DE102004049622A1 - Novel para-sulfonamido-substituted 2-anilinopyrimidines are useful in a wide range of medicaments and as cell cycle kinase inhibitors or receptor tyrosine kinase inhibitors - Google Patents

Abstract

Para-sulfonamido-substituted 2-anilinopyrimidines and their isomers, diastereomers, enantiomers and/or salts are new. Para-sulfonamido-substituted 2-anilinopyrimidines of formula (I) and their isomers, diastereomers, enantiomers and/or salts are new. A and D : halogen, hydroxy, cyano, -OR 5>, 3-6C cycloalkyl or 1-4C optionally branched alkyl optionally once or more substituted by halogen, hydroxy or OR 5>; X : -NH-, -N(1-3C optionally branched alkyl)- or -O-; R 1>halogen or cyano; R 2>1-3C alkoxy-substituted hydroxy-1-8C optionally branched alkyl or hydroxy- or 1-3C alkyl-substituted 3-7C cycloalkyl; R 3>and R 4>H or 1-3C optionally branched alkyl optionally once or more substituted by hydroxy, -OR 5>or -NR 6>R 7>; R 5>optionally halogen-substituted 1-4C optionally branched alkyl; and R 6>and R 7>1-3C alkyl optionally substituted by hydroxy or -OR 5>. Independent claims are also included for (1) the use in the preparation of (I) of acetylaminobenzene sulfonyl halides or acetylaminobenzene sulfonamides of formula (IIa) and (IIb) respectively or their isomers, diastereomers, enantiomers and/or salts; and (2) pharmaceuticals including (I). [Image] ACTIVITY : Cytostatic; Ophthalmological; Immunosuppressive; Dermatological; Antiinflammatory; Cardiant; Nephrotropic; Virucide; Vulnery; Hepatropic; Antiarteriosclerotic; Vasotropic; Antidiabetic; Antipsoriatic; Thrombolytic; Antiparkinsonian; Nootropic; Neuroprotective; Anti-HIV; Anticonvulsant; Antirheumatic. MECHANISM OF ACTION : (I) can inhibit proliferation of tumor cells and/or tumor angiogenesis with simultaneous reduction of side effects by a carboanhydrase effect. Proliferation tests of 4-[5-Bromo-4-((R)-2-hydroxy-1-dimethylethylamino)-pyrimidin-2-ylamino]-2,6-dimethylbenzenesulfonamide with mamma carcinoma cells (MCF7), prostate carcinoma cells (DU145) and multiply-resistant mamma carcinoma cells (MaTu-MDR) gave proliferation values (IC 50) of below 0.1, 0.09 and 0.07 mu M respectively, the corresponding values for a control comprising the pyrimidine compound of Example 10 of being 0.4, 0.7 and 0.8 mu M.

Description

The The present invention relates to substituted 2-anilino-pyrimidines as cell cycle kinase and / or receptor tyrosine kinase inhibitors, their preparation and use as medicaments for treatment or prophylaxis of various diseases.

The cyclin-dependent Kinases (cyclin-dependent kinase, CDK) are an enzyme family that plays an important role in the regulation of the cell cycle and thus a particularly interesting target for the development of small inhibitory molecules represents. Selective inhibitors of CDKs may be used to treat cancer or other disorders that disrupt cell proliferation to the cause have to be used.

receptor and their ligands, specifically the function of endothelial cells regulate, are critically important to the physiological, as well as the pathogenic angiogenesis involved. Really important Here is the Vascular Endothelial Growth Factors (VEGF) / VEGF receptor System. In pathological situations with increased neovascularization go along, such. Tumor disease, was an increased expression of angiogenic growth factors and their receptors. Inhibitors of the VEGF / VEGF receptor system may be the formation of a blood vessel system in the tumor inhibit, thus the tumor from the oxygen and nutrient supply and thus inhibit tumor growth.

Pyrimidines and analogues have already been described as active substances, for example the 2-anilino-pyrimidines as fungicides ( DE 4029650 ) or substituted pyrimidine derivatives for the treatment of neurological or neurodegenerative diseases (WO 99/19305). As CDK inhibitors a wide variety of pyrimidine derivatives are described, for example bis (anilino) pyrimidine derivatives (WO 00/12486), 2-amino-4-substituted pyrimidines (WO 01/14375), purines (WO 99/02162), 5-cyano Pyrimidines (WO 02/04429), anilinopyrimidines (WO 00/12486) and 2-hydroxy-3-N, N-dimethylaminopropoxypyrimidines (WO 00/39101).

In particular, WO 02/096888 and WO 03/7076437 have disclosed pyrimidine derivatives which have inhibitory effects on CDKs. Compounds which contain a phenylsulfonamide group are known as inhibitors of human carbonahydrases (in particular carbonahydrase-2) and are used as diuretics, inter alia, for the treatment of glaucoma. The nitrogen atom and the oxygen atoms of the sulfonamide bind by hydrogen bonding with the zinc ²⁺ ion and the amino acid Thr 199 in the active site carbonic anhydrase-2 and thereby block their enzymatic function (A. Casini, F. Abbate, A. Scozzafava, CT Supuran, Bioorganic Med Med Chem 2003, 1, 2759.3).

A increase of specificity the known CDK inhibitors by reducing or eliminating the inhibitory properties in terms of carbonic anhydrases might be too an improvement of pharmacological properties and a change lead the side effect spectrum.

The The object of the present invention is to provide compounds the improved pharmaceutical properties, in particular a Reduction of carbonic anhydrase-2 inhibition, as already known Have CDK inhibitors.

in der
A und D jeweils unabhängig voneinander für Halogen, Hydroxy, Cyano oder für die Gruppe -O-R⁵ oder für gegebenenfalls ein- oder mehrfach, gleich oder verschieden mit Halogen oder Hydroxy oder mit der Gruppe -O-R⁵ substituiertes C₁-C₄-alkyl oder C₃-C₆-Cycloalkyl, wobei der Alkylrest gegebenenfalls verzweigt sein kann, stehen,
X für -NH-, -N(C₁-C₃-alkyl)- oder -O-, wobei der Alkylrest gegebenenfalls verzweigt sein kann, steht,
R¹ für Halogen oder Cyano steht,
R² für gegebenenfalls ein- oder mehrfach, gleich oder verschieden mit C₁-C₃-alkoxy substituiertes Hydroxy-C₁-C₈-alkyl, wobei der Alkylrest gegebenenfalls verzweigt sein kann, steht,
R³ und R⁴ jeweils unabhängig voneinander für Wasserstoff oder für gegebenenfalls ein- oder mehrfach, gleich oder verschieden mit Hydroxy oder der Gruppe -O-R⁵ oder -NR⁶ substituiertes C₁-C₃-alkyl, wobei der Alkylrest gegebenenfalls verzweigt sein kann, stehen,
R⁵ für gegebenenfalls mit Halogen substituiertes C₁-C₄-alkyl, wobei der Alkylrest gegebenenfalls verzweigt sein kann, steht und
R⁶ für gegebenenfalls ein- oder mehrfach, gleich oder verschieden mit Hydroxy oder der Gruppe -O-R⁵ substituiertes C₁-C₃-alkyl steht,
bedeuten, sowie deren Isomeren, Diastereomeren, Enantiomeren und/oder Salze, zyklin-abhängige Kinasen und VEGF-Rezeptortyrosinkinasen inhibieren, wobei überraschenderweise die erfindungsgemäßen Substanzen gleichzeitig eine stark reduzierte bis keine nachweisbare Carboanhydrase Inhibierung aufweisen und
gleichzeitig eine verbesserte Inhibierung der VEGF-Rezeptortyrosinkinasen gegenüber der Inhibition von Aminopyrimidinen, die unsubstituiert oder einfach in Orthostellung zu der Sulfonamidsubstituenten am Phenylring der 2-Anilinopyrimidin substituiert sind, aufweisen.It has now been found that compounds of general formula I

