WO2004035580A1

WO2004035580A1 - Reactivation of wild type p53 in human tumour cells by a low molecular weight compound

Info

Publication number: WO2004035580A1
Application number: PCT/SE2003/001635
Authority: WO
Inventors: Galina Selivanova; Natalia Issaeva
Original assignee: Aprea Ab
Priority date: 2002-10-21
Filing date: 2003-10-21
Publication date: 2004-04-29
Also published as: AU2003272175A1

Abstract

The present invention provides low molecular weight compounds of general formula I (X, Z, R1, R2, R3, R4, R5 and R6 have the meanings indicated in the description), having the property of restoring the apoptosis-inducing function of wild type p53 in tumour cells. The compounds reactivate the apoptosis-inducing function of p53, prevent its degradation by HDM-2 and induce p53 levels in cells, and rescue its transactivation function in living cells. The invention relates to the use of the compounds in cancer therapy.

Description

REACTIVATION OF WILD TYPE P53 IN HUMAN TUMOUR CELLS BY A LOW MOLECULAR WEIGHT COMPOUND

Field of the invention

The present invention relates to low molecular weight compounds, which are able to restore the apoptosis inducing function of wild type (wt) p53. The present invention also relates to pharmaceutical compositions comprising the new compounds, as well as methods for treating cancer comprising adπiinistrating said new compounds to a mammal in need thereof.

Background

p53 is a potent tumour suppressor gene and one of the key players in signalling apop- tosis: a number of stress signals converge on p53, which responds to them by triggering cell cycle arrest and/or cell death by apoptosis. This function of p53 is crucial for the prevention of tumour development as well as for the response to anticancer therapy. p53 mutations occur in about 50% of various types of human tumours with the consequent loss of the wild type functions of the p53 protein, such as the sequence- specific DNA binding and transcriptional transactivation functions, thus resulting in reduced ability to suppress cell growth and induce apoptosis. Other classes of tumours, however, retain wt p53 protein, but demonstrate alternative mechanisms of its inactivation. Such inactivation of p53 can for example result from binding of the onco- gene protein HDM2, encoded by the hdm2 (human double minute 2) gene, sometimes also referred to in the art as mdrn2 (murine double minute 2). HDM2 binds to the p53 transactivation domain and inhibits its transactivation function. Moreover, HDM2 functions as an E3 ubiquitin ligase that transports p53 from a nucleus to a cytoplasm targeting it for the proteasomal degradation. Thirty percent of human sarcomas show no p53 mutations but have an amplified hdm2 gene. Agents increasing the intracellu- lar concentration of active p53 in tumour cells by interfering with the HDM2-p53 interaction therefore are considered having therapeutic utility in sensitizing tumour cells for chemo- or radiotherapy. In tumour types particularly sensitive to increases in functional p53 agents of this type are believed to be sufficient to induce apoptosis on their own. Accordingly, activation of the p53 pathway, in order to specifically stimulate the above pathway of cell death in human tumours, by inhibiting the p53-HDM2 interaction has been suggested as an approach for cancer therapy.

Thus, WO9847525, WO9602642 and WO9801467 describe peptide inhibitors of the p53/MDM2 interaction. More recently, low molecular weight compounds capable of interrupting the p53/MDM2 interaction have also been described in EP-A-0947511 and WO0015657.

The present inventors have now found low molecular weight compounds of the below general formula to be capable of interrupting the p53/HDM2 interaction.

Summary of the invention

The present invention relates to compounds corresponding to general formula (I)

*1 R. wherein

X is a hetero atom selected from N and S;

Z is independently selected from N and C, the total number of nitrogen atoms represented by Z being 0 to 3, of which maximally 2 are contained in one and the same ring; the optional substituent Rl is selected from hydrogen, arnino, aliphatic C1-5 al- kyl, aliphatic C1-5 alkoxy, -C(O)O(CH2)_nCH3, optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, -NH(CH2)mN((CH2)nCH₃)₂, and optionally substituted phenyl, benzyl and benzoyl, any second substituent coordinated to same ring atom being hydrogen; the optional substituent R2 is selected from hydrogen, a ino, aliphatic C₁-5 alkyl, aliphatic C1-5 alkoxy, -C(O)O(CH₂)_nCH3, optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, -NH(CH2)mN((CH2)nCH3)2, and optionally substituted phenyl, benzyl and benzoyl, any second substituent coordinated to same ring atom being hydrogen; the optional substituent R3 is selected from hydrogen, amino, aliphatic C2-5 alkyl, aliphatic C1-5 alkoxy, -C(O)O(CH2)_nCH3, optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, -NH(CH2)mN((CH2)nCH₃)2, and optionally substituted phenyl, benzyl and benzoyl; the optional substituent R4 is selected from hydrogen, keto, and aliphatic C₁-₅ alkyl, aliphatic C1-5 alkoxy, -C(O)O(CH₂)nCH₃, optionally C1.3 dialkyl substituted aliphatic C1-5 aminoalkyl, -NH(CH2)__,N((CH2)nCH₃)2, any second substituent coordinated to same ring atom being hydrogen;

