WO2007139499A1

WO2007139499A1 - Compounds for use as a medicament

Info

Publication number: WO2007139499A1
Application number: PCT/SE2007/050369
Authority: WO
Inventors: Jacob Westman; Nina Mohell
Original assignee: Aprea Ab
Priority date: 2006-05-31
Filing date: 2007-05-29
Publication date: 2007-12-06

Abstract

A compound of formula (I) or (II) for use as a medicament. A pharmaceutical composition the compound. Use of the compound for preparing a medicament for the treatment of a disorder selected from hyperproliferative diseases, autoimmune diseases, and heart diseases.

Description

Compounds for use as a medicament

Field of the invention

The present invention relates to cyclohepta[c]pyrroliαm and cyclohepta[c]pyrrol derivatives for use in therapy. More particularly, the present invention relates to cyclohepta[c]pyrrolium and cyclohepta[c]pyrrol derivatives for the treatment and prevention of disorders and diseases such as, for example cancer, autoimmune diseases and heart diseases.

Background of the invention

The most common target for mutations in tumors is the p53 gene. The fact that around half of all human tumors carry mutations in this gene is solid testimony as to its critical role as tumor suppressor. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko and Prives, 1996; Sherr, 1998). Upon activation, p53 initiates the p53-dependent biological responses through transcriptional transactivation of specific target genes carrying p53 DNA binding motifs. In addition, the multifaceted p53 protein may promote apoptosis through repression of certain genes lacking ρ53 binding sites and transcription-independent mechanisms as well (Bennett et ah, 1998; Gottlieb and Oren, 1998; Ko and Prives, 1996). Analyses of a large number of mutant p53 genes in human tumors have revealed a strong selection tor mutations that inactivate the specific DNA binding function of ρ53; most mutations in tumors are point mutations clustered in the core domain of p53 (residues 94-292) that harbours the specific DNA binding activity (Beroud and Soussi, 1998).

Both p53-induced cell cycle arrest and apoptosis could be involved in p53-mediated tumor suppression. While p53-induced cell cycle arrest could conceivably be reversed in different ways, p53-induced cell death would have the advantage of being irreversible. There is indeed evidence from animal in vivo models (Symonds et al, 1994) and human tumors (Bardeesy et ah, 1995) indicating that ρ53-dependent apoptosis plays a major role in the elimination of emerging tumors, particularly in response to oncogenic signaling. Moreover, the ability of p53 to induce apoptosis often determines the efficacy of cancer therapy (Lowe et al, 1994). Taking into account me fact that more than 50% of human tumors carry p53 mutations, it appears highly desirable to restore the function of wild type p53-mεdiated growth suppression in tumors. The advantage of this approach is that it will allow selective elimination of tumor cells, carrying mutant p53. Tumor cells are particularly sensitive to p53 reactivation, suppos- edly for two main reasons. First, tumor cells are sensitized to apoptosis due to oncogene activation (reviewed in (Evan and Littlewood, 1998)). Second, mutant p53 proteins tend to accumulate at high levels in tumor cells. Therefore, restoration of the wild type function to the abundant and presumably "activated" mutant p53 should trigger a massive apoptotic response in already sensitized tumor cells, whereas normal cells that express low or undetectable levels of p53 should not be affected. The feasibility of p53 reactivation as an anticancer strategy is supported by the fact that a wide range of mutant p53 proteins are susceptible to reactivation. A therapeutic strategy based on rescuing p53-induced apoptosis should therefore be both powerful and widely applicable.

It may be generally shown that malfunctioning of the p53 pathway is generally involved in a number of diseases, such as those enumerated herein above.

Taken together, these findings suggest that pharmacological restoration of p53 function would result in elimination of tumor cells. Consequently, there is a need within this field to achieve methods and substances for use therein, which enable such a restoration.

It was earlier found that the compound PRIMA-I (i.e. 2,2-bis(hydroxymethyl)-l- azabicyclo[2.2.2]octan-3-one) (disclosed in WO0224692), is able to induce apoptosis of cells carrying mutant p53 and also in melanoma cells carrying wt p53 which are inactivated (WO 04084893). Later some other analogues to PRIMA-I that showed similar results were disclosed in WO05090341.

There however remains a need of still further compounds capable of providing a therapeutic benefit in the treatment of disorders of the kind that are susceptible of being related - directly or indirectly - to p53 abnormality and one object of the present invention is to provide such compounds.