in the
A and D are each independently halogen, hydroxy, cyano or the group -OR ⁵ or optionally mono- or polysubstituted, identically or differently with halogen or hydroxy or with the group -OR ⁵ substituted C ₁ -C ₄ alkyl or C ₃ -C ₆ -cycloalkyl, where the alkyl radical may optionally be branched,
X is -NH-, -N (C ₁ -C ₃ -alkyl) - or -O-, where the alkyl radical may optionally be branched,
R ^{1 is} halogen or cyano,
R ^{2 represents} optionally mono- or polysubstituted, identically or differently C ₁ -C ₃ -alkoxy-substituted hydroxy-C ₁ -C ₈ -alkyl, where the alkyl radical may optionally be branched,
R ³ and R ^{4 are} each independently hydrogen or optionally mono- or polysubstituted by identical or different hydroxy or the group -OR ⁵ or -NR ⁶ substituted C ₁ -C ₃ -alkyl, wherein the alkyl radical may optionally be branched, stand,
R ^{5 is} optionally substituted by halogen C ₁ -C ₄ alkyl, wherein the alkyl radical may optionally be branched, and
R ^{6 represents} optionally mono- or polysubstituted, identically or differently with hydroxy or the group -OR ⁵ substituted C ₁ -C ₃ -alkyl,
and inhibit their isomers, diastereomers, enantiomers and / or salts, cyclin-dependent kinases and VEGF receptor tyrosine kinases, wherein surprisingly the substances according to the invention simultaneously have a strongly reduced to no detectable carbonic anhydrase inhibition and
simultaneously an improved inhibition of VEGF receptor tyrosine kinases against inhibition of aminopyrimidines which are unsubstituted or substituted in ortho position to the sulfonamide substituents on the phenyl ring of 2-anilinopyrimidine.

Consequently thus possess the compounds of the invention improved pharmaceutical properties, in particular by the Reduction of carbonic anhydrase inhibition and improved by the VEGF receptor tyrosine kinase Inhibition, whereby substances of the invention the proliferation can inhibit the tumor cells and / or tumor angiogenesis in simultaneous reduction of side effects due to carbonic anhydrase effect.

Under Alkyl is in each case a straight-chain or branched alkyl radical, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec. Butyl, tert. Butyl, pentyl, isopentyl, hexyl, heptyl, octyl, Nonyl or Decyl, to understand.

Under Alkoxy is in each case a straight-chain or branched alkoxy radical, such as methyloxy, ethyloxy, propyloxy, isopropyloxy, Butyloxy, isobutyloxy, sec. Butyloxy, pentyloxy, isopentyloxy, hexyloxy, Heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy or dodecyloxy to understand.

Under Cycloalkyl is in each case cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

heterocycloalkyl stands for a 3-12 Carbon atoms comprising alkyl ring instead of the carbon one or more, identical or different heteroatoms, such as. B. Contains oxygen, sulfur or nitrogen.

When Heterocycloalkyls are z. B. called: oxiranyl, oxethanyl, aziridinyl, Azetidinyl, tetrahydrofuranyl, pyrrolidinyl, dioxolanyl, imidazolidinyl, Pyrazolidinyl, dioxanyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, Piperazinyl, trithianyl, quinuclidinyl, etc.

Under the ring systems, in which optionally one or more possible double bonds can be contained in the ring, are, for example, cycloalkenyls such as cyclopropenyl, cyclobutenyl, Cyclopentenyl, cyclohexenyl, cycloheptenyl to understand, where the linking both at the double bond and at the single bonds can.

Under Halogen is to be understood in each case fluorine, chlorine, bromine or iodine.

The Alkenyl substituents are each straight-chain or branched, wherein For example, the following radicals are meant: vinyl, propen-1-yl, Propen-2-yl, but-1-en-1-yl, but-1-en-2-yl, but-2-en-1-yl, but-2-en-2-yl, 2-methyl prop-2-en-1-yl, 2-methylprop-1-en-1-yl, But-1-en-3-yl, ethynyl, prop-1-yn-1-yl, but-1-yn-1-yl, but-2-yn-1-yl, But-3-en-1-yl, allyl.

Of the Aryl radical has 6-12 each Carbon atoms such as naphthyl, biphenyl and in particular Phenyl.

Under Heteroaryl is to be understood as a heteroaryl radical, which also includes may be benzo-fused. For example, as 5-ring heteroaromatics called: thiophene, furan, oxazole, thiazole, imidazole, pyrazole, triazole, Thia-4H-pyrazole and benzo derivatives thereof and as 6-membered heteroaromatic pyridine, pyrimidine, Triazine, quinoline, isoquinoline and their benzo-fused derivatives.

Under Isomers are chemical compounds of the same molecular formula but different chemical structure to understand. One differentiates in general, constitutional isomers and stereoisomers.

constitutionally have the same molecular formula, but differ by the way of linking their atoms or atomic groups. These include functional isomers, Positional isomers, tautomers or valence isomers.

stereoisomers basically have the same structure (constitution) - and thus the same empirical formula - different but through the spatial Arrangement of the atoms.

you generally distinguishes configuration isomers and conformational isomers. Configuration isomers are stereoisomers that only undergo bond breaking can be converted into each other. These include enantiomers, Diastereomers and E / Z (cis / trans) isomers.

enantiomers are stereoisomers that are like image and mirror image to each other behave and have no symmetry plane. All stereoisomers, which are not enantiomers are called diastereomers. One Special case are E / Z (cis / trans) isomers on double bonds.

conformational are stereoisomers resulting from the rotation of single bonds can be converted into each other.

to Differentiation of the isomerism types from each other see also the IUPAC rules Section E (Pure Appl. Chem. 45, 11-30, 1976).

is contain an acidic function, are as salts the physiological acceptable Salts of organic and inorganic bases suitable, such as the well soluble Alkali and alkaline earth salts and N-methyl-glucamine, dimethylglucamine, Ethyl glucamine, lysine, 1,6-hexadiamine, ethanolamine, glucosamine, Sarcosine, serinol, tris-hydroxy-methyl-amino-methane, aminopropanediol, Sovak base, 1-amino-2,3,4-butanetriol.

is contain a basic function, are the physiologically acceptable Salts of organic and inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid and the like are suitable.