R5 is selected from hydrogen, amino, aliphatic C1-5 alkyl, aliphatic C1-5 alkoxy, - C(O)O(CH2)nCH3, and optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, - NH(CH2)_mN((CH2)nCH3)2, and optionally substituted phenyl, benzyl and benzoyl; and

R6 is selected from hydrogen, amino, aliphatic C1-5 alkyl, aliphatic C1-5 alkoxy, - C(O)O(CH2)nCH₃, optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, - NH(CH2)_mN((CH2)nCH₃)2, phenyl, and optionally substituted furanyl, benzyl and benzoyl; n is 0, 1 or 2; m is 2, 3, or 4; and at least one of the substituents Rl to R6 is selected to be other than hydrogen,

or pharmaceutically acceptable salts, prodrugs or solvates thereof, capable of disrupt- ing the wt p53/HDM2 interaction, for use as a pharmaceutical.

In another aspect the invention relates to the pharmaceutical compositions comprising at least one of the above-mentioned compounds.

In a third aspect the present invention also relates to the use of the above compounds for the preparation of a pharmaceutical composition for use in cancer therapy.

The present compounds have been found to exhibit marked selectivity to the interruption of wild type p53-HDM2 interaction. Also, the present compounds have been dem- onstrated to induce cell death in a variety of different types of human tumour cells, such as lung, colon and breast carcinoma cells, as well as in osteosarcoma and fi- brosarcoma derived cells. Moreover, since the activity of the compounds will preferentially affect tumour cells as these are particularly sensitive to p53 activation, the present compounds are expected to exhibit reduced non-specific toxicity. The present compounds are also believed to affect GST to a lower extent than previously known low molecular weight compounds inhibiting the p53-HDM2 interaction.

The present compounds have also been shown to induce p53 levels and prevent its degradation in living cells.

Restoration of p53 transcriptional activity has also been confirmed by experimentation.

The term compound 8 will be used herein to denote the compound 5H-pyrido[4,3- b]indole-5-propanam_-ne-N,N-dirnethyl.

As used herein the term "substituted" is intended to mean carrying a substituent selected from halo, hydroxi, metoxi, amino, nitro, or -CH2OH, preferably hydroxi metoxi, amino, nitro, or -CH2OH, most preferably hydroxi, nitro, or -CH2OH. The term halo, as used herein, refers to chloro, bromo or iodo.

Brief description of the drawings

FIGURE 1 shows preferred compounds of the present invention. FIGURES 2 A and B illustrate the growth suppression of tumour cells expressing wild type p53 by compound 8 according to the invention.

FIGURES 3 A-C illustrate how compound 8 according to the invention induced apoptosis in human tumour cells in a p53 dependent manner. FIGURES 4 A-C describe how the treatment with compound 8 resulted in stabilization and induction of p53 in tumor cells and prevention of its interaction with HDM2 and ubiquitination.

FIGURES 5 A-B show how compound 8 restored the transcription transactivation function to p53 in tumor cells and induction of expression of p53 target genes. FIGURE 6 demonstrates the dependence of compound 8-induced growth suppression on protein synthesis. FIGURE 7 A-C describe how the compound 8 according to the invention prevented the interaction of the purified p53 protein with its E3 ubuquitin ligase HDM2. FIGURE 8 shows the stability of the compound 8 at 37°C.

Detailed description of the invention

The present compounds are able to disrupt the wt p53/HDM2 interaction in tumour cells, and thereby reactivate the transcriptional transactivation and apoptosis- inducing function of wt p53.

The low molecular weight synthetic compounds of the present invention has a different pharmacophore from the ones disclosed in WOOO 15657 and EP-A-094751 1.

Especially preferred compounds are those wherein: the optional substituent Rl is selected from -H, -PI1NO2, and -Me; the optional substituent R2 is selected from -H, -NH₂, -C(O)Ph; the optional substituent R3 is selected from -C(O)OEt, -CH₂Ph, -H; the optional substituent R4 is selected from -C(O)OEt, -H, =O, -NH(CH₂)₃N(Et)₂;

R5 is -H or -NH₂; and

1 1

R6 is selected from -H, -Me, -(CH₂)₃NH₂, -CH_H->-CH(OH)-CH(OH)-CH(CH₂OH), -

C(O)Ph, and -(CH₂)₃N(Me)2, and salts, prodrugs or solvates thereof.

Examples of such preferred compounds of the invention are shown in Figure 1. and salts, prodrugs or solvates thereof.

A suitable pharmaceutically acceptable salt of a compound of formula (I) is, for exa ple, in the case of a compound which is sufficiently basic, an acid- addition salt with an inorganic or organic acid such as hydrochloric, hydrobromic, sulphuric, trϋluoroacetic, citric or maleic acid; or in the case where the compound is sufficiently acidic, an alkali or alkaline earth metal salt such as a calcium or magnesium salt , or an ammonium salt , or a salt with an organic base such as methyla ine, dimethyl- a ine, trimet-hylamine, piperidine, morpholine or tris-(2-hydroxyethyl)arnine. Various forms of prodrugs are known in the art, for example in-vivo hydrolysable esters, and have been described in, for example, Design of Prodrugs, edited by H. BundsgaaRD, (Elsevier, 1985).