Summary of the invention

The present inventors now surprisingly have found new compounds capable of exerting an advantageous biological activity that is susceptible of providing them with a usefulness in the treatment of disorders wherein malfunctioning of the p53 pathway may be involved, and this discovery forms the basis of the present invention. Consequently, according to one aspect, the present invention provides a compound of formula (I) or (II)

wherein

R¹ is selected from branched or unbranched, saturated or unsaturated, unsusbstituted or substituted C1-C6 alkyl and C3-C12 cycloalkyl, and non-susbstituted or substituted C6-C10 aryl;

R² is selected from H; non-susbstituted or substituted C1-C6 alkyl; unsubstituted or substituted C6-C10 aryl; unsubstituted or substituted benzyl, and unsubstituted or substituted het- erocycles or heteroaryl;

R³ is absent or is selected from branched or unbranched, saturated or unsaturated, unsusbstituted or substituted C1-C6 alkyl and C3-C12 cycloalkyl, or non-susbstituted or substituted C6-C10 aryl;

R⁴ is selected from H and C1-C6 alkyl; unsubstituted or substituted C6-C10 aryl, unsubstituted or substituted heterocycles or heteroaryl;

X^" is a pharmaceutically acceptable anion;

as well as pharmaceutically acceptable salts or prodrugs thereof, for use as a medicament.

In one embodiment of the invention, Rl is selected from C1-C6 alkyl. In another embodiment of the invention, Rl is selected from C1-C3 alkyl. In one embodiment of the invention, R3 is absent.

In one embodiment of the invention, R4 is is selected from C1-C6 alkyl. In another embodiment, R4 is selected from C1-C3 alkyl.

In one embodiment of the invention Rl, R3 and R4 are unsubstituted.

In one embodiment of the invention, the substituents, when present on any of R1-R4, are selected from C1-C6 alkyloxy and 4-6 membered heterocycles.

In one embodiment of the invention, the compound of formula (I) is a compound wherein Rl is ethyl, R3 is absent, R4 is methyl and R2 is selected from H; unsubstituted or substituted C1-C6 alkyl; unsubstituted or substituted C6-C10 aryl, unsubstituted or substituted benzyl, unsubstituted or substituted heterocycles or heteroaryl.

According to a further aspect, the present invention provides the use of the compounds of formula (I) or pharmaceutically acceptable salts or prodrugs thereof for the treatment of diseases associated with a malfunctioning p53 signalling pathway.

According to a still further aspect, the invention provides pharmaceutical compositions comprising compounds of formula (I), or salts or prodrugs thereof.

According to a still further aspect, the invention provides a method of medical treatment by administration of a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof to a mammal in the need of such treatment.

According to one aspect, the invention provides the use of the inventive compounds, or salts or prodrugs thereof in the manufacture of a medicament for the treatment or prevention of a disorder selected from hypeproliferative diseases, autoimmune diseases and heart diseases.

Any further aspects are as defined in the claims. Brief description of the drawings

Fig. 1 Fluorometric microculture cyo toxicity assay (FMCA): Percent fluorescence signal intensity vs. concentration of inventive compound 4,8-diethoxy-l,3-dimethyl-2-benzyl- cyclohepta[c]pyrrolium perchlorate in microtiter plates containing various human tumor cell lines.

Fig. 2 Fluorometric microculture cyo toxicity assay (FMCA): Percent fluorescence signal intensity vs. concentration of inventive 4,8-diethoxy-l,3-dimethyl-2-benzyl-cyclohepta[c] pyr- rolium perchlorate in microtiter plates containing various human tumor cell lines.

Figure 3 is a bar diagram showing caspase activation by 4,8-diethoxy-l,3-dimethyl-2-pentyl- cyclohepta[c]pyrrolium perchlorate.

Detailed description of the invention

In the pyrrolium compound according to the invention the positive charge on the nitrogen atom is associated with a negatively charged pharmaceutically acceptable anion. The anion may be derived from a mineral acid such as, for example, an anion selected from chloride, bromide, iodide, sulfate, nitrate, phosphate, and perchlorate. The anion also may be derived from an organic acid such as, for example, an anion selected from acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p- to luenesulfonate .

It should be realized that the compound of formula (I), as well as the compound of formula (II) may additionally comprise other functional moieties that may likewise form pharmaceutically acceptable salts, e.g. with pharmaceutically acceptable acids or bases..