Under Cancer is solid tumors or leukemia, especially ataxia angiography, Basal cell carcinoma, bladder carcinoma, brain tumor, breast cancer, cervix Carcinoma, tumors of the central nervous system, colorectal carcinoma, Endometrial carcinoma, gastric carcinoma, gastrointestinal carcinoma, Head and neck tumors, Acute lymphocytic leukemia, Acute myelogenous leukemia, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Hairy cell leukemia, Liver carcinoma, lung tumor, non-small cell lung carcinoma, Small cell lung carcinoma, B-cell lymphoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, T-cell lymphoma, Melanoma, mesothelioma, myeloma, fibroid, tumors of the esophagus, oral Tumors, ovarian carcinoma, pancreatic tumors, prostate tumors, renal carcinoma, Sarcoma, Kaposi's Sarcoma, leiomyosarcoma, skin cancer, squamous cell carcinoma, testicular cancer or thyroid cancer.

Particularly effective are those compounds of the general formula I in which
A and D are each independently halogen, hydroxy, cyano or the group -OR ⁵ or optionally mono- or polysubstituted, identically or differently with halogen or hydroxy or with the group -OR ⁵ substituted C ₁ -C ₄ alkyl or C ₃ -C ₆ -cycloalkyl, where the alkyl radical may optionally be branched,
X is -NH-, -N (C ₁ -C ₃ -alkyl) - or -O-, where the alkyl radical may optionally be branched,
R ^{1 is} halogen or cyano,
R ^{2 represents} optionally mono- or polysubstituted, identically or differently C ₁ -C ₃ -alkoxy-substituted hydroxy-C ₁ -C ₈ -alkyl, where the alkyl radical may optionally be branched,
R ³ and R ^{4 are} each independently hydrogen and
R ^{5 is} -CF ₃ or C ₁ -C ₄ -alkyl, where the alkyl radical may optionally be branched,
and their isomers, diastereomers, enantiomers and / or salts.

Furthermore, such Verbindunge the general formula I are of great importance, in the
A and D are each independently of the other halogen, hydroxy, cyano or the group -OR ⁵ or optionally mono- or polysubstituted by identical or different halogen-substituted C ₁ -C ₄ -alkyl or C ₃ -C ₆ -cycloalkyl,
X is -NH- or -O-,
R ^{1 is} halogen,
R ^{2 is} hydroxy-C ₁ -C ₈ -alkyl which is optionally substituted by the group -OR ⁵ ,
R ³ and R ^{4 are} hydrogen and
R ^{5 is} C ₁ -C ₄ -alkyl,
and their isomers, diastereomers, enantiomers and / or salts.

Of particular importance are compounds of the general formula I in which
A and D are each independently of one another C ₁ -C ₄ -alkyl,
X is -NH- or -O-,
R ^{1 is} halogen,
R ^{2 is} hydroxy-C ₁ -C ₈ -alkyl which is optionally substituted by the group -OR ⁵ ,
R ³ and R ^{4 are} hydrogen and
and their isomers, diastereomers, enantiomers and / or salts.

The Compounds of the invention essentially inhibit cyclin-dependent Kinases, as well as their effect against, for example, cancer solid tumors and leukemia, Autoimmune diseases such as psoriasis, alopecia, and multiple sclerosis, Chemotherapeutic-induced alopecia and mucositis, cardiovascular diseases, such as stenoses, atherosclerosis and restenosis, infectious diseases, such as B. by unicellular Parasites, such as Trypanosoma, Toxoplasma or Plasmodium, or by Fungi, nephrological diseases such. B. glomerulonephritis, chronic neurodegenerative diseases, such as Huntington's disease, amyotrophic Lateral sclerosis, Parkinson's disease, AIDS dementia and Alzheimer's disease, acute neurodegenerative diseases, such as brain ischaemia and neurotrauma, viral infections such. As cytomegalovirus infections, herpes, hepatitis B and C, and HIV-based illnesses.

Of the eukaryotic cell division cycle represents the duplication of the genome and his distribution to the daughter cells safely by having a coordinated and undergoes a regulated sequence of events. The cell cycle is in divided into four consecutive phases: the G1 phase represents the time before DNA replication in which the cell is growing and for external Stimuli receptive is. In the S phase, the cell replicates its DNA, and in the G2 Phase prepares them for entry into mitosis. In the Mitosis (M phase) separates the replicated DNA and cell division completed.

The cyclin-dependent Kinases (CDKs), a family of serine / threonine kinases whose members bind of a cyclin (Cyc) as a regulatory subunit for its activation need, drive the cell through the cell cycle. Different CDK / Cyc Couples are active in different phases of the cell cycle. For the basic Function of the cell cycle significant CDK / Cyc pairs are for example CDK4 (6) / CycD, CDK2 / CycE, CDK2 / CycA, CDK1 / CycA and CDK1 / CycB. Some Members of the CDK enzyme family have a regulatory function by the activity affect the aforementioned cell cycle CDKs, while other members of the CDK enzyme family still no specific function could be assigned. One of This, CDK5, is characterized by being an atypical, possesses a different regulatory subunit from the cyclins (p35), and their activity highest in the brain is.

Entry into the cell cycle and passage through the restriction point, which marks the independence of a cell from further growth signals to complete the initiated cell division, are controlled by the activity of the CDK4 (6) / CycD and CDK2 / CycE complexes. The major substrate of these CDK complexes is the retinoblastoma protein (Rb), the product of the retinoblastoma tumor suppressor gene. Rb is a transcriptional co-repressor protein. In addition to other mechanisms that are largely misunderstood, Rb binds and inactivates E2F-type transcription factors and forms transcriptional repressor complexes with histone deacetylases (HDAC) (Zhang HS et al., 2000. Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI / SNF and Rb-hSWI / SNF, Cell 101, 79-89). The phosphorylation of Rb by CDKs releases bound E2F transcription factors and lead to transcriptional activation of genes whose products are required for DNA synthesis and progression through S-phase. In addition, Rb phosphorylation causes the dissolution of the Rb-HDAC complexes, thereby activating additional genes. The phosphorylation of Rb by CDKs is equivalent to exceeding the "restriction point". For the progression through the S-phase and its completion, the activity of the CDK2 / CycE and CDK2 / CycA complexes is necessary, e.g. For example, the activity of E2F-type transcription factors is turned off by CDK2 / CycA phosphorylation as soon as the cells enter S-phase. Upon complete replication of the DNA, CDK1 in complex with CycA or CycB controls the entry and passage of G2 and M ( 1 ).

Corresponding the extraordinary Importance of the cell division cycle is strictly going through the cycle regulated and controlled. The enzymes responsible for progression through the Cycle are necessary, too activated at the right time, and also switched off again as soon as the appropriate phase has passed. Appropriate Control points ("checkpoints") lock the Progression through the cell cycle in case of DNA damage be detected, or DNA replication, or the structure of the Spindle apparatus is not finished yet.