Certain compounds of the present invention may exist in solvated, for example hy- drated, as well as unsolvated forms.

The pharmaceutical compositions according to the invention may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, buffer or stabilizer, or any other material well known to those skilled in the art and appropriate for the intended application. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. Examples of techniques and protocols to this end may e.g. be found in Remingtonis Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

The composition according to the invention may be prepared for any route of a<_lrnini- stration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular or intraperitoneal. The precise nature of the carrier or other material will depend on the route of administration. For a parenteral administration, a parenterally acceptable aqueous solutions is employed, which is pyrogen free and has requisite pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions and numerous methods are described in the literature (for a brief review of methods of drug delivery, see Langer, Science 249:1 527-1533 (1990)). Preservatives, stabilizers, buffers, antioxidants and/ or other additives may be included, as required. Dosage lev- els can be determined by those skilled in the art, taking into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors. Examples of the techniques and protocols mentioned above can be found in Remingtonis Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

As previously mentioned, the present invention also relates to the use of substances defined above as a medicament. In particular, the invention relates to the use of these substances as medicaments for treatment of human tumours of different origin containing wild type p53 protein. Finally, the present invention also relates to methods of medical treatment wherein the substances according to the invention are used.

EXPERIMENTAL

Below, the present invention will be described in more detail by way of examples that are not intended to limit the scope of the invention in any way. All references given below and elsewhere in the present specification are hereby included herein by reference.

Materials and Methods

The chemical compounds of the present invention can be obtained by synthesis methods generally known in the art of preparative organic chemistry, and will not be de- scribed in any further detail herein.

Plasmids

The plasmids encoding the GST-human wild type p53 (1-393) fusion protein and the

GST-deletion mutant p53 protein N( 1-100) and have been described earlier by Seli- vanova et al., Nucleic Acids Res, 24, 3560-7 (1996). GST-deletion mutant p53 protein deltaN(l-63)-encoding construct was obtained by digestion of the vector pGEX-2TK with Bsml and BamHI, followed by ligation. The HDM-2 encoding plasmid was as described in Bottger et al., Curr Biol, 7, 860-9 (1997). The p53-lacZ plasmid containing synthetic p53 consensus DNA binding site in front of the lacZ coding sequence was stably transfected into LIM1215 colon carcinoma cells and in HT1080 fibrosarcoma cells. pSUPERp53siRNA expressing plasmid was from OligoEngine, Seattle, WA.

Growth suppression assays

HCT116 cells and HCT116p53-/ -cells were obtained from B. Vogelstein, John Hopkins University, Baltimore, USA (MTA). p53-expressing and p53-null HCT116 cells, stable clones expressing pSUPERp53siRNA and human tumor cell lines listed in Table II were grown in 96-well plates at a density of 3000 cells per well and treated with 25μM or otherwise indicated concentrations of the present compounds. After 48 hours of incubation the proliferative cell reagent WST-1 (Roche) was added to the cells. Reduction of WST-1, which reflects cell viability, was measured by microplate reader at λ 490 nm according to the manufacturer (Roche). Stable clones expressing pSUPERp53siRNA were obtained by transfection of U2OS osteosarcoma cells with pSUPERp53siRNA vector followed by selection of Neo-resistant clones.

Apoptosis assays

Cells were placed on 12-well plate at a density of 30000/cm² and treated with compounds. After 24h of incubation with 20 μM of compound 8 cells were harvested by trypsinization, fixed with 70% ethanol, treated with RNase A (0.25 mg/ml) and stained with propidium iodide (0.02 mg/ml). Samples were analyzed on a Becton Dickinson FACScan. Data were analyzed by the CellQuest software, version 3.2.1. TUNEL and annexin staining were performed according to standard procedures.

Colony formation assay

Cells were treated with compound 8 and seeded in plates at 500 cells per plate. Colo- nies were stained with Giemsa and counted 14 days after seeding.

β-galactozidase assays

Transactivation assays were performed using p53-responsive promoter constructs linked to the lacZ reporter gene stably transfected into HT1080 and LIM1215 cell lines. Cells were treated with compound 8 at concentration of 25 μM. β-galactozidase assay was performed 16 hours post- treatment by the standard procedure.

