DE10233412A1 - New compounds as histone deacetylase inhibitors - Google Patents

Abstract

The present invention relates to compounds of the general formula (I) DOLLAR F1 or pharmaceutically suitable salts or physiologically functional derivatives thereof, in which: DOLLAR A n is a non-aromatic ring system which contains 2 to 6 carbon atoms, in which the ring system can contain 1 or 2 double bonds ; DOLLAR A X is C, CH, or CH¶2¶; DOLLAR AY under C, CH, CH¶2¶, S, NR, CH¶2¶-CH¶2¶, DOLLAR AH¶2¶C- -CH, HC - CH¶2¶, C - CH¶2 ¶, H¶2¶C - C or C - C is selected; wherein one or more of the hydrogen atoms are optionally substituted by one or more of the substituents R '; DOLLAR A where each of the dashed lines represents a single bond, a double bond or a triple bond, a combination of a triple bond with a triple bond and a double bond with a triple bond being excluded; DOLLAR A R 'is independently H, -CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, -OH, -SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkoxy; DOLLAR A R H, is an alkyl or cycloalkyl group; DOLLAR A Z is CH, C or P; DOLLAR A p is 0 or 1.

Description

The present invention relates to Compounds as inhibitors of enzymes with histone deacetylase activity, method for the preparation of these compounds and their use for treatment of diseases associated with hypoacetylation of histones and / or other molecules related, or where the induction of hyperacetylation a useful one Has an effect, for example by inhibiting proliferation and / or Induction of differentiation and / or induction of apoptosis in transformed cells like cancer. In addition, the connections for the Treatment of other proliferative diseases, for therapy or prophylaxis of states useful, that are related to abnormal gene expression.

BACKGROUND THE INVENTION

The local rearrangement of chromatin is an important step in gene transcription activation. Dynamic changes The nucleosomal packaging of DNA must occur for the transcription proteins can come into contact with the DNA template. One of the most important Mechanisms affecting chromatin rearrangement and gene transcription are the post-translational modification of histones and other cellular Proteins by acetylation and subsequent change in the chromatin structure (Davie, 1998, Curr Opin Genet Dev 8, 173-8; Kouzarides, 1000, Curr Opin Genet Dev 0, 40-8; Ray and Allis, 2000, Nature 403, 41-4). In the case of histone hyperacetylation to lead changes electrostatic attraction for DNA and steric disabilities, which are introduced through the hydrophobic acetyl group, for destabilization the interaction of histones with DNA. Thus, acetylation of histones leads to dissolve the nucleosomes and makes the DNA for the transcription machinery accessible. Removal of the acetyl groups allows the histones to themselves bind more tightly to the DNA and neighboring nucleosomes, so that one suppressing transcription Chromatin structure is retained. The acetylation is carried out by a Series of enzymes mediated with histone acetyltransferase (HAT) activity. Conversely, acetyl groups are replaced by specific histone deacetylase (HDAC) enzymes away. The disturbance of this Mechanisms to an incorrect regulation of the transcription and can lead to a lead to tumorigenic transformation.

In addition, other molecules change how Transcription factors, their activity and stability depending of their state of acetylation. For example, PML-RAR inhibits the fusion protein associated with acute promyelocytic leukemia (APL) related, p53 by mediating deacetylation and by breaking down p53, so that APL herd the p53-dependent Because cancer surveillance no longer subject to. Expression of PML-RRR in hematopoietic precursors leads to Repression of p53-mediated transcription activation and to Protection from the p53 dependent Apoptosis caused by genotoxic stress (X-rays, oxidative stress). The However, p53 function in the presence of HDAC inhibitors restored, indicating activation of HDAC versus p53 by PML-RAR as a mechanism indicating which of the p53 inhibition is the basis (Insinga et al. 2002, manuscript submitted). Therefore Factor acetylation plays a crucial role in the anti-tumor activity of HDAC inhibitors.

Hormone receptors in the core are ligand-dependent transcription factors, which the development and homeostasis through both positive and negative control of gene expression check. There are defects in these regulatory processes the cause of many diseases and play an important role in development of cancer. Many core receptors, including T3R, RAR, and PPAR, can use the Corepressors N-CoR and SMRT interact in the absence of ligands and thereby inhibit transcription. It has also been reported that N-CoR interact with antagonist-filled progesterone and estrogen receptors. It has been shown that N-CoR and SMRT in large Protein complexes occur that also contain mSin3 proteins and histone deacetylases included (Pazin and Kadonaga, 1997; Cell 89, 325-8). Consequently reflects the ligand-induced switching of nuclear receptors from Repression to activate the exchange of the corepressor and the Coactivator complexes with antagonistic enzymatic activities reflected.