The activity The CDKs is driven by different mechanisms, such as synthesis and degradation the cyclins, complexing of the CDKs with the corresponding cyclins, Phosphorylation and dephosphorylation of regulatory threonine and tyrosine residues, and the bond of natural inhibitory proteins, directly controlled. While the protein amount of CDKs is relatively constant in a proliferating cell, oscillates the amount of each cyclone as it goes through the cycle. For example, the expression of CycD during the early G1 phase stimulated by growth factors, and the expression of CycE will pass after of the "Restriction Point "through the Activation of the transcription factors induced by the E2F type. The Cyclins themselves are degraded by ubiquitin-mediated proteolysis. Activating and inactivating phosphorylations regulate the activity CDKs, for example, phosphorylate CDK activating kinases (CAKs) Thr160 / 161 of CDK1, whereas the family of Wee1 / Myt1 Inactivate kinases CDK1 by phosphorylation of Thr14 and Tyr15. These inactivating phosphorylations can be caused by cdc25 phosphatases be lifted again. Very important is the regulation of activity of the CDK / Cyc complexes by two families of natural CDK inhibitor proteins (CKIs), the protein products of the p21 gene family (p21, p27, p57) and the p16 gene family (p15, p16, p18, p19). Members of the p21 Family bind to cyclin complexes of CDKs 1,2,4,6, but inhibit only complexes containing CDK1 or CDK2. Members of the p16 family are specific inhibitors of the CDK4 and CDK6 complexes.

Above this complex direct regulation of the activity of the CDKs is the level the checkpoint regulation. Control points allow the cell the orderly progression of the individual phases during the cell cycle follow. The most important checkpoints are at the transition from G1 to S and from G2 to M. The G1 checkpoint ensures that the cell does not begin DNA synthesis if it does not correspond nourished is, interacts correctly with other cells or the substrate, and their DNA is intact. The G2 / M checkpoint represents complete replication the DNA and the construction of the mitotic spindle sure before the Cell enters mitosis. The G1 checkpoint is from the Gene product of the p53 tumor suppressor gene activated. p53 will be after Detection of changes is activated in the metabolism or genomic integrity of the cell either trigger a stop of cell cycle progression or apoptosis. there plays the transcriptional activation of the expression of the CDK Inhibitor protein p21 by p53 plays a crucial role. A second Branch of the G1 checkpoint involves the activation of the ATM and Chk1 kinases after DNA damage by UV light or ionizing radiation and finally the Phosphorylation and the subsequent proteolytic degradation of cdc25A phosphatase (Milan N. et al. (2000). Rapid destruction of human cdc25A in response to DNA damage. Science 288, 1425-1429). This results in a locking of the cell cycle, since the inhibitory Phosphorylation of the CDKs is not removed. After activation of the G2 / M checkpoint by DNA damage are both mechanisms in a similar way Involved in stopping the progression through the cell cycle.

Loss of cell cycle regulation and loss of control point function are characteristics of tumor cells. The CDK-Rb signaling pathway is affected by mutations in over 90% of human tumor cells. These mutations eventually leading to inactivating phosphorylation of RB include overexpression of D and E cyclins by gene amplification or chromosomal translocations, inactivating mutations or deletions of p16-type CDK inhibitors, as well as increased (p27) or decreased (CycD ) Protein breakdown. The second set of genes hit by mutations in tumor cells encodes components of the control points. Thus, p53, which is essential for the G1 and G2 / M control points, is the most frequently mutated gene in human tumors (approximately 50%). In tumor cells that express p53 without mutation, it is often inactivated due to greatly increased protein degradation. In similar Thus, the genes of other proteins necessary for the function of the control points are affected by mutations, for example ATM (inactivating mutations) or cdc25 phosphatases (overexpression).

Convincing experimental data suggest that CDK2 / cyc complexes are a crucial Position during of cell cycle progression: (1) Both dominant-negative Forms of CDK2, such as transcriptional repression of CDK2 expression anti-sense oligonucleotides cause cell cycle progression to stop. (2) The inactivation of the CycA gene in mice is lethal. (3) The malfunction of the function of the CDK2 / CycA complex in cells using cell-permeable peptides led for tumor cell-selective apoptosis (Chen Y.N.P. et al., 1999) killing of transformed cells by cyclin / cyclin-dependent kinase 2 antagonists. Proc. Natl. Acad. Sci. USA 96, 4325-4329).

changes Cell cycle control is not limited to cancer a role. The cell cycle is affected by a number of viruses, both by transforming, as by non-transforming, activated to allow the proliferation of viruses in the host cell. The fake one Entry into the cell cycle of normally post-mitotic cells is associated with various neurodegenerative diseases brought. The mechanisms of cell cycle regulation, its changes in diseases and a variety of approaches to the development of inhibitors The cell cycle progression and especially the CDKs have already been reported in several publications in detail in summary (Sielecki T.M. et al., (2000), Cyclin-dependent kinase inhibitors: useful targets in cell cycle regulation. J. Med. Chem. 43, 1-18; Fry D.W. & Garrett M.D. (2000). Inhibitors of cyclin-dependent kinases as therapeutic agents for the treatment of cancer. Curr. Opin. Oncol. Endo. Metab. Invest. Drugs 2, 40-59; Rosiania G.R. & Chang Y.T. (2000). Targeting hyperproliferative disorders with cyclin dependent kinase inhibitors. Exp. Opin. Ther. Patents 10, 215-230; Meijer L. et al. (1999). Properties and potential applications of chemical inhibitors of cyclin-dependent kinases. Pharmacol. Ther. 82, 279-284; Senderowicz A.M. & Sausville E.A. (2000). Preclinical and clinical development of cyclin-dependent kinase modulator. J. Natl. Cancer Inst. 92, 376-387).

to Use of the compounds of the invention As medicines, these are in the form of a pharmaceutical preparation brought in addition to the active ingredient for enteral or parenteral Application suitable pharmaceutical, organic or inorganic inert carrier materials, such as, water, gelatin, gum arabic, lactose, Strength, Magnesium stearate, talc, vegetable oils, polyalkylene glycols, etc. contains. The pharmaceutical preparations can in solid form, for example as tablets, dragees, suppositories, Capsules or in liquid Shape, for example as solutions, Suspensions or emulsions are present. If necessary included her about it In addition, adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers; Salts for change osmotic pressure or buffer.

These pharmaceutical preparations are also the subject of the present invention.

For the parenteral In particular, use is made of injection solutions or suspensions, in particular aqueous solutions the active compounds in polyhydroxyethoxylated castor oil.

When carrier systems can also surface-active Excipients such as salts of bile acids or animal or vegetable Phospholipids, but also mixtures thereof and liposomes or their Components are used.

For the oral In particular, tablets, dragees or capsules are used Talc and / or hydrocarbon carriers or binders, such as Lactose, corn or potato starch, suitable. The application can also be done in liquid form, such as Example as juice, which may be accompanied by a sweetener.

The enteral, parenteral and oral applications are also Subject of the present invention.

The Dosage of active ingredients may vary depending on the route of administration, age and Weight of the patient, type and severity of the disease to be treated and similar Factors vary. The daily Dose is 0.5-1000 mg, preferably 50-200 mg, where the dose is to be administered once Single dose or divided into 2 or more daily doses can be.