ELISA

50 ng of GST-p53(l-393), GST-N(l-lOO) and GST-deltaN(l-63) or 20 ng of His-HDM2 protein were preincubated without or with compound 8 at room temperature for 30 or 90 min. The ELISA analyses were performed according to standard procedures. Briefly, after the treatment with compound 8 samples were diluted with coating buffer (CB, 150 mM NaCL, 25 mM HEPES) supplemented with 1 μM PMSF and 10 μM DTT and coated on ELISA plates (MaxiSorp, Nunc) at +4°C overnight. The wells were washed with coating buffer and blocked with 5% skim milk in CB at room temperature (RT) for 3 h. Wells were rinsed twice with CB followed by addition of either recombi- nant HDM2 or p53, incubated for lh, washed 4x15 min with CB and incubated with primary antibodies (anti-HDM2 2A10 monoclonal mouse, Santa Cruz, dilution 1:250; or anti-p53 C5 polyclonal rabbit, Santa Cruz, dilution 1:500). Samples were incubated at RT for 40 min. After washing a secondary antibody (horse radish peroxidase (HRP) conjugate, anti-mouse, or anti-rabbit, respectively) was incubated with samples at RT for 40 min. Then plates were washed 4x15 min with CB and a peroxidase substrate was added. An absorbance at λ 405 nm was monitored by ELISA reader.

Complex formation between p53 and HDM2 in living cells was monitored by two-site ELISA. Briefly, either p53 or HDM2 proteins from lysates of cells treated or untreated with compound 8 were captured by anti-p53 or anti-HDM2 antibodies, respectively, which were immobilized on ELISA plates. The presence of a partner binding protein (HDM-2 or ρ53, respectively) was detected by probing with corresponding primary antibodies followed by secondary HRP-conjugated antibodies as described above. The amount of p53 in complex with HDM-2 before and after treatment was calculated according to the levels of p53 or HDM2 in lysates.

Preparation of cell extracts and Western blotting were performed according to standard procedures.

Detailed description of the drawings

Figure 1 shows structural formula of 5H-F^do[4,3-b]indole-5-propanamine-N,N- dimethyl and compounds 1, 4, 5, 6, and 7 that inhibit the growth of human tumour cells by preventing p53/HDM2 interaction.

Figure 2 illustrates how compound 8 suppressed preferentially the growth of cells expressing wild type p53, but not the growth of cells lacking p53 expression at a range of concentrations. More specifically, Figure 2A shows how compound 8 suppressed the growth of HCT116 cells expressing wild type p53. In contrast, the effect of treatment on HCT116p53-/- cells lacking p53 expression was rather minor. The graph illustrates the difference between viability of cells treated by compound compound 8 in the presence and absence of p53, expressed as the percentage of reduction of WST-1 cell proliferation reagent by untreated versus treated cells. The degree of WST-1 reduction, which reflects a number of living cells, was measured by microplate reader at λ 490 nm according to manufacturer (Roche). The growth suppression was calculated as a difference in absorbance at λ 490 nm between un- treated and treated cells and expressed in a percent from untreated control. Growth Suppression = 100% X (control absorbance - treated absorbance)/ Control absorbance.

Compound 8 suppressed the growth of cells expressing wild type p53 but did not significantly affect the growth of cells lacking p53 expression. Figure 2B demonstrates how inhibition of p53 expression by siRNA in U2OS cells confers resistance to compound 8-mediated growth suppression, as assessed by WST assay described in A.

Figure 3 illustrates the p53-dependent induction of apoptosis by compound 8 in wild type p53 expressing tumor cell lines. More specifically, Figure 3A shows that compound 8 induced the appearance of subGl fraction, indicating apoptosis. Induction of apoptosis was determined by FACS analysis of ethanol fixed cells stained with propidium iodide (PI) as percentage of a sub-Gl population. Cells were treated with compound 8 at concentration 20μM for 24 h. Figure 3B presents an- nexin exposure, the hallmark of apoptosis, on the surface of HCT116 cells treated with compound 8 at a concentration of 20 μM for 24 h. Figure 3C shows how compound 8 induces DNA fragmentation during cell death by apoptosis as assessed by morphology of Hoescht-stained cell nuclei (right) and TUNEL staining (left). The treatment conditions were as described in B.

Figure 4 illustrates the induction of p53 levels, inhibition of p53/HDM2 interaction and prevention of p53 ubiquitination by compound 8 in living cells. Figure 4A, Treatment with compound 8 results in prominent increase in p53 levels in HCTl 16 cells, but not in nontransformed human fibroblasts. Figure 4B, compound 8 inhibited complex formation between p53 and HDM2 by 83%. HCTl 16 cells were treated by 50 μM of compound 8 for 18 hours, cells lysed and the amount of p53/HDM2 complex was estimated using ELISA. Figure 4C, compound 8 prevented ubiquitination of p53. HCTl 16 cells were treated with proteasome inhibitor MG132 in order to get accumulation of ubiquitinated p53 and then incubated with or without compound 8 (50 μM). The amount of ubiquitinated forms of p53 decreases dramatically upon treatment with compound 8.

