8 6 hours 99 133.6 85.7 48.8

-31 · · · 9 9 9.... 9

9 9 9 9 9

9 9 9 9

Claims (10)

Patent claims Use of compounds of formula (I): 10 where: the -OC (R 1) (R 2) - (CH 2) _p -A group may be in the ortho, meta or para position; A is selected from -COOH, COO- (C 1 -C ₄ ) alkyl, -CN or a group of the formula 15 λίκ \ c .. '' NN wherein R 'is hydrogen or (C 1 -C ₄ ) alkyl; or A- (CH 2) _p -C (R 1) (R 2) - is selected from phenyl, benzyl or (indolyl) methyl 20 groups which may be substituted with R 4 groups; p is 0, 1 or 2; R1 and R2 are independently selected from hydrogen or (C1- _C8 ) alkyl or form together 25 having a carbon atom to which they are attached, a (C 3 -C 7) cycloalkyl group; R 4 are from 0 to 2 substituents independently selected from chloro, bromo, iodo, fluoro, linear or branched (C 1 -C 4) alkyl, hydroxy, (C 1 -C ₄ ) alkoxy, (C 1 -C ₄ ) acyl 30 groups; -32 · · · • 99 99 9 9 9 · 999,999 Or the group in formula (I) is a naphthyl group which may be substituted with R 4 groups; n is an integer from 1 to 4; m is 0 or 1; B is selected from linear or branched C 1 -C 10 alkyl, -CO-C (R 3) = CH-R, -CH = C (R 3) -CO-Ar, -CO-CH (R 3) -CH ₂ -R or 15 -CO-CH (R 3) -CH 2 -NR 5 R 6 when m is 0, or -CH = C (R 3) -CO-Ar when m is 1; -R is selected from hydrogen, -Ar or -CO-Ar; 20 -R 3 is hydrogen or (C 1 -C 6) alkyl; -R 5 and R 6 are independently a (C 1 -C 4) alkyl group or form together with the nitrogen atom to which they are attached a piperidine, piperazine, (C 1 -C 6 alkylpiperazine, morpholine or thiomorpholine group); -Ar is a phenyl group which may be unsubstituted or substituted with from 1 to 3 groups independently selected from chlorine, bromine, iodine, fluorine, linear or branched (C 1 -C 6 alkyl, hydroxy, (C 1 -C 6 alkoxy), (C 1 -C 6) acyl groups, stereoisomers of these groups, or salts of these groups with pharmaceutically acceptable acids 30 or bases, for the preparation of a medicament having MDM2 antagonist activity. -33 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 2. Use of Step 1 for the treatment of tumors. Iwýl / č. Use of claim 2, wherein the tumor being treated is a sarcoma. 5 4. Compounds of formula (I): where: the -OC (R 1) (R 2) - (CH ₂ ) _p -A group may be in the ortho, meta or para position; 15 - A is selected from -COOH, COO- (C 1 -C 4) alkyl, -CN or a group of the formula R ’ N-N wherein R 'is hydrogen or (C 1 -C 4 alkyl); or the group A- (CH ₂ ) _p -C (R 1) (R 2) - is selected from phenyl, benzyl or (indolyl) methyl group, which may be substituted with R 4 groups; p is 0, 1 or 2; R 1 and R 2 are independently selected from hydrogen or (C 1 -C 8) alkyl or form together with A carbon atom to which they are attached a (C 3 -C ₇ ) cycloalkyl group; -34 # ··· · * ·· R4 are from 0 to 2 substituents independently selected from chlorine, bromine, iodine, fluorine, linear or branched (Ci-C) _alkyl, hydroxy, (C _t -C ₄₎ alkoxy, _(Ci-C4) acyl groups; 5 - or a group in formula (I) is a naphthyl group which may be substituted with R 4 groups; n is an integer from 1 to 4; m is 0 or 1; B is selected from linear or branched C 1 -C ₁₀ alkyl, -CO-C (R 3) = CH-R, -CH = C (R 3) -CO-Ar, -CO-CH (R 3) -CH ₂ -R or -CO-C (R 3) -CH ₂ -NR 5 R 6 wherein m is 0, or is -CH = C (R 3) -CO-Ar when m is 1; -R is selected from hydrogen, -Ar or -CO-Ar; 25 -R 3 is hydrogen or (C 1 -C 8) alkyl; -R 5 and R 6 are independently (C 1 -C ₄ ) alkyl groups or form together with the nitrogen atom to which they are attached a piperidine, piperazine, (C ₁ -C ₆ ) alkylpiperazine, morpholine or thiomorpholine group; ··· 0 <00 ·· «· 00 0« «00 ·· 0 0000 ··· ·· · 0000 000 000 · ·· 000 0 · «<040 0000 0« -Ar is a phenyl group which may be unsubstituted or substituted with from 1 to 3 groups independently selected from chlorine, bromine, iodine, fluorine, linear or branched (C 1 -C 8) alkyl, hydroxy, (C 1 -C ₄ ) alkoxy, ( C 1 -C ₄ ) acyl groups, 5 stereoisomers of these groups or salts of these groups with pharmaceutically acceptable acids or bases, except when m = 0, A is selected from -COO- (C 1 -C ₄ ) alkyl, CN or a group of formula R ’ NN wherein R ¹ is hydrogen or (C 1 -C ₄ ) alkyl; and when m = 0 and A is -COO- (C 1 -C ₄ ) alkyl, B can only be -CO-C (R 3) -CH ₂ -NR 5 R 6. Compounds according to claim 4, wherein m is 0, R is hydrogen and A is a tetrazole group. Compounds according to claim 4, wherein Ar is a phenyl substituent of from 1 to 2 chlorine atoms. Compounds according to any one of claims 4 to 6, wherein R 4 is from 1 to 2 chlorine atoms. Compounds according to any one of claims 4 to 7, wherein the -OC (R 1) (R 2) - (CH ₂ ) _p -A group is in the para position. 9. Compounds of one of the following formulas: C N-N F Pharmaceutical composites comprising at least one compound according to claims 4 to 9 in admixture with pharmaceutically acceptable carriers.