On the other hand, compounds of the general formula I according to the invention also inhibit receptor tyrosine kinases and their ligands which specifically inhibit the function of endothelial cells. Receptor tyrosine kinases and their ligands that specifically regulate the function of endothelial cells are in decisively involved in physiological as well as pathogenic angiogenesis. Of particular importance here is the VEGF / VEGF receptor system. In pathological situations associated with increased neovascularization, increased expression of angiogenic growth factors and their receptors has been found. Thus, most solid tumors express high levels of VEGF, and expression of the VEGF receptors is preferably significantly increased in the endothelial cells that are adjacent to or pass through the tumors (Plate et al., Cancer Res. 53, 5822 -5827, 1993). Inactivation of the VEGF / VEGF Receptor System by VEGF Neutralizing Antibodies (Kim et al., Nature 362, 841-844, 1993), retroviral expression of dominant negative VEGF receptor variants (Millauer et al., Nature 367, 576-579, 1994), recombinant VEGF-neutralizing receptor variants (Goldman et al., Proc Natl Acad Sci., USA 95, 8795-8800, 1998), or low molecular weight inhibitors of VEGF receptor tyrosine kinase (Fong et al., Cancer Res. 99-106, 1999; Wedge et al., Cancer Res. 60, 970-975, 2000; Wood et al., Cancer Res. 60, 2178-2189, 2000) resulted in decreased tumor growth and tumor vascularization. Thus, inhibition of angiogenesis is a potential mode of treatment for tumor diseases.

Accordingly, compounds according to the invention can be either cyclin-dependent kinases, such as CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9, as well as glycogen synthase kinase (GSK-3β) and VEGF receptor tyrosine kinases or cyclin-dependent kinases or inhibiting VEGF receptor tyrosine kinases. These effects contribute to the compounds of the present invention being useful in the treatment of cancer, angiofibribroma, arthritis, ocular diseases, autoimmune diseases, chemotherapeutic-induced alopecia and mucositis, Crohn's disease, endometriosis, fibrotic disorders, hemangiomas, cardiovascular diseases, infectious diseases , nephrological disorders, chronic and acute neurodegenerative disorders, as well as nerve tissue injuries, viral infections, to inhibit reocclusion of vessels after balloon catheterization, in vascular prosthetics or after insertion of mechanical devices to hold vessels open, such as. As stents, as immunosuppressants, to support the scar-free wound healing, in age spots and contact dermatitis, wherein
cancer, solid tumors, tumor or metastasis growth, Kaposis sarcoma, Hodgkin's disease and leukemia,
arthritis, rheumatoid arthritis, eye diseases, diabetic retinopathy, neovascular glaucoma,
psoriasis, alopecia and multiple sclerosis in autoimmune diseases,
fibrotic diseases, liver cirrhosis, mesangial cell proliferative disorders, arteriosclerosis,
infectious diseases caused by unicellular parasites,
cardiovascular diseases include strictures, such as Stent-induced restenosis, arteriosclerosis and restenosis,
nephrological disorders include glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndromes, transplantation rejection and glomerulopathy,
Chronic Neurodegenerative Diseases Huntington's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, AIDS Dementia and Alzheimer's Disease
ischemia of the brain and neurotraumata in acute neurodegenerative diseases,
and viral infections are cytomegalovirus infections, herpes, hepatitis B or C, and HIV disorders.

Also The present invention relates to medicaments for treatment the above listed Diseases which contain at least one compound according to the general formula (I) as well as drugs with suitable formulation and Excipients.

The Compounds of the invention of general formula I are, inter alia, excellent inhibitors the cyclin-dependent Kinases such as CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9, and glycogen synthase kinase (GSK-3β) or VEGF receptor tyrosine kinases.

The Isomer mixtures can according to usual Methods such as crystallization, chromatography or Salt formation are separated into the enantiomers or E / Z isomers.

The Preparation of the salts is carried out in the usual manner by adding a solution the compound of formula I with the equivalent amount or an excess of one Base or acid, which is possibly in solution, offset and the precipitate separated or worked up in the usual way, the solution.

in der A, D, R³ und R⁴ die in der allgemeinen Formel (I) angegebenen Bedeutungen haben, sowie deren Isomeren, Diastereomeren, Enantiomeren und/oder Salze sind ebenfalls Gegenstand der vorliegenden Erfindung.For the preparation of the compounds of general formula I according to the invention preferably used intermediates of the general formulas (IIa) or (IIb)

in which A, D, R ³ and R ^{4 have} the meanings given in the general formula (I), and their isomers, diastereomers, enantiomers and / or salts are likewise provided by the present invention.

So far the preparation of the starting compounds is not described, these are known or analogous to known compounds or here produced method produced. It is also possible, all here described reactions in parallel reactors or by means combinatorial working techniques.

The Isomer mixtures can according to usual Methods such as crystallization, chromatography or Salt formation are separated into the enantiomers or E / Z isomers.

The Preparation of the salts is carried out in the usual manner by adding a solution the compound of formula I with the equivalent amount or an excess of one Base or acid, which is possibly in solution, offset and the precipitate separated or worked up in the usual way, the solution.

manufacturing the compounds of the invention

The explain the following examples the preparation of the compounds according to the invention, without the To limit the scope of the claimed compounds to these examples.

Process variant 1

The substituents R ¹ , R ² , A and D have the meaning given in the general formula (I).

Ex. 1) Preparation of 4- [5-bromo-4 - ((R) -2-hydroxy-1-methyl-ethylamino) -pyrimidin-2-ylamino] -2,6-dimethylbenzenesulfonamide

180 mg (0.9 mmol) of 4-amino-2,6-dimethyl-benzenesulfonamide in 3 ml of acetonitrile are mixed with 0.25 ml of a 4 molar solution of hydrogen chloride in dioxane and 0.5 ml of water. 266 mg (1.0 mmol) of (R) -2- (5-bromo-2-chloro-pyrimidin-4-ylamino) -propan-1-ol are added and the batch is stirred under reflux for 16 h. After cooling, the precipitated solid is filtered off with suction and washed successively with acetonitrile, ethanol, water and diisopropyl ether. After drying, 349 mg (0.8 mmol, corresponding to 88% of theory) of the product are obtained.
¹ H-NMR (DMSO-D6): 10.53 (s, 1H), 8.29 (s, 1H), 7.70 (d, 1H), 7.44 (s, 2H), 7.20 (s, 2H), 4.25 (m, 1H ), 3.53 (m, 2H), 2.45 (s, 6H), 1.20 (d, 3H).
MS: 430 (ES).