The N-CoR corepressor complex doesn't just mediate. the repression by nuclear receptors, but also interacts with additional transcription factors, including Mad-1, BCL-6 and ETO. Many of these proteins play a key role in disorders of cell proliferation and differentiation (Pazin and Kadonaga, 1997; Cell 89, 325-8; Huynh and Bardwell, 1998, Oncogene 17, 2473-84; Wang, J. et al., 1998, Proc Natl Acad Sci USA 95, 10860-5). For example, T3R was originally identified on the basis of its homology with the viral oncogene v-erbA, which, in contrast to the wild-type receptor, does not bind ligands and acts as a constitutive transcription repressor. Mutations in RARs are also associated with a number of human cancers, particularly acute promyelocytic leukemia (APL) and hepatocellular carcinoma. In APL patients, RAR fusion proteins resulting from chromosomal translocations contain either the promyelocytic leukemia protein (PML) or the promyelocytic protein Zinc finger protein (ZIP F) involved. Although both fusion proteins can interact with components of the corepressor complex, the addition of retinoic acid leads to the detachment of the corepressor complex from PML-RAR, whereas PLZF-RAR has a constitutive interaction. These results explain why PML-RAR APL patients recover completely after treatment with retinoic acid, whereas PLZF-RAR APL patients hardly respond (Grignani et al., 1998, Nature 391, 815-8; Guidez et al., 1998, Blood 91, 2634-42; He et al., 1998, Nat Genet 18, 126-35; Lin et al., 1998, Nature 391, 8911-4). In addition, a PML-RAR patient who had suffered multiple relapses after treatment with retinoic acid was recently treated with the HDAC inhibitor phenylbutyrate, leading to a complete recovery from leukemia (Warrell et al., 1998, J, Natl. Cancer Inst. 90, 1621-1625).

So far, attempts at clinical Phase II with the closely related butyric acid derivative Pivanex (Titan Pharmaceuticals) completed as monotherapy, which activity in stage III / IV at not showed small cell lung cancer (Keer et al., 2002, ASCO, Abstract No. 1253). Several HDAC inhibitors have been identified, whereby NVP-LAQ824 (Novartis) and SAHA (Aton Pharma Inc.) members of the Structural class of the hydroxamic acids that have been tested in clinical phase I trials (Marks et al., 2001, Nature Reviews Cancer 1, 194-202). Another class includes cyclic ones Tetrapeptides, such as depsipeptide (FR901228 - Fujisawa), have been successful in phase II trials for the treatment of T cell lymphoma was used (Piekarz et al., 2001, Blood 98, 2865-8). Besides, will currently MS-27-275 (Mitsui Pharmaceuticals), a compound that is related to the class of benzamides in experimental patients Phase I with hematological malignities tested.

In Int. J. Chem. Kinet. 1997, 29, 729-735 become 3-cyclopentyl-N-hydroxypropionamide, 4-cyclohexyl-N-hydroxybutyramide and 2-cyclohexyl-N-hydroxyacetamide (see also Berndt et al., 1992, Int. J. Chem. Kinet., 24, 695-701).

The crystal structure of a histone deacetylase-like Protein from the hyperthermophile Bacterium aquifex aeolicus, the was cocrystallized with the two inhibitors TSA and SAHA by Finnin et al., 1999, Nature, 401, 188-193.

Hydroxamic acids with at least one aromatic Ring or ring system as histone deacetylase inhibitors are from Lavoie et al., 2001, Bioorg. Med. Chem. Letters 11, 2847-2850; Remiszewski et al., 2002, J. Med. Chem. 45, 4, 753-757; Massa et al., 2001, J. Med. Chem. 44, 2069-2072; Sternson et al. 2001, org. Lett. 3, 26, 4239-4242; Mai et al., 2002, J. Med. Chem. 45, 1778-1784; Woo et al., 2002, J. Med. Chem. 45, 2877-2885, described.

In EP 1174438 , WO 0052033, WO 0118045, WO 0118171, WO 0138322, WO 0170675, WO 9735990, WO 9911659, WO 0226703, Wo 0230879 and WO 0226696 describe hydroxamic acids as histone deacetylase inhibitors.

In WO 9805635 and WO 9533709 hydroxamic acids described as matrix metalloproteinase inhibitors.

The compounds according to the invention are hydroxamic acids, which Inhibitors of enzymes with histone deacetylase activity are. Due to their HDAC inhibitory activity, they induce from the following three reasons differentiation and / or apoptosis in many cancer cells: (1) these enzymes are present in all cells, (2) pilot studies with model compounds, such as butyrate or TSR, which differ from those according to the invention, have shown that HDAC inhibitors induce differentiation in many cells, and (3) clinical Effectiveness was for several other HDAC inhibitors, which with the compounds of the invention are not shown in the treatment of cancer patients.

The activity to induce differentiation and / or apoptosis in many transformed cells is one biological activity much more complex than just inhibiting the Proliferation. In the latter case, it would not be clear why the proliferation of transformed cells (tumor cells), however should not be inhibited by normal cells. The induction apoptosis, differentiation or, more precisely, redifferentiation in undifferentiated tumor cells explains why the compounds of the invention advantageous by induction of differentiation and / or apoptosis Effect on a large Range of tumors.