Unless otherwise specified, the alkyl groups that are considered useful in the compounds according to the invention generally may be selected from unbranched or branched, cyclic, saturated or unsaturated (alkenyl or alkynyl) hydrocarbyl radicals. Where cyclic, the alkyl group is preferably C3 to C 12, more preferably C5 to ClO, most preferably C5-C7. Where acyclic, the alkyl group is preferably Cl to ClO, more preferably Cl to C6, more preferably methyl, ethyl, propyl (n-propyl, isopropyl), butyl (branched or unbranched) or pentyl, most preferably methyl. As used herein, the term "C6-C10 aryl" means phenyl or naphthyl.

As used herein, the term "heteroaryl" means an aromatic group containing one or more het- eroatom(s) preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxa- zolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thio- phenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzo imidazolyl, benzthiazolyl, purinyl, cinnolinyl, pterdinyl.

As used herein, the term "heterocycle" means a non-aromatic cyclic group containing one or more heteroatom(s) preferably selected from N, O and S, such as a cyclic amino group such as pyrrolidinyl, piperidyl, piperazinyl, morpholinyl or a cyclic ether such as tetrahydrofuranyl, monosaccharide.

As used herein the term "halogen" means a fluorine, chlorine, bromine or iodine.

As used herein, and unless specified otherwise, the term "substituted" means that the entity is substituted with at least one moiety selected from saturated or unsaturated, branched, un- branched or cyclic alkyl, or at least one functional group such as hydroxyl, amine, sulfide, silyl, carboxylic acid, halogen, aryl, etc.

The inventive compounds will be useful for treating or preventing various diseases such as hyperproliferative diseases, e.g. cancer, autoimmune diseases, such as rheumatoid arthritis and Sjogren's syndrome, and heart diseases such as hereditary idiopatic cardiomyopathy. The treatment may be preventive, palliative or curative.

It should be realized that the compound of formula (I), as well as the compound of formula (II) may additionally comprise other functional moieties that may likewise form pharmaceutically acceptable salts, e.g. with pharmaceutically acceptable acids or bases.

Thus, examples of pharmaceutically acceptable acid addition salts for use in the pharmaceutical compositions of the present invention include those mentioned herein above, e.g. derived from mineral acids, such as hydrochloride, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids. Examples of pharmaceutically acceptable base addition salts are ammonium, alkali metal (e.g. sodium or potassium) or alkaline earth metal (e.g.magnesium or calcium) salts.

The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. The pharmaceutically acceptable carrier may be one which is chemically inert to the active compounds and which have no detrimental side effects or toxicity under the conditions of use. Pharmaceutical formulations are found e.g. in Remington: The Science and Practice of Pharmacy. A. R. Gennaro, Editor. Lippincott, Williams and Wilkins, 20th edition (2000).

Prodrugs of the compounds of formulae (I) and (II) may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formulae (I) and (II) wherein a hydroxy, amino, sulfhydryl, carboxy or car- bonyl group in a compound of formula (I) or (II) is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, or sulfhydryl group, respectively.

Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxy functional groups, esters groups of carboxyl functional groups, N-acyl derivatives, N-Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. "Design of Prodrugs" pl-92, Elesevier, New York-Oxford (1985).

The composition according to the invention may be prepared for any route of administration, 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 solution is suitably 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. A brief review of methods of drug delivery is also found in e.g. Langer (1990). The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable time frame. One skilled in the art will recognize that dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease. The dose will also be determined by the route (administration form), timing and frequency of administration. In the case of oral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula (I) or (II) or the corresponding amount of a pharmaceutically acceptable salt or prodrug thereof.

The compounds of the present invention may be used or administered in combination with one or more additional drugs useful in the treatment of diseases mediated by mutant p53, or wherein a malfunction of the p53 signalling pathway is involved, such as cytostatic drugs. The components may be in the same formulation or in separate formulations for administration simultaneously or sequentially. The compounds of the present invention may also be used or administered in combination with other treatment such as irradiation for the treatment of cancer.

Examples of cytotstatic compounds for use as indicated herein above are DNA alkylating compounds, topoisomerase I inhibitors, topoisomerase II inhibitors, compounds interfering with RNA and DNA synthesis, compounds polymerising the cytoskeleton, and compounds depolymerising the cytoskeleton.

Compounds according to formula (I) and (II) are commercially available, or may be prepared by methods that are well within the knowledge of the person skilled in the art.