Figure 5 demonstrates the restoration of transcriptional transactivation activity of p53 by compound 8. Figure 5A, compound 8 induced the wild-type p53-responsive

LacZ reporter in HT1080 (upper panel) and in LIM1215 (lower panel) cells carrying wild type p53. Figure 5B demonstrates the induction of p53 target genes p21 HDM2, and PUMA by compound 8 in HCTl 16 cells, but not in the absence of p53 expression in HCT116p53-/- cells. The expression of proteins was analysed using Western blot. Compound 8 did not induce p53 target genes in nontransformed human diploid fibroblasts carrying wild-type p53.

Figure 6 describes the dependence of compound 8-induced growth suppression on protein synthesis. Pretreatment of cells with protein synthesis inhibitor cyclo- heximide inhibited the growth suppressor activity of compound 8 by two-fold.

Figure 7 shows how compound compound 8 prevents interaction between purified p53 and HDM2 proteins. More specifically, Figure 7A illustrates how compound compound 8 inhibits interaction between recombinant p53 and HDM2. Preincuba- tion of p53 or HDM2 with compound 8 for 30 (white bars) or 90 (black bars) min at RT resulted in inhibition of protein-protein interaction. Absorbance of the control sample without treatment was taken as 100%. Figure 7B demonstrates how compounds 1, 4, 5, 6, and 7 prevent interaction between purified p53 N-terminal(l- 109) protein and HDM2 protein. Hatched bars, preincubation with HDM2, black bars, preincubation with p53. Experimental design as in A. Figure 7C shows how compound 8 prevents binding of HDM2 to different recombinant p53 proteins. In- teraction between full length p53 (1-393), N-terminal (N, 1-109), and delta - N terminal (dN, 1-63) was inhibited in a dose-dependent manner, whereas the binding to the core domain (100-300) was not affected by compound 8. The experimental design is as in Fig. 4A.

Figure 8 shows the stability of compound 8 in PBS upon incubation at 37°c for 4 and 10 hours. The growth suppressor activity of compound 8 was not affected by incubation at 37°C.

Results and discussion

Growth suppression by compound 8 depends on wtp53 expression In HCTl 16 colon carcinoma line expressing wild type p53 protein, p53 is nonfunctional due to rapid degradation by HDM2. In a derivative of HCTl 16, HCT116p53-/- cell line both alleles of the p53 gene were deleted by means of homologous recombina- tion. This pair of cell lines was used for testing the inventive compounds. HCTl 16 and HCTl 16p53-/- cells were grown in parallel in 96-well plates at a density of 3000 cells per well. The treatment was performed at a concentration of 25 μM of each of the compounds tested. After 48 hours of incubation the proliferative cell reagent WST-1 (Roche) was added to the cells. The degree of WST-1 reduction, which is proportional to the cell viability, was measured by a microplate reader at λ 490 nm according to the manufacturer (Roche). Growth suppression = 100 % x (controlabsorbance - treatedabsorbance) / controlabsorbance. Compounds which were identified as being able to suppress the growth of HCTl 16 cells expressing p53, but which did not affect the growth of HCT116p53-/- cells without p53 expression are shown in Fig. 1, and the p53- and dose-dependent growth suppression by compound 8 in HCTl 16 and HCT116p53-/- cells is shown in Fig. 2A.

The ability of the compound (designated compound 8) to suppress the growth of mutant p53-expressing cells was further evaluated using a colony formation assay. HCTl 16 cells or HCT116p53-/- cells were treated with 10 μM compound 8 or left untreated and seeded in plates. The cells were Giemsa stained and scored for the appearance of colonies after 14 days. As shown in the below Table I, treatment with 10 μM of compound 8 dramatically reduced the number of colonies formed by HCTl 16 cells expressing p53 (24% of untreated control), but did not affect the growth of HCTl 16 p53-/- cells lacking p53 (94% of untreated control). Treatment with compound 8 of another colon carcinoma cells, A431, which express His273 mutant p53, did not have inhibitory effect either.

Table 1. Compound 8 inhibited colony formation in a wild type p53-dependent man- ner.

Cell line p53 status number of colonies % of untreated control

+cmpd 8 -cmpd 8

HCTl 16 wild type 55±5 243±13 23

HCT116p53-/- null 212±9 225±33 94

A431 mutant 174±9 170±15 102 Thereafter, the ability of compound 8 to suppress the growth of tumour cells in a wild type p53-dependent manner was tested using a series of human-derived cell lines with different p53 status (p53 null, wild type p53, mutant p53). p53 null lines included Saos-2 osteosarcoma and HI 299 lung carcinoma. We used wild type p53 expressing lines HT1080 fibrosacroma, LIM1215 colon carcinoma, U2OS osteosarcoma, MCF7 breast carcinoma and three different lines of nontransformed human diploid fibro- blasts: primary human fibroblasts (HDF and KF) and HTERT fibroblasts immortalized by expression of a telomerase gene hTERT. Mutant p53 expressing lines included colon carcinoma SW480 and A431, both expressing His-273 mutant p53, Saos-2-His- 273 stably transfected with His-273 mutant p53, and H1299-His-175 stably transfected with His- 175 mutant p53. As shown in Table II below, compound 8 was much more efficient as a growth suppressor in wtp53-expressing cells, but not in p53-null and mutant p53-carrying cells. IC50 values for all wtp53-expressing tumour cell lines are at least twice lower that that for p53-null and mutant p53-expressing lines. Nota- bly, the growth of nontransformed cells was not suppressed even at doses higher than 50 μM. Importantly, suppression of p53 expression by siRNA in human osteosracoma cells U2OS conferred resistance to compound 8 (Fig.2B). This shows that compound 8 targets specifically tumour cells expressing wild type p53.