The histone deacetylase inhibitory activity New connections can be made using a range of state of the art technologies the technology, such as by a transcriptional repression test, Western blot analysis showing the acetylation of histone H3 and / or Detects histone H4, or by an enzymatic in vitro test. Histone deacetylase inhibitors can also about her cytotoxic and growth inhibitory effects on tumor cell lines and their ability characterized for modulating gene expression patterns in cells become.

oder pharmazeutisch geeignete Salze oder physiologisch funktionelle Derivate davon, worin:
n ein nicht aromatisches Ringsystem ist, das 2 bis 6 Kohlenstoffatome enthält, worin das Ringsystem 1 oder 2 Doppelbindungen enthalten kann;
X C, CH oder CH2 ist;
Y unter C, CH, CH₂, S, NR, CH₂-CH₂,
H₂C- -CH, HC--CH₂, C--CH₂, H₂C--C oder C--C ausgewählt ist; wobei ein oder mehrere der Wasserstoffatome gegebenenfalls durch einen oder mehrere der Substituenten R' substituiert sind;
wobei jede der gestrichelten Linien eine Einfachbindung, eine Doppelbindung oder eine Dreifachbindung bedeutet, wobei eine Kombination einer Dreifachbindung mit einer Dreifachbindung und einer Doppelbindung mit einer Dreifachbindung ausgeschlossen ist;
R' unabhängig H, -CN, Alkyl, Cycloalkyl, Aminoalkyl, Alkylamino, Alkoxy, -OH, -SH, Alkylthio, Hydroxyalkyl, Hydroxyalkylamino, Halogen, Halogenalkyl, Halogenalkoxy ist;
R H, eine Alkyl- oder Cycloalkylgruppe ist;
Z CH, C oder P ist;
p 0 oder 1 ist; und
mit der Maßgabe, daß die folgenden Verbindungen ausgeschlossen sind:

The present invention relates to compounds of the general formula (I):

or pharmaceutically acceptable salts or physiologically functional derivatives thereof, wherein:
n is a non-aromatic ring system containing 2 to 6 carbon atoms, in which the ring system may contain 1 or 2 double bonds;
X is C, CH or CH2;
Y under C, CH, CH ₂ , S, NR, CH ₂ -CH ₂ ,
H ₂ C- -CH, HC-CH ₂ , C-CH ₂ , H ₂ C-C or C-C is selected; wherein one or more of the hydrogen atoms are optionally substituted by one or more of the substituents R ';
wherein each of the dashed lines represents a single bond, a double bond or a triple bond, a combination of a triple bond with a triple bond and a double bond with a triple bond being excluded;
R 'is independently H, -CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, -OH, -SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkoxy;
RH is an alkyl or cycloalkyl group;
Z is CH, C or P;
p is 0 or 1; and
with the proviso that the following connections are excluded:

An alkyl group, unless otherwise is preferably a linear or branched chain with 1 to 6 carbon atoms, preferably a methyl, ethyl, propyl, Isopropyl, butyl, t-butyl, isobutyl, pentyl or hexyl group, where a methyl, Most preferred are ethyl, isopropyl or t-butyl group.

The term "alkyl" if not otherwise stated, also means that those derivatives of alkyl are included, which are further defined below as "unsaturated alkyl". An unsaturated one Alkyl group is one with one or more double bonds or Triple bonds, preferably vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and the higher Homologues and isomers.

The alkyl group in the compounds of formula (I) can optionally with one or more of those defined above Substituents R 'may be substituted.

Designated a cycloalkyl group a non-aromatic ring system containing 3 to 8 carbon atoms, wherein one or more of the carbon atoms in the ring by a group X can be replaced, where X is as defined above.

An alkoxy group denotes one O-alkyl group, the alkyl group being as defined above.

An alkylthio group denotes one S-alkyl group, the alkyl group being as defined above.

Denoted a hydroxyalkyl group an HO alkyl group, the alkyl group as defined above is.

A haloalkyl group denotes an alkyl group which is substituted by 1 to 5, preferably 3, halogen atoms, the alkyl group being as defined above, CF _{3 being} preferred.

Haloalkyloxy group denotes an alkoxy group which is substituted by 1 to 5, preferably 3 halogen atoms, the alkoxy group being as defined above, OCF _{3 being} preferred.

A hydroxyalkylamino group denotes a (HO-alkyl) ₂ -N group or a HO-alkyl-NH group, the alkyl group being as defined above.

Denotes an alkylamino group an HN-alkyl or N-dialkyl group, wherein the alkyl group is as defined above.

An aminoalkyl group denotes an H ₂ N-alkyl group, monoalkylaminoalkyl or dialkylaminoalkyl group, the alkyl group being as defined above.

A halogen group is chlorine, bromine, Fluorine or iodine, with fluorine being preferred.