Biology

HTS screening

Cell lines

The human SAOS-2 (human osteosarcoma) cell line lacking p53 expression and its trans- fected clone SAOS-2-His273 that carries tetracycline-regulated mutant p53 constructs were used for the HTS screen. Cells were cultured in Iscove's Modified Dulbecco 's Medium supplemented with 10% fetal bovine serum, L-glutamine and gentamycin. WST-I assay

Cells were plated on 96-well plates at a density of 3000 cells per well per 100ml medium, cultured overnight and treated with compounds at a concentration of 1, 5, 10, 25, or 50μM, respectively. After 96h lOμl of WST-I -cell proliferation reagent were added to each well. Samples were incubated at 37⁰C for 1-2 h and absorbance of samples was measured at 490nm. Survival of the untreated cells was taken as 100%.

A number of compounds according to the invention, that all showed inhibition of cell proliferation at a cone, ranging from 1 to 50 μM, are illustrated in Table 1 herein below, wherein the negative counterion is not represented. In the present experiments, the counterion was perchlorate (ClOzf). However, it is to be understood that the counterion may be any pharmaceutically acceptable counterion.

Table 1. Some selected results from IC50 determination and the comparison between derivative in human SAOS-2 (human osteosarcoma) cell line lacking p53 expression and in a SAOS-2-His273 cell line that carries tetracycline-regulated mutant p53 constructs.

Human tumor cell-line panel

To evaluate the activity patterns of the drugs a human cell line panel of four sensitive parental cell lines, five drug resistant sublines, representing different mechanisms of resistance, and one cell line with primary resistance was used. The cell lines included were the myeloma cell line RPMI 8226/S and its sublines 8226/Dox40 and 8226/LR-5, the lymphoma cell lines U- 937 GTB and U-937-Vcr, the SCLC cell line NCI-H69 and its subline H69AR, the renal adenocarcinoma cell line ACHN (ATCC) and the leukaemic cell line CCRF-CEM and its subline CEM/VM-1. The 8226/Dox40 cell line was selected for doxorubicin resistance and shows the classical MDR phenotype with overexpression of P-glycoprotein 170 (Pgp-170). The 8226/LR-5 cell line was selected for melphalan resistance, proposed to be associated with increased levels of GSH. The U-937-Vcr cell line was selected for vincristine resistance, proposed to be tubulin associated. The H69AR cell line, selected for doxorubicin resistance, expresses a MDR phenotype proposed to be mediated by MRP. The CEM/VM-1 cell line, selected for teniposide resistance, expresses an atypical MDR, which is proposed to be topoi- somerase II (topoll) associated. The exact mechanism of resistance for the primary resistant ACHN cell line is not known and may be multifactorial.

The cell lines were grown in complete culture medium consisting of carbonate buffered culture medium RPMI- 1640 (HyClone, Cramlington, UK) supplemented with 10% inactivated FCS, 2mM glutamine, 50 μg/ml of streptomycin and 60 μg/ml of penicillin, at 37°C in hu- midified atmosphere containing 5% CO₂ . The 8226/Dox40 cell line was treated once a month with doxorubicin at 0.24 μg/ml and the 8226/LR-5 cell line at each change of medium with melphalan at 1.53 μg/ml. The U-937-Vcr cell line was continuously cultured in presence of 10 ng/ml of vincristine and the H69AR cell line was alternately fed with drug free medium and medium containing 0.46 μg/ml of doxorubicin. The CEM/VM-1 cell line was cultured in drug free medium without any loss of resistance for a period of 6-8 months. The resistance patterns of the cell lines were routinely confirmed in control experiments.

Table 2 Human tumor cell lines used in the study

The fluorometric microculture cytotoxicity assay (FMCA)

The fluorometric microculture cytotoxicity assay (FMCA) is based on measurement of fluorescence generated from hydrolysis of FDA to fluorescein by cells with intact plasma membranes and has been described in detail previously in the literature.

Tumor cells were seeded in the drug prepared 384-well micro titer plates at a cell density of 5,000 cells/well. The plates were incubated at 37°C in humidified atmosphere containing 5% CO₂ for 72 hrs. At the end of the incubation period the medium was removed by aspiration. After one wash in PBS, 50 μl/well of FDA dissolved in a physiological buffer (10 μg/ml) was added. The plates were incubated for 45 minutes and the generated fluorescence from each well was measured in a 384-well scanning fiuorometer. The fluorescence is proportional to the number of intact cells in the well.