Table H Wild type p53-dependent suppression of tumour cell growth by compound 8.

Type of tumour Cell line p53 status IC50

Colon carcinoma HCTl 16 wt 10

HCT116p53-/- null 48

LIM1215 wt 16

A431 mutant >50

SW480 mutant >50

Breast carcinoma MCF7 wt 26

Fibrosarcoma HT1080 wt 23

Osteosarcoma U2OS wt 12

Saos-2 null 46

Saos-2-His273 mutant 46

Lung carcinoma H1299 null 42

H1299-Hisl75 mutant 44

Hontransformed human HTERT wt >50 diploid fibroblasts HDF1 wt >50

KF wt >50

Growth suppression was determined as described in Fig. 2.

Restoration of the apoptosis-inducing function ofwtp53 by compound 8 To address the question whether growth suppression induced by compound 8 is due to the induction of cell death, we performed FACS analysis. Cell death was monitored in HCTl 16 ethanol-fixed cells stained with propidium iodide (PI) as percentage of sub- Gl population, the appearance of which is indicative of cell death. As is evident from Figure 3A, treatment with compound 8 (20 μM, 24 h) resulted in 73% of HCTl 16 cells in subGl fraction. In contrast, no significant increase in subGl fraction was observed in HCT116p53-/- cells. It can therefore be concluded that compound 8 induce cell death in a wild type p53-dependent manner. In addition, apoptotic morphology was detected in HCTl 16 cells stained with DNA-binding dye Hoechst (Fig. 3C). Annexin staining and TUNEL staining of HCTl 16 cells treated with compound 8 (20 μM, 24 h) also coiifirmed apoptosis induction (Fig. 3B and C). Taken together, these results clearly indicate that growth suppression by compound 8 occurs via induction of apoptosis in a wild type p53-dependent manner and is not due to the nonspecific cellular toxicity.

Compound 8 induces p53 accumulation in human tumour cells lines Next we addressed a question whether the stability of the p53 protein was affected by compound 8. Since inactivation of p53 in many tumour cells occurs due to enhanced degradation of p53 by HDM2, we tested whether treatment with compound 8 will lead to an increase in p53 levels. Results presented in Figure 4A demonstrate that treat- ment with compound 8 resulted in a prominent increase in p53 levels in HCTl 16 cells. Notably, compound 8 did not induce p53 in normal human fibroblasts.

In order to examine whether induction of p53 occurs due to the inhibition of ubu- quitin-dependent proteasomal degradation of p53, we tested how treatment with com- pound 8 affects ubiquitination of p53. Cells were incubated with proteasome inhibitor MG132 in order to get accumulation of ubiquitinated forms of p53 and then incubated with or without compound 8 (50 μM). As shown in Fig.4C, a ladder of high molecular forms of p53 accumulated in cells upon incubation with MG132, indicating continuous p53 ubiquitination. However, treatment with compound 8 prevented formation of ubiquitinated forms of p53.

Next we tested whether compound 8 affects the complex formation between p53 and E3 ubiquitin ligase HDM2 using ELISA. p53 from HCTl 16 cell lysates treated or untreated with compound 8 (50 μM, 18 hours) was captured by immobilized anti-p53 an- tibody on ELISA plates. The amount of HDM2 bound to captured p53 was detected by probing with anti-HDM2 antibody followed by standard ELISA procedure. As shown in graph presented in Fig. 4B, treatment with 50 μM of compound 8 inhibited the complex formation between p53 and HDM2 in living cells by 83%. Taken together, our data show that compound 8 induced p53 stabilization and prevented degradation of p53 by blocking p53/HDM2 interaction in human tumour cells.

Compound 8 restored the transcriptional transactivation function of wild type p53 in living cells Having established that compound 8 can prevent interaction between p53 and its de- structor protein HDM2 in living cells, we addressed the question whether compound 8 can restore the transcriptional transactivation function of p53. HT1080 cells carrying a p53-responsive lacZ reporter gene were treated with compound 8. β-galactosidase activity was measured using the standard procedure. As shown in Fig. 5A, upper panel, compound 8 stimulated the transcription of the p53-responsive lacZ reporter in HT1080 cells, harboring wild type p53. Similarly, treatment of LIM1215 cells that carry endogenous p53 and a transfected p53-responsive lacZ reporter with 25 μM of compound 8 for 16 hours resulted in the appearance of lacZ-positive cells whereas untreated cells were negative (Fig. 5A, lower panel).