The invention also provides a pharmaceutical composition ready which a compound of formula (I) in free form or in in the form of pharmaceutically acceptable salts and physiologically functional derivatives or drug precursors together with a pharmaceutical suitable diluent or carrier for that contains.

The expression "physiologically functional Derivative "denotes compounds such as ethers, esters, N-alkylated or acetylated hydroxamic acids, 2,5-dihydro- [1,2,4] -oxodiazolyl or 4,5-dihydro- [1,2,4] -oxodiazolyl, which are not themselves pharmaceutical, but which are in vivo, i.e. in the patient to whom the compound is administered, in their pharmaceutical effective form are transferred.

In another aspect, the present invention also a method of treatment or prophylaxis a state where it is advantageous to inhibit histone deacetylase activity, the method comprising administering an effective amount of a Compound of formula (I) and of physiologically suitable salts or physiologically functional derivatives thereof.

The invention also relates to the use of compounds of formula (I) and their pharmacologically suitable Salts or physiologically functional derivatives for the production a drug for prevention and treatment of diseases in which the inhibition of histone deacetylase is advantageous.

In addition, the present Invention Process for the preparation of the desired hydroxamic acids Formula (I) ready.

A method of synthesizing the compounds of formula (I) the conversion of an acid (formula II) in the corresponding acid chloride (Formula III) and reacting the acid chloride with hydroxylamine (Watanabe et al., 1989, J. Org. Chem., 54, 17, 4088-4097; Shishido et al., 1992, J. Org. Chem., 57, 10, 2876-2883).

Coupling reactions of acids Formula (II) with hydroxylamine, other processes for the preparation of the Compounds of formula (I) are described by Woo et al., 2002, J. Med. Chem. 45, 2877-2885; Knorr et al., 1989, Tetrahedron Lett., 30, 1927-1930, Carpino, 1993, J. Am. Chem. Soc., 115, 4397-4398 and Albericio et al., 1998, J. Org. Chem, 63, 9678-9683.

Another manufacturing process of the compounds of formula (I) is the reaction of the corresponding one Hydroxylamine esters as described by Stowell et al., 1995, J. Med.

Chem, 38, 8, 1411-1413.

In a preferred embodiment in the compounds of formula (I) the ring n, including Z, cyclopentyl; Cyclohexyl, cycloheptyl, cyclopent-1-enyl, cyclohex-1-enyl, cyclohept-1-enyl, Cyclopent-2-enyl, cyclohex-2-enyl, cyclohept-2-enyl, cyclohex-3-enyl, Be cyclohept-3-enyl.

In a further preferred embodiment, in the compounds of formula (I) according to the invention the ring, including Z, cyclopentyl or cyclohexyl, and Y is selected from CH, CH ₂ , CH ₂ -CH ₂ , S, NR, or p = 0 and Z is CH or P.

In a further, more preferred embodiment, in the compounds of the formula (I) according to the invention the ring n, including Z, cyclopentyl or cyclohexyl, Y is selected from CH, CH ₂ and CH ₂ -CH ₂ , or p = 0 and Z CH.

In another, more preferred embodiment is not one of the carbon atoms of the alkyl groups by a group A replaced.

Preferred compounds according to the invention are:
3-cyclohexyl-N-hydroxypropionamide;
2-cycloheptyl-N-hydroxyacetamide.

The compounds of the formula according to the invention (I) can form salts with inorganic or organic acids or bases. Examples of such salts are alkali metal salts, especially sodium and potassium salts, or ammonium salts.

The compounds of formula (I) can by different procedures can be obtained. In preferred embodiments the inventive method the following two synthetic methods are used.

acids or acid chlorides of α-β-unsaturated Compounds of formula (I) can by hydrolysis of the corresponding ester to form the Acid preserved (Bojic et al., 1998, 354, 289-299), which are then treated with chlorinating agents (Oxalyl chloride, thionyl chloride, phosphorus pentachloride) in the acid chloride can be converted. ? -β-Unsaturated esters can be made from the corresponding aldehyde by reaction with (carbethoxymethylene) triphenylphosph? ran (PhP = CHCOOEt) can be synthesized (Maryanoff et al., 1989, Chem. Rev. 89, 863-927).

Other manufacturing processes different acids are described by Mancuso et al., 1981, Synthesis, 165-185; or Bal et al., 1981, Tetrahedron, 37, 2091-2096 described.

The compounds of the invention can be used for various human and animal diseases, preferably human Diseases are used in which the inhibition of histone deacetylase activity is advantageous is.

Therefore, the compounds of the invention and the drugs made therefrom in general used to differentiate and / or apoptose cells, such as indifferentiated tumor cells. The therapeutic Effect of the invention results from one or more mechanisms, including, but not limited on the regulation of cell proliferation, cell activation, cell survival, Cell differentiation, cell cycle, cell maturation and cell death, or the Induction of systemic changes in metabolism, like changes in sugar, lipid or protein metabolism, the inhibition of Formation of new blood vessels (anti-angiogenesis), the inhibition of tumor spread to other organs (anti-metastatic), inhibiting tumor spread to neighboring normal structures (anti-invasive) or promotion programmed cell death (apoptosis).