Quality criteria for a successful analysis included a fluorescence signal in the control wells of more than five times mean blank value, a mean coefficient of variation (CV) in the control wells of less than 30%. Experiments were performed twice (2 for ten- fold dilutions and 2 for 5 -fold), mean values are used throughout.

In Figs.1 and 2 test results are shown for two inventive compounds, viz. compound No. 6 (cf. Table 1): 4,8-diethoxy-l,3-dimethyl-2-benzyl-cyclohepta[c]pyrrolium perchlorate (Fig. 1); and compound No. 7 (cf. Table 1): 4,8-diethoxy-l,3-dimethyl-2-pentyl- cyclohepta[c]pyrrolium perchlorate (Fig. 2). Results are expressed as percent fluorescence signal intensity as a function of the concentration of inventive compound in the medium of microtiter plate well and taking the blank signal as 100%. In Table 3, the corresponding EC50s calculated for the two compounds are shown.

Table 3 Activity in the cell line panel presented as EC50 in μM.

The FLICA apoptosis assay

The Carboxyfluorescein FLICA Apoptosis Detection Kit Caspase Assay, Poly-Caspases FLICA (FAM-VAD-FMK) (Immunohistochemistry Technologies, LLC) detects activated caspases. In this assay, the cell lines used were human SAOS-2 (human osteosarcoma) cell line lacking p53 expression and its transfected clone SAOS-2-His273 that carries tetracycline- regulated mutant p53 constructs. 0.038 x 10⁶ cells/ml were seeded out in cell medium in 6 well plates, 2.5 ml/well, and kept at 37°C, 5% CO₂. 24 h later 2-10 μl of substance or solvent control were added. The following day the cells were harvested, transferred to FACS tubes, and incubated for one hour with FAM-VAD-FMK FLICA. After washing, the cells were analyzed by FACS (Becton Dickinson) in detector FLl. In Fig. 3 the results, in terms of caspase activation, obtained for inventive compound No. 7 (4,8-Diethoxy-l,3-dimethyl-2-pentyl- cyclohepta[c]pyrrolium perchlorate) are shown.

References

Bardeesy et al., Cancer Res 55, 215-9 (1995).

Bennett, M., et al Science 282, 290-293 (1998).

Beroud, C. & Soussi, T., Nucl. AcidsRes. 26, 200-204 (1998).

Elkin et al (US 3726877).

Evan, G. & Littlewood, T., Science. 281, 1317-1322 (1998).

Gottlieb and Oren, Semin Cancer Biol 8, 359-68 (1998),

Ko, LJ. & Prives, C, Genes Dev. 10, 1054-1072 (1996).

Langer, Science 249:1527-1533 (1990).

Lowe et al, 1994 Science 266, 807-10 (1994).

Claims

1. A compound of formula (I) or (II)

wherein

X^" is a pharmaceutically acceptable anion;

2. The compound according to claim 1, wherein Rl is selected from C1-C6 alkyl.

3. The compound according to claim 2, wherein Rl is selected from C1-C3 alkyl.

4. The compomound according to any of the claims 1-3, wherein R3 is absent.

5. The compound according to any of the claims 1-4, wherein R4 is is selected from C1-C6 alkyl.

6. The compound according to any of the claims 1-5, wherein R4 is selected from C1-C3 alkyl.

7. The compound according to any of the claims 1-6, wherein Rl, R3 and R4 are unsubsti- tuted.

8. The compound according to any of the claims 1-7, wherein the substituents, when present on any of R1-R4, are selected from C1-C6 alkyloxy and 4-6 membered heterocycles.

9. The compound according to any of the claims 1-8, having the formula

wherein R2 is selected from H; unsubstituted or substituted C1-C6 alkyl; unsubstituted or substituted C6-C10 aryl, unsubstituted or substituted benzyl, unsubstituted or substituted heterocycles orheteroaryl.

10. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any of the preceding claims, or a pharmaceutically acceptable salt or prodrug thereof, and at least one pharmaceutically acceptable excipient.

11. Use of a compound according to any of the claims 1-9, for preparing a medicament for the treatment of a disorder selected from hyperproliferative diseases, autoimmune diseases, and heart diseases.

12. The use according to claim 11, wherein the disorder is a cancer.

13. A method of treatment of a disease selected from hyperproliferative diseases, autoimmune diseases, and heart diseases by administration of a therapeutically effective amount of a compound according to any of the claims 1-11 or a pharmaceutically acceptable salt or prodrug thereof to a mammal in the need of such treatment.