As a final confirrnation that compound 8 can rescue transcriptional transactivation of mutant p53, we examined whether compound 8 was able to induce two classical p53 target genes, p21 and MDM2. Treatment of HCTl 16 cells expressing p53 with 20 μM of compound 8 resulted in a solid induction of both HDM2 and p21 (Figure 5B). In contrast, treatment with compound 8 of HCTl 16p53-/- cells did not cause any induction of HDM2 nor p21 (Figure 5B). This demonstrates that compound 8 induced expression of p21 and HDM2 is p53-dependent. Notably, compound 8 but did not cause any significant changes of HDM2 and p21 protein levels in nontransformed human diploid fibroblasts that carry wild type p53. Importantly, compound 8 induced expression of PUMA, a p53 target gene that is involved in apoptosis induction (Figure 5B, lower panel).

Compound 8-induced apoptosis depends on the transactivation function ofp53 To test whether compound 8 exerts its growth suppressor effect through p53-mediated activation of gene expression and de novo protein synthesis, we tested the effect of cyclohexLmide on compound 8 -induced growth inhibition. Pretreatment of HCTl 16 cells with cycloheximide before addition of compound 8 caused a 2-fold increase in cell survival according to the WST-1 proliferation assay (Fig.6). These results suggest that de novo gene expression is necessary for compound 8 -induced cell death.

Compound 8 prevents interaction between purified recombinant p53 and HDM2 proteins To get insight into the molecular mechanism of compound 8-mediated reactivation of p53, we tested whether compound 8 targets directly p53 and/ or HDM2. Purified re- combinant HDM2 or p53 proteins were incubated with compound 8 for 30 min or 90 min at room temperature and then applied to ELISA plates contaming immobilized purified p53 or HDM2 protein, respectively. Preincubation of proteins with compound 8 for 90 minutes more efficiently inhibited interaction between p53 and HDM2, than 30 min incubation. No significant difference in the degree of inhibition was observed upon preincubation with either p53 or HDM2 (Fig.7A). These data suggest that com- pound 8 targets directly purified p53 or HDM2 proteins. Compounds 1, 4, 5, 6, and 7 also prevented interaction between purified N-terminal p53 proteins and HDM as assessed by two-site ELISA (Fig.7B).

It has been shown previously, that HDM2 interacts with two different domains of p53. One binding site for HDM2 comprises residues 17-25 of the p53 N-terminus, whereas another binding site is located in the core domain of p53. We tested which binding site is affected by compound 8. HDM2 binds full length GST-53( 1-393) and its deletion mutants GST-N(1-109), GST-deltaN (1-63), as well as GST-core domain (100-309). Pre- incubation of HDM2 with compound 8 prevented its binding to the full length GST- p53(l-393), GST-p53-N( 1-100), and GST-p53-delta N(l-63) in dose-dependent manner. In contrast, the interaction of HDM2 with the core domain was not affected by compound 8 (Fig.7C). Thus, compound 8 prevents HDM2 interaction with the N- terminal binding site in p53. The experimental design was as described in connection with Fig. 4.

Induction of p53 levels and rescue of transcriptional transactivation function of p53 in living cells by compound 8 correlated with the data obtained in ELISA and demonstrates that compound 8 can work both on purified proteins and in living cells as reac- tivator of p53.

Stability of compound 8 in growth medium.

Compound 8 was incubated in phosphate-buffered saline at 37°C for a different periods of time (4 hours and 10 hours) and then tested in cell proliferation assay. As shown in Fig. 8, the growth suppressor activity of compound 8 was not affected by incubation at 37°C.

Testing of the ability of the preferred compounds to specifically suppress growth of wild type p53-containing cells. HCTl 16 and HCT116p53-/- cells were treated with the respective compounds and analyzed using WST test as described above. The effect of a specific compound on cell growth was examined using different concentrations of the compounds, ranging from 2.5 to 50 μM. IC50 values are presented in Table III. Table III Growth suppression by the preferred compounds of wild type p53 expressing cells in comparison to p53 null cells

IC50 (μM)

No. HCT116 HCT116p53-/-

_

1 7.3

2 48 >100 3 3 1 188 >50

4 3.1 17

5 1.8 5.6

6 <1 43

7 1.1 12

As can be seen from the Table, the tested compounds were able to suppress the growth of HCTl 16 without significant inhibition of HCT116p53-/- cell growth (Fig.l). The compounds No. 1, 4, 5, 6, and 7 were found to especially efficient. The most selec- tive and potent compound was No. 6, with IC50 < 1 μM in p53-containing cells and 43 μM in p53 null cells.