They can also be used to support cell formation, including blood cell growth and blood cell formation (prohaemotopoietic Effect) after reducing the number or destroying the Cells, which are caused, for example, by toxic agents, radiation, Immunotherapy, growth defects, malnutrition, malabsorption, immune deficiency regulation , Anemia and the like, or they can have therapeutic control tissue formation and degradation and therapeutic modification provide cell and tissue maintenance and blood cell homeostasis.

The compounds according to the invention and the pharmaceuticals that are made with it are also for the treatment of diseases useful, those with gene-specific hypoacetylation of histones or others molecules like p53, related. In addition, the compounds of the invention also in the treatment of conditions are used in which the suppression of anti-apoptotic Genes like BCL-XL and other members of the BCL family, or induction of tumor suppressor activity of molecules such as p21 and / or p53, is required.

The compounds and medicaments according to the invention, that are made with it are also for the treatment of diseases suitable where the induction of hyperacetylation of histones a cheap one Has effect, which leads to differentiation and / or apoptosis of tumor cells leads in patients resulting in a clinical improvement in the patient's condition is effected. Examples of such diseases include, but not limited to this are skin cancer, melanoma, estrogen receptor dependent and independent breast cancer, Ovarian cancer, testosterone receptor-dependent and independent prostate cancer, stomach cancer, Colorectal and colon cancer, pancreatic cancer, bladder cancer, esophageal cancer, Stomach cancer, urinary tract cancer, gastrointestinal cancer, uterine cancer, astrocytomas, Gliomas, basal cell cancer and sponge cell cancer, sarcomas such as Kaposi's Sarcoma and osteosarcoma, head and neck cancer, small cell or non-small cell lung cancer, leukemia, lymphoma and other blood cell cancers.

The combinatorial treatment according to the invention is particularly minimal for the treatment of remaining tumor diseases or tumor metastases are useful.

In addition, the invention can thereby useful be that one inappropriate gene expression in diseases that differ from the ordinary form Recruitment of histone deacetylase activity, such as thyroid resistance syndrome, or in other conditions, associated with abnormal gene expression is reversed as in Inflammatory diseases Diabetes, thalassemia, Cirrhosis, protozoan infections or the like, and in all types autoimmune diseases, especially rheumatic arthritis, rheumatic spondylitis, all forms of rheumatism, osteoarthritis, Gouty arthritis, multiple sclerosis, insulin dependent diabetes mellitus and not insulin dependent Diabetes, arthritis, runny nose, uveithis, lupus erythematoidis, ulcerative Colitis, Crohn's disease, inflammatory Abdominal disease, as well as other chronic inflammation and chronic diarrhea.

In addition, the compounds of the invention and medicines made therewith for treatment other proliferative diseases useful, such as psoriasis, fibrosis and other dermatological disorders. The expressions "Proliferative disease" and "cell proliferation" are mutually exclusive here used interchangeably and affect an unwanted or uncontrolled Cell proliferation of excess or abnormal cells, such as neoplastic and hyperplastic growth, be it in vitro or in vivo. Examples of proliferative conditions include but are not limited to precancerous and cancerous cell proliferation, including malicious Neoplasms and tumors, crayfish, leukaemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. connective tissue) and arteriosclerosis. Anyone Cell type can be treated, including but not limited to limited, Cells of the lungs, intestines, breasts, ovaries, the Prostate, liver, pancreas, brain and skin, and any treatment of disorders related to T cells like aplastic anemia and DiGeorge syndrome and Graves disease can be done.

The invention also includes Compounds of formula (I) used in the patient in a compound the formula of the invention are metabolized. Those described in the present invention embodiments are also applicable to such connections.

The present invention relates to also a diagnostic method for the identification of tumors, which includes the stage in which it is tested in vitro whether a tumor is on treatment either with compounds of formula (I) or in combination with addresses established tumor therapeutics. The method preferably comprises the process of identifying genes through these treatments be regulated. In a particular embodiment, the diagnostic includes Process the use of nucleic acid technology, preferably hybridization or polymerase chain reaction for detection. Other types of nucleic acid technologies can however, can also be used. In another embodiment comprises the procedure for Evidence of the use of specific antibodies against differently regulated Proteins: For this purpose, proteins are made by these genes are coded, which are expressed in terms of their expression in combinatorial Treatment using formulations and derivatives according to the invention thereof are deregulated, for example in recombinant expression systems expressed, and antibodies, that are directed against these proteins. After that, such Antibodies (or Patterns of antibodies) used to determine the condition of a tumor or of tumor cells for diagnostic and / or to characterize prognostic purposes.