In an analogous procedure to the process variant described above, the following compounds are also prepared:

Example 4) Preparation of 4- [5-bromo-4 - ((1R, 2R) -2-hydroxy-1-methyl-propoxy) -pyrimidin-2-ylamino] -2,6-dimethylbenzenesulfonamide

202 mg (1.01 mmol) of 4-amino-2,6-dimethylbenzenesulfonamide in 3 ml of acetonitrile are treated with 0.28 ml of a 4 molar solution of hydrogen chloride in dioxane. A solution of 310 mg (1.10 mmol) of (2R, 3R) -3- (5-bromo-2-chloro-pyrimidin-4-yloxy) -butan-2-ol in 1.5 ml of acetonitrile is added and The mixture is stirred under reflux for 75 h. After cooling, the precipitated solid is filtered off with suction and then purified by chromatography (DCM / EtOH 95: 5). The crude product obtained is finally recrystallized from methanol. 39 mg (0.09 mmol, corresponding to 9% of theory) of the product are obtained.
¹ H-NMR (DMSO-D6): 9.91 (s, 1H), 8.40 (s, 1H), 7.51 (s, 2H), 7.10 (s, 2H), 5.20 (m, 1H), 4.90 (d, 1H ), 3.82 (m, 1H), 2.60 (s, 6H), 1.30 (d, 3H), 1.15 (d, 3H).
MS: 445 (ES).

The following examples can be obtained analogously to the method variants described above, including methods obvious to the person skilled in the art:

Production of intermediates

1) Preparation of aniline derivatives

The Substituents A and D have the meanings given in the general formula (I) Importance.

1a) Preparation of 4-amino-2,6-dimethyl-benzenesulfonamide

7.53 g (31.1 mmol) of N- (3,5-dimethyl-4-sulfamoyl-phenyl) -acetamide are stirred in 100 ml of a 2N NaOH solution for 2 hours under reflux. The batch is extracted after cooling against ethyl acetate (3x). The combined organic phases are filtered through a Whatman filter and concentrated. This gives 1.17 g (5.9 mmol, corresponding to 19% of theory) of the product.
¹ H-NMR (DMSO-D6): 6.81 (s, 2H), 6.25 (s, 2H), 5.55 (br, 2H), 2.40 (s, 6H).

1b) Preparation of N- (3,5-dimethyl-4-sulfamoyl-phenyl) -acetamide

A solution of 8.54 g (32.6 mmol) of 4-acetylamino-2,6-dimethylbenzenesulfonyl chloride in 100 ml of DCM is saturated with ammonia gas. The mixture is then stirred for a further 10 minutes at room temperature. The precipitate formed is filtered off and recrystallized from hot methanol. This gives 3.29 g (13.6 mmol, corresponding to 42% of theory) of the product.
¹ H-NMR (DMSO-D6): 10.05 (s, 1H), 7.37 (s, 2H), 7.15 (s, 2H), 2.55 (s, 6H), 2.03 (s, 3H).

1c) Preparation of 4-acetylamino-2,6-dimethylbenzenesulfonyl chloride

21.0 g (129 mmol) of N- (3,5-dimethyl-phenyl) -acetamide become cautious to 35 ml of chlorosulfonic acid given and then the approach 3 hours at 90 ° C touched. After cooling, the mixture is slowly added to a mixture of ethyl acetate / hexane (1: 1) and ice. The organic phase is separated, over a Whatman filter is filtered and concentrated. This gives 15.1 g (58 mmol, corresponding to 45% of theory) of the crude product, without further purification is used in the following experiments.

1d) Preparation of N- (3,5-dimethyl-phenyl) -acetamide

20 ml (160 mmol) of 3,5-dimethylaniline are added slowly while stirring with 30 ml (317 mmol) of acetic anhydride. The reaction mixture is stirred for 2 hours, then the formed solid is filtered off with suction and washed with diisopropyl ether. After drying, 21.1 g (129 mmol, corresponding to 81% of theory) of the product.
¹ H-NMR (DMSO-D6): 9.75 (s, 1H), 7.18 (s, 2H), 6.65 (s, 1H), 2.21 (s, 6H), 2.02 (s, 3H).

2) Preparation of 2-chloro-pyrimidine derivatives

Process Variant 2.1: Preparation of 4-N Derivatives

The Substituents R1 and R2 have the meanings given in the general formula (I) given meaning.

2.1a) Preparation of (R) -3- (5-bromo-2-chloro-pyrimidin-4-ylamino) -2-methyl-butan-2-ol

An ice-cooled solution of 2.95 g (10.0 mmol) of methyl (R) -2- (5-bromo-2-chloro-pyrimidin-4-ylamino) -propionate in 150 ml of THF is added dropwise with 30 ml (90 mmol) of a 3 molar solution of methylmagnesium bromide in diethyl ether. After 2.5 hours at room temperature, the batch is mixed with 30 ml of saturated ammonium chloride solution. Dilute with water and extract against ethyl acetate (3x). The combined organic phases are dried (Na ₂ SO ₄ ), filtered and concentrated. The remaining residue is purified by chromatography (hexane / ethyl acetate: 4: 1-1: 1). This gives 2.81 g (9.5 mmol, corresponding to 95% of theory) of the product.
¹ H-NMR _(CDCl3): 8.1 (s, 1H), 5.9 (d, 1H), 4.2 (m, 1H), 1.8 (br, 1H), 1.2 (m, 9H).

2.1 b) Preparation of methyl (R) -2- (5-bromo-2-chloro-pyrimidin-4-ylamino) -propionate

22.8 g (100 mmol) of 5-bromo-2,4-dichloro-pyrimidine and 14.0 g (100 mmol) of methyl D-alanate hydrochloride are dissolved in 300 ml of THF and 75 ml of DMF. The ice-cooled mixture is admixed with 33.5 ml (240 mmol) of triethylamine and then slowly warmed to room temperature. After 48 hours, the solvent is removed on a rotary evaporator and the remaining residue is purified by chromatography (hexane / ethyl acetate: 4: 1-2: 1). This gives 25.5 g (86.1 mmol, corresponding to 86% of theory) of the product.
¹ H-NMR _(CDCl3): 8.2 (s, 1H), 6.1 (d, 1H), 4.8 (m, 1H), 3.8 (s, 3H), 1.6 (d, 3H).

2.1c) Preparation of (2R, 3R) -3- (5-bromo-2-chloro-pyrimidin-4-ylamino) -butan-2-ol

32.7 g (159 mmol) of copper (I) bromide dimethyl sulfide complex are initially charged under nitrogen atmosphere in 1000 ml of diethyl ether and cooled to -78 ° C. Over a period of about 25 minutes, 200 ml of a 1.6 molar solution of methyl lithium in diethyl ether are added dropwise and then removed the cooling bath. The mixture is stirred for 40 minutes, the temperature rises to -35 ° C. It is cooled to -55 ° C and added 18.9 g (71.5 mmol) of (R) -2- (5-bromo-2-chloro-pyrimidin-4-ylamino) propionaldehyde over a period of 20 minutes. It is stirred for 6 hours at -55 ° C, then the cooling bath is again filled with dry ice, covered with aluminum foil and the mixture stirred overnight. 200 ml of a saturated ammonium chloride solution are added dropwise and the batch is warmed to room temperature. It is diluted with 500 ml of diethyl ether, the organic phase separated and the aqueous phase extracted with diethyl ether. The combined organic phases are washed with saturated ammonium chloride solution and saturated NaCl solution, dried (Na ₂ SO ₄ ), filtered and concentrated. The remaining residue is purified by chromatography (hexane / ethyl acetate: 4: 1-1: 1). This gives 8.4 g (30.0 mmol, corresponding to 42% of theory) of the product.
¹ H-NMR _(CDCl3): 8.1 (s, 1H), 5.8 (d, 1H), 4.2 (m, 1H), 3.9 (m, 1H), 2.0 (d, 1H), 1.3 (d, 3H) , 1.2 (d, 3H). HPLC analysis: Pillar: Chiralpak AD-H 5μ Length × ID: 150 × 4.6 mm eluent: A = hexane, C = ethanol River: 1.0 ml / min Gradient: Isocratic 5% C Detector: UV 254nm Temperature: 25 ° C RT in minutes: 6.04