Claims

1. A compound having the following general formula:

wherein

X is a hetero atom selected from N and S;

Z is independently selected from N and C, the total number of nitrogen atoms represented by Z being 0 to 3, of which maximally 2 are contained in one and the same ring; the optional substituent Rl is selected from hydrogen, amino, aliphatic C1.5 alkyl, aliphatic C1-5 alkoxy, -C(O)O(CH2)nCH3, optionally C1-3 dialkyl substituted aliphatic Cι_5 aminoalkyl, -NH(CH2)mN((CH₂)nCH3)2, and optionally substituted phenyl, benzyl and benzoyl, any second substituent coordinated to same ring atom being hydrogen; the optional substituent R2 is selected from hydrogen, amino, aliphatic C₁-5 alkyl, aliphatic C1-5 alkoxy, -C(O)O(CH2)_nCH3, optionally C1-3 dialkyl substituted aliphatic Ct-5 aminoalkyl, -NH(CH2)mN((CH2)nCH3)2, and optionally substituted phenyl, benzyl and benzoyl, any second substituent coordinated to same ring atom being hydrogen; the optional substituent R3 is selected from hydrogen, amino, aliphatic C2-5 al- kyl, aliphatic C1-5 alkoxy, -C(O)O(CH2)nCH3, optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, -NH(CH2)mN((CH2)_nCH3)2, and optionally substituted phenyl, benzyl and benzoyl; the optional substituent R4 is selected from hydrogen, keto, and aliphatic C₁-5 alkyl, aliphatic C1.5 alkoxy, -C(O)O(CH2)nCH3, optionally C1-3 dialkyl substituted ali- phatic Ci-5 aminoalkyl, -NH(CH₂)_mN((CH₂)_nCH3)₂, any second substituent coordinated to same ring atom being hydrogen;

R5 is selected from hydrogen, amino, aliphatic C1-5 alkyl, aliphatic C1-5 alkoxy, - C(O)O(CH₂)_πCH3, and optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, - NH(CH₂)_mN((CH₂)nCH3)2, and optionally substituted phenyl, benzyl and benzoyl; and

R6 is selected from hydrogen, amino, afiphatic C1-5 alkyl, aliphatic C1-5 alkoxy, - C(O)O(CH₂)_nCH₃, optionally C1-3 dialkyl substituted aliphatic C1-5 aminoalkyl, - NH(CH₂)_mN((CH₂)nCH3)2, phenyl, and optionally substituted furanyl, benzyl and benzoyl; is O, 1 or 2; m is 2, 3, or 4; and at least one of the substituents Rl to R6 is selected to be other than hydrogen,

or pharmaceutically acceptable salts, prodrugs or solvates thereof, capable of disrupting the wt p53/HDM2 interaction, for use as a pharmaceutical.

2. The compound of claim 1, wherein:

the optional substituent Rl is selected from -H, -Me, -PhNO₂; the optional substituent R2 is selected from -H, -NH₂, -C(O)Ph; the optional substituent R3 is selected from -H, -C(O)OEt, -CH₂Ph; the optional substituent R4 is selected from -H, =O, -C(O)OEt, -NH(CH₂)3N(Et)₂;

R5 is -H or -NH₂; and

R6 is selected from -H, -Me, -(CH₂)₃NH₂,

1 1

-CH2-O-CH(OH)-CH(OH)-CH(CH₂OH), -C(O)Ph, -(CH₂)₃N(Me)₂.

3. The compound of claim 1 or 2, wherein Z represents C in all positions, or represents N either in position 6, in position 4, in position 2, or in positions 1, 6 and 8, as defined in the general formula (I).

4. The compound of any of the previous claims, wherein: X is N;

Z is C; and Rl, R2, R4, and R5 are -H; and R6 is -(CH₂)₃N(Me)₂.

5. The compound of any of the claims 1-3, wherein: X is N; only Z in position 4 is N; Rl, R2 and R5 are -H; R3 and R4 are both -C(O)OEt; and R6 is -Me.

6. The compound of any of the claims 1-3, wherein: X is N; Z is C;

Rl, R2, R4, R5 and R6 are -H; and R3 is -CH₂Ph.

7. The compound of any of the claims 1-3, wherein:

X is S;

Z is C; and

Rl, R2, R4, R5 and R6 are -H.

8. The compound of any of the claims 1-3, wherein: X is N;

Z is C; and

Rl, R2, R4, and R5 are -H; and

9. The compound of any of the claims 1-3, wherein: X is N;

Z in position 1, 6 and 8 is N; R2 and R5 are -NH₂; R3 is -H; R4 is =O (keto); and

! r

R6 is -CH2-O-CH(OH)-CH(OH)-CH(CH₂OH).

10. The compound of any of the claims 1-3, wherein: X is N; only Z in position 2 is N; Rl is -PhNO₂; R2 and R6 are -C(O)Ph; R3 and R5 are -H; and R4 is =O (keto).

11. The compound of any of the claims 1-3, wherein: X is N; only Z in position 6 is N; Rl is -Me;

R2, R5 and R6 are -H; and R4 is -NH(CH₂)₃N(Et)₂.

12. The compound of any of the previous claims for use in cancer therapy.

13. A pharmaceutical composition comprising a substance according to any one of claims 1-11 and a pharmaceutically acceptable carrier.

14. Use of a substance according to any one of claims 1-11 in the manufacture of a medicament for treatment of cancer in a human being.