In general, the present Invention new opportunities ready to treat various human diseases. The Applicants found that the HDAC inhibitory and cell differentiation-inducing activity of the compounds of the formula (I) successful in combination with well-established and clinical investigated therapeutic drugs for the treatment of tumor cells of different origins can be used. Such a combinatorial Treatment based on these compounds leads to better therapeutic Success in human tumor patients than in the corresponding ones therapeutic drugs that are used alone. It is an inventive subject, combinatorial therapeutic approaches using the compounds of the invention for the To provide treatment for cancer. Such combinatorial treatments can to reduce therapeutic doses, for example necessary chemotherapeutic agents and can therefore monitor the currently observable sometimes considerable side effects of frequently used therapies limit.

• – chemotherapeutische oder zytotoxische Arzneimittel (wie 5-FU, Taxol, cisPlatin, Camptothecin, Gemcitabin, Doxorubicin, Irinothecan)
• – Differenzierungs-induzierende Arzneimittel (wie Vitamin D, Retinirsäure, Cytokine, wie II-3, II-6, SCF, G-CSF, GM-CSF, TNF)
• – Bestrahlungstherapie (wie Röntgenstrahlen oder gamma- Strahlen)
• – immunologische Ansätze (Antikörpertherapie, Impfung)
• – kombinierte immuntherapeutische/zytotoxische Ansätze (wie Antikörper konjugiert mit zytotoxischen Komponenten)
• - anti-Angiogeneseansätze.

Aspects of the present invention are the combination of compounds of formula (I) with, but not limited to, therapeutic principles currently in clinical use or development, such as

• - chemotherapeutic or cytotoxic drugs (such as 5-FU, taxol, cisplatin, camptothecin, gemcitabine, doxorubicin, irinothecan)
• Differentiation-inducing drugs (such as vitamin D, retinic acid, cytokines such as II-3, II-6, SCF, G-CSF, GM-CSF, TNF)
• - radiation therapy (such as x-rays or gamma rays)
• - immunological approaches (antibody therapy, vaccination)
• - combined immunotherapeutic / cytotoxic approaches (such as antibodies conjugated with cytotoxic components)
• - anti-angiogenic approaches.

The compounds and salts thereof can as pharmaceutical compositions (such as liquids, suspensions, emulsions, Pastilles, gelatin capsules, ampoules, suppositories, pessaries, ointments, gels, Pastes, sprays, lotions, oils, size Pills, latwerge, aerosols, powders, granules, tablets, pills, Capsules, syringes, solutions, foams, Creams, enemas and the like), which have at least one such connection alone or in a mixture with pharmaceutically suitable carriers, corrections and / or diluents contain. The drug compositions can be formulated according to conventional methods his.

Specific dose concentrations be for the individual patients depending on various factors, including age, body weight, the general condition, gender, diet and previous diseases, the Severity of the patient's specific illness and activity of the specific compounds used, the time of administration, the route of administration, the excretion rate, the duration of treatment, of other drugs, of Compounds and / or materials used in combination, used. It is appreciated that the suitable dosage of the active compounds and the compositions, which contain the active compounds from patient to patient can vary. Determining the optimal dosage generally requires a balance between therapeutic benefits and any Risk or harmful Side effects of the treatments according to the invention.

Administration in vivo can be in one dose, continuously or interrupted over the treatment period. Procedures for determining the most effective procedures and dosages for administration are well known to the person skilled in the art and vary with the for the formulation used, the purpose of the therapy, the target cell to be treated and the patient to be treated. Single or multiple administrations can be made with dosages and dosage patterns carried out selected by the attending physician.

In general is an appropriate one Doses of the active compound range from about 0.1 to about 500 mg per kg body weight, preferably 0.1 to 100 mg per kg of patient body weight per Day. If the active ingredient is a salt, an ester, a precursor drug or the like, the amount administered is based the output connection is calculated so that the weight actually used accordingly increases.

assays

1. Transcription reporter gene assay

The transcription assay uses for repressor activity activation and depression of a Gal4-dependent reporter gene from (Hildebrand et al., 2001, J Biol Chem 276, 9889-95; Maurer et al., 2002, Blood 99, 2647-52). This assay is made using specially designed permanent cell lines carried out. Transcription factors, such as the thyroid hormone receptor, PPARδ, retinoic acid receptor, N-CoR and AML / ETO, repress transcription when attached to one Promoter containing UAS elements as fusion proteins with the heterologous DNA binding domain of the Gal4 protein bind from yeast. In the absence of the Gal4 fusion protein, this has Reporter gene due to the presence of binding sites for others A high level of transcription factors in the thymidine kinase promoter basal transcription activity. The Gal4 fusion proteins repress this activity up to 140-fold. HDAC inhibitors induce the release of this repression, resulting in an increase in the reporter gene activity (for example, by proof of luciferase activity) can.