2.1d) Preparation of (R) -2- (5-bromo-2-chloro-pyrimidin-4-ylamino) -propionaldehyde

A solution of 40.0 g (135.8 mmol) of methyl (R) -2- (5-bromo-2-chloro-pyrimidin-4-ylamino) -propionate in 800 ml of toluene is stirred at -78 ° C Added 310 ml of a 1.2 molar solution of diisobutylaluminum hydride. After 30 minutes, it is carefully quenched with methanol. The mixture is warmed to room temperature and diluted with 1000 ml of tert-butyl methyl ether. It is washed successively with 1 N HCl (3 × 100 ml), saturated sodium bicarbonate solution (3 ×) and saturated NaCl solution (3 ×). The organic phase is dried (MgSO ₄ ), filtered and concentrated. The remaining residue is purified by chromatography (hexane / ethyl acetate: 4: 1-1: 1). This gives 22.5 g (83.9 mmol, corresponding to 62% of theory) of the product.
¹ H-NMR _(CDCl3): 9.6 (s, 1H), 8.2 (s, 1H), 6.3 (d, 1H), 4.8 (m, 1H), 1.5 (d, 3H).

2.1e) Preparation of (2R, 3R) -3- (5-bromo-2-chloro-pyrimidin-4-ylamino) -4-methoxy-butan-2-ol

311 mg (2.6 mmol) of (2R, 3R) -3-amino-4-methoxy-butan-2-ol hydrochloride (preparation according to AI Meyers, D. Hoyer, Tet. Lett. 1985, 26, 4687) in 2 ml of acetonitrile are mixed with 0.28 ml of triethylamine and shaken. Filter and wash the filter cake with 2 ml of acetonitrile. The filtrate is added dropwise to a solution of 455 mg (2.0 mmol) of 5-bromo-2,4-dichloro-pyrimidine in 26 ml of acetonitrile at -30 ° C. By removing the cooling bath is slowly warmed to room temperature with stirring. After 16 hours, the solvent is removed on a rotary evaporator and the remaining residue is purified by chromatography (hexane / ethyl acetate: 4: 1-1: 1). This gives 509 mg (1.6 mmol, corresponding to 80% of theory) of the product.
¹ H-NMR (CDCl ₃ ): 8.1 (s, 1H), 6.3 (d, 1H), 4.3 (m, 1H), 4.2 (m, 1H), 3.8 (d, 2H), 3.4 (s, 3H) , 3.1 (d, 1H), 1.2 (d, 3H).

Process Variant 2.2: Preparation of 4-O Derivatives

The substituents R ¹ and R ² have the meaning given in the general formula (I).

2.2a) Preparation of (2R, 3R) -3- (5-bromo-2-chloro-pyrimidin-4-yloxy) -butan-2-ol

A solution of 2.7 g (30.0 mmol) of (2R, 3R) -butane-2,3-diol in 120 ml of THF is added in portions with 0.96 g (24.0 mmol) of sodium hydride while cooling with an ice bath and then 30 Stirred at room temperature for min. The reaction is added to an ice-cooled solution of 4.46 g (20.0 mmol) of 5-bromo-2,4-dichloro-pyrimidine in 40 ml of THF. The batch is slowly warmed to room temperature with stirring. After 12 hours, the batch is concentrated on a rotary evaporator and the remaining residue is purified by chromatography (hexane / ethyl acetate: 4: 1-1: 1). 3.18 g (11.3 mmol, corresponding to 57% of theory) are obtained.
¹ H-NMR _(CDCl3): 8.4 (s, 1H), 5.2 (m, 1H), 3.9 (m, 1H), 2.0 (br, 1H), 1.4 (d, 3H), 1.2 (d, 3H) ,

example 1

CDK1 / CycB kinase assay

recombinant CDK1 and CycB GST fusion proteins purified from baculovirus-infected Insect cells (Sf9) were purchased from ProQinase GmbH, Freiburg. The histone IIIS used as a kinase substrate is about the Fa. Sigma for sale to acquire.

CDK1 / CycB (200 ng / measuring point) was for 15 min at 22 ° C in the presence of various concentrations of test substances (0 μM, as well as within the range 0.01-100 μM) in assay buffer [50mM Tris / HCl pH8.0, 10mM MgCl2, 0.1mM Na ortho-vanadate, 1.0 mM dithiothreitol, 0.5 μM Adenosine trisphosphate (ATP), 10 μg / measuring point histone IIIS, 0.2 μCi / measuring point 33P-gamma ATP, 0.05% NP40, 12.5% dimethylsulfoxide]. The Reaction was by addition of EDTA solution (250 mM, pH 8.0, 14 μl / measuring point) stopped.

From each reaction was 10 μl applied to P30 filter strips (Wallac), and non-built-in 33P-ATP was prepared by washing the filter strips for 10 times three times min in 0.5% phosphoric acid away. After drying the filter strips for 1 hour at 70 ° C were the filter strips with scintillator strips (MeltiLex ™ A, Fa. Wallac) and covered for 1 hour at 90 ° C baked. The amount of incorporated 33P (substrate phosphorylation) was determined by scintillation measurement in a gamma radiation meter (Wallac) certainly.

Example 2

CDK2 / CycE kinase assay

recombinant CDK2 and CycE GST fusion proteins purified from baculovirus-infected Insect cells (Sf9) were purchased from ProQinase GmbH, Freiburg. Histone IIIS, which was used as a kinase substrate, was added purchased from the company Sigma.

CDK2 / CycE (50 ng / measuring point) was incubated for 15 min at 22 ° C in the presence of various concentrations in test buffer (50 μM Tris / HCl pH8.0, 10 mM MgCl ₂ , 0.1 mM Na ortho-vanadate, 1.0 mM dithiothreitol, 0.5 μM adenosine trisphosphate (ATP), 10 μg / measuring point histone IIIS, 0.2 μCi / measuring point ³³ P-gamma ATP, 0.05% NP40, 12.5% dimethyl sulfoxide]. The reaction was stopped by addition of EDTA solution (250 mM, pH 8.0, 14 μl / measurement point).

10 μl of each reaction was applied to P30 filter strips (Wallac Co.) and unincorporated ³³ P-ATP was removed by washing the filter strips three times each for 10 minutes in 0.5% phosphoric acid. After drying the filter strips for 1 hour at 70 ° C, the filter strips were covered with scintillator strips (MeltiLex ^™ A, Wallac) and baked at 90 ° C for 1 hour. The amount of incorporated ³³ P (substrate phosphorylation) was determined by scintillation measurement in a gamma radiation meter (Wallac).