2. Induction of histone hyperacetylation

Induce histone deacetylase inhibitors the accumulation of N-terminal hyperacetylated histones H3 and H4. These acetylated histones can by Western blot analysis of the whole cell extract or of histone preparations from histone deacetylase inhibitor-treated cells using of antibodies, the specific for Acetylated N-terminal lysine residues of histones H3 and H4 have been detected become (Göttlicher et al., 2001, EMBO J 20, 6969-78).

3. Measurement of cellular metabolic activity after treatment with HDAC inhibitors

The viability of tumor cell lines after treatment with various concentrations of histone deacetylase inhibitors can be determined by a cell titer 96® AQ _ueous cell proliferation test (Promega) be true. Tumor cell lines placed in a 96-well microtiter plate are treated with histone deacetylase inhibitors for 48 hours. After adding the "One Solution Reagent", a tetrazolium compound (MTS) is bioreduced into a soluble colored farmazan product by the dehydrogenase enzymes in metabolically active cells. The life activity and metabolic activity of histone deacetylase inhibitor-treated cells can be calculated in comparison to untreated cells, since the MTS conversion is directly proportional to the number of living cells in the culture.

4. Protein expression profile

Histone hyperacetylation is related to an open, unfolded chromatin structure and gene activation, while hypoacetylation is related to chromatin compression and transcriptional repression. Treatment of tumor cell lines with histone deacetylase inhibitors leads to histone hyperacetylation and transcription regulation of target genes. The expression pattern induced by histone deacetylase inhibitors can be monitored by Western blot analysis from whole cell extracts, for example by inducing the expression of the cell cycle inhibitor / tumor suppressor p21 and suppressing the expression of the anti-apoptotic molecule BCL-X _L.

5. Measurement of histone deacetylase activity

Inhibition of histone deacetylase activity in nuclear extracts by histone deacetylase inhibitors can be monitored by in vivo enzymatic assays become. Nuclear extracts, immunoprecipitates or recombinant Histone deacetylases are made with different concentrations of Histone deacetylase inhibitors incubated. The histone deacetylase activity is determined by Deacetylation of a fluorogenic substrate (Fluor de Lys, Biomol) observed. The ICSO values can can be calculated from the dose-response curves.

6. Growth inhibition of tumor cell lines

The reduction of cellular biomass was through SRB assays measured. For This assay was performed on cells in 96-well culture plates at densities between 3000 and given 8000 cells per hole. After 24 hours, the cells 48 to 72 hours in the absence or presence of the specified Concentrations of compounds cultivated. The synergistic reduction total cell biomass was added by an SRB assay from 1 to 500 μM of the chemotherapeutic agent cisPlatin in combination with the Compounds in the medium, or by irradiating the cells with X-rays (2 Gray) 24 hours after treatment with the compounds and followed by Cultivation during Tested for 48 hours.

The cells were fixed with cold trichloroacetate (TCA) to give a final TCA concentration of 10%. After 1-hour incubation at 4 ° C., the cells were washed 5 times with water and air-dried. The fixed cells were stained with 0.4% (w / v) sulforhodamine B (SRB) dissolved in 1% acetic acid for 30 minutes and washed 4 times with 1 acetic acid to remove unbound dye. After air drying, bound dye was dissolved with 10 mM unbuffered Tris base (pH 10.5) for 5 minutes. The optical densities (OD) were read with a Molecular Devices Versa Max microplate reader at 520 to 550 nm. Four test holes were set in parallel with 12 control holes per cell line for each dose-dependent response. The measurement of the cell population density at time 0 (T ₀ ; the time at which the drug was added) was also carried out with 12 reference holes for cells which were fixed with TCA immediately before the drug was added to the test plates. The optical density of the background of the complete medium with 5 FBS, fixed and stained as described above, was also determined in 12 separate holes. From the unprocessed optical density data for each microtiter plate, the measurement of the background optical density (ie the optical density of the complete medium including the coloration and the optical density of the cells at time T ₀ ) was subtracted, thereby reducing the cell biomass of the cells was obtained.

7. Cell cycle analysis

Analysis of the effect of the compounds cell cycle distribution was assessed by flow cytometry after propidium iodide staining Cancer cell lines performed. Cells were placed in 6-well culture plates at 100,000 cells / well. The day after they were treated with the compounds and then Cultivated for 48 or 72 hours. The cells were filled with propidium iodide stained after permeabilization and ethanol fixation. The Cell cycle analysis results are as a percentage (%) of cells in the cell cycle phases G1, S, G2 + M and sub-G1 (apoptotic cells) specified. All of these assays were performed in duplicate.

8. Measurement of apoptosis

Cells were placed in 6-well culture plates at 100,000 cells / well. The day after they became treated with the compounds and then cultured for 48 or 72 hours. Induction of apoptosis was measured using the Vybrant® Apoptosis Assay Kit from Molecular Probes Inc. Apoptotic cells were stained with the green fluorescent dye Yo-Pro-1® and with propidium iodide to exclude necrotic cells. The analysis of the fractions of the viable, apoptotic and necrotic cells was carried out by flow cytometry.

Synthesis of 3-cyclohexyl-N-hydroxypropionamide

To a solution of 3-cyclohexylpropionyl chloride (12.1 ml, 12.6 g, 72.0 mmol) in dichloromethane (50 ml) were added hydroxylamine hydrochloride (5.00 g, 72.0 mmol) and sodium bicarbonate (12.0 g. 144 mmol) was given. After stirring for 24 hours at room temperature, the reaction was stopped by adding a saturated ammonium chloride solution (50 ml). The layers were separated. The water layer was extracted with ethyl acetate (4 x 50 ml). The solvent of the combined organic layers was removed in vacuo. Precipitation of the crude product from ethyl acetate by the addition of petroleum ether gave hydroxamate (7.21 g, 42 mmol, 58%) as a white solid. ¹ H NMR (300 MHz, [D ₆ -DMSO]: δ = 0.95-1.12 (m, 2H), 1.38-1.69 (m, 6H), 1.62-1.77 (m, 3H), 1.94 (t, J = 7.6 Hz, 2H), 8.58 (s, 1H) and 10.28 (s, 1H) (NH and OH); ¹³ C-NMR ( 75 MHz, [D ₆ -DMSO]? = 25.0, 31.8, 32.0, 32.3, 39.5, 169.5 (C (=) NHOH); MS: m / z calculated for ( C ₉ H ₁₇ NO ₂ ) [M + H] ⁺ 172; found 172.

Biological data

The active concentrations for an exemplary compound of the invention, determined using the assays described above, are shown in Table 1 below. Table 1

Claims (17)

Compounds of the general formula (I)

or pharmaceutically acceptable salts or physiologically functional derivatives thereof, wherein: n is a non-aromatic ring system containing 2 to 6 carbon atoms, in which the ring system has 1 or 2 double bonds. may contain; X is C, CH or CH ₂ ; Y under C, CH, CH ₂ , S, NR, CH ₂ -CH ₂ , -H ₂ C- -CH, HC - CH ₂ , C - CH ₂ , H ₂ C - C or C - C is selected; wherein one or more of the hydrogen atoms are optionally substituted by one or more of the substituents R '; wherein each of the dashed lines represents a single bond, a double bond or a triple bond, a combination of a triple bond with a triple bond and a double bond with a triple bond being excluded; R 'is independently H, -CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, -OH, -SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkoxy; R is H, an alkyl or cycloalkyl group; Z is CH, C or P; p is 0 or 1; and with the proviso that the following connections are excluded:

A compound according to claim 1, wherein n is cyclopentyl or cyclohexyl is. A compound according to claim 1, wherein n is cyclopentyl or cyclohexyl is and Z is CH. Pharmaceutical composition, which is a compound according to a of claims 1 to 3 in free form or in the form of pharmaceutically suitable ones Contains salts or physiologically functional derivatives. A compound according to any one of claims 1 to 3, including the Compounds excluded in claim 1 for use as a medicine. Use of a compound according to claim 5 for the manufacture a drug for use in the treatment of a disease or therapeutic Indication as inhibitors of enzymes with histone deacetylase activity. Use of a compound according to claim 5 in the manufacture a drug for use in the treatment of a disease or therapeutic indication, where inhibition of histone deacetylase activity is useful. Use according to claim 6, wherein the human histone deacetylase selected from the group those from HDACs 1-10 or a member of the SIR2 protein family. Use of a compound according to claim 5 in the manufacture a drug for the induction of cell differentiation. Use of a compound according to claim 5 in the manufacture a drug for induction of differentiation from transformed cells. Use of a compound according to claim 5 in the manufacture a drug for induction of apoptosis from transformed cells. Use of a compound according to claim 5 in the manufacture a drug for inhibiting the proliferation of transformed cells. Use of a compound according to claim 1 for inhibition of histone deacetylase activity. Use of a compound of formula (I) according to claim 5 in the manufacture of a drug, the induction of hyperacetylation of histones a useful one Has an effect. Use of a compound according to claim 5 in the manufacture a drug for use in the treatment of a disease or therapeutic Indication selected from the group consisting of skin cancer, melons, estrogen receptor dependent or -unabhängigem Breast cancer, ovarian cancer, prostate cancer, stomach cancer, colon cancer and colon cancer, pancreatic cancer, head or semi-cancer, small cell or non-small cell lung cancer, leukemia and other types of blood cell cancer and endocrine diseases that on a change histone deacetylase, like thyroid resistance syndrome, consists. Use of a compound according to claim 5 in the manufacture a drug for the use in the inhibition of abnormal gene expression, such as in inflammatory diseases, Diabetes, thalassemia, Cirrhosis or protozoan infection. A process for the preparation of a compound as claimed in claim 1, which comprises the step in which an acid of formula (II)

(wherein n, X, Y, Z and p are as defined in claim 1) or an acid chloride of the formula (III)

(wherein n, X, Y, Z and p are as defined in claim 1) is reacted with hydroxylamine.