Example 3

VEGF receptor-2 kinase assay

recombinant VEGF receptor tyrosine kinase-2 was derived from baculovirus as a GST fusion protein Insect cells (Sf9) purified. Poly (Glu4Tyr) acting as a kinase substrate was purchased from Sigma. VEGF receptor tyrosine kinase (90 ng / measuring point) was for 10 minutes at 22 ° C in the presence of various concentrations of test substances (0 μM, as well as within the range 0.001-30 μM) in 30 μl of assay buffer [40 mM Tris / HCl pH 5.5, 10 mM MgCl 2, 1 mM MnCl 2, 3 μM Na ortho-vanadate, 1.0 mM dithiothreitol, 8 μM Adenosine trisphosphate (ATP), 27 μg / assay poly (Glu4Tyr), 0.2 μCi / assay 33P-gamma ATP, 1% dimethylsulfoxide]. The reaction was by addition from EDTA solution (250 mM, pH 7.0, 10 μl / measuring point) stopped.

From each reaction was 10 μl applied to P30 filter strips (Wallac), and non-built-in 33P-ATP was prepared by washing the filter strips for 10 times three times min in 0.5% phosphoric acid away. After drying the filter strips for 1 hour at 70 ° C were the filter strips with scintillator strips (MeltiLex ™ A, Fa. Wallac) and covered for 1 hour at 90 ° C baked. The amount of incorporated 33P (substrate phosphorylation) was determined by scintillation measurement in a gamma radiation meter (Wallac) certainly. The IC50 values are determined from the inhibitor concentration, which is necessary for the phosphate incorporation on 50% of the uninhibited Incorporation after deduction of the blank value (EDTA-stopped reaction) to inhibit.

Example 4

proliferation assay

cultivated human tumor cells (MCF7, hormone-independent human breast carcinoma cells, related from ATCC HTB22; NCl-H460, human non-small cell lung carcinoma cells, ATCC HTB-177, ATCC CCL-247; DU 145, hormone independent human prostate carcinoma cells, ATCC HTB-81; MaTu-MDR, hormone-independent, multiple drugs resistant human breast carcinoma cells, EPO GmbH, Berlin) in a density of approx. 5000 cells / measuring point, depending on the growth rate of the respective cells in a 96-well multititer plate in 200 μl of the corresponding Growth medium plated. After 24 hours, the cells were a plate (zero plate) with crystal violet colored (s.u.), while the medium of the other plates by fresh culture medium (200 ul), the Test substances in different concentrations (0 μM, as well as in the range 0.01-30 μM; the final Concentration of the solvent Dimethylsulfoxide was 0.5%) were added. The cells were for Incubated for 4 days in the presence of the test substances. Cell proliferation was by staining of the cells with crystal violet: the cells were passed through Add 20 μl / measuring point an 11% glutaraldehyde solution Fixed at room temperature for 15 min. After washing three times The cells were fixed with water at room temperature dried. The cells were supplemented by adding 100 μl / measuring point a 0.1% crystal violet solution (pH by adding acetic acid adjusted to pH3). After washing three times the dyed Cells with water, the plates were dried at room temperature. The dye was dissolved by adding 100 μl / measuring point of a 10% acetic acid solution. The Absorbance was determined photometrically at a wavelength of 595 nm. The percentage change Cell growth was measured by normalizing the readings on the Absorbance values of the zero point plate (= 0%) and the extinction of the untreated (0 μM) Cells (= 100%) calculated.

Example 5

Carboanhydraseassay

The The principle of the assay is based on the hydrolysis of 4-nitrophenyl acetate by carbonic anhydrases (Pocker & Stone, Biochemistry, 1967, 6, 668.), followed by photometric determination the resulting dye 4-nitrophenolate at 400 nm using a 96-channel spectrophotometer.

2μl of test compounds, solved in DMSO (100x the final concentration), in a concentration range from 0.03-10 μM (final) were pipetted into the wells of a 96-well microtiter plate as quadruplicate determinations. holes, the solvents without test compounds contained al reference values (1st holes without Carbonic anhydrase for correcting non-enzymatic hydrolysis of the substrate, and 2. holes with carbonic anhydrase to determine the activity of the non-inhibited enzyme).

188 μl assay buffer (10mM Tris / HCl pH7.4, 80mM NaCl) with no or 3 units / well Carbonic anhydrase I or II were pipetted into the wells of the microtiter plate. The enzymatic reaction was monitored by the addition of 10 μl of the substrate solution (1 mM 4-nitrophenyl acetate (Fluka # 4602) dissolved in anhydrous acetonitrile, started (final substrate concentration: 50 μM). The plate was at room temperature Incubated for 60 min. The extinctions were photometrically at a wavelength measured from 400 nm. The enzyme inhibition was after normalization the measured values on the extinction of the reactions in the holes without enzyme (= 100% inhibition) and on the extinction of the reactions in the holes with non-inhibited Enzyme (= 0% inhibition) calculated.

The Results from the examples and the comparative data are in the given below Tables 1 to 3. To prove the superiority the compounds of the invention Ex. 1 to 4 opposite the known compounds were the compounds of the invention with structure-like known compound Ex. 10, 47, 144, 255, 271 and 275 of WO 02/096888 compared in enzyme assays. The result is in the following tables 1 and 2 listed. Table 3 gives the improved data of the compounds of the invention in comparison to the compound from WO 02/096888 and acetazolamide (Diuretic).

Table 1

Table 2

Table 3

Out the above-described tables, the skilled artisan recognize that in simultaneous, equal or improved inhibition of cell cycle kinases in comparison to the known structure-like compounds (Tablelle 1), the compounds of the invention at the same time a greatly reduced to undetectable Carbonic anhydrase inhibition (Table 3) and an improved VEGF receptor tyrosine kinase Inhibition (Table 2).

Claims (24)

Compounds of the general formula I

in the A and D each independently represent halogen, hydroxy, cyano or the group -OR ⁵ or optionally mono- or polysubstituted, identically or differently with halogen or hydroxy or with the group -OR ⁵ substituted C ₁ -C ₄ - Alkyl or C ₃ -C ₆ -cycloalkyl, wherein the alkyl radical may optionally be branched, X is -NH-, -N (C ₁ -C ₃ -alkyl) - or -O-, wherein the alkyl radical may optionally be branched R ^{1 is} halogen or cyano, R ^{2 is} optionally mono- or polysubstituted by identical or different C ₁ -C ₃ -alkoxy-substituted hydroxy-C ₁ -C ₈ -alkyl, where the alkyl radical may optionally be branched, R ³ and R ^{4 are} each independently hydrogen or optionally mono- or polysubstituted by identical or different hydroxy or the group -OR ⁵ or -NR ⁶ substituted C ₁ -C ₃ alkyl, wherein the alkyl radical may be optionally branched may be, R ⁵ is optionally substituted with halogen C ₁ -C ₄ -alkyl, where the alkyl radical may optionally be branched, and R ^{6 is} C ₁ -C ₃ -alkyl which is optionally mono- or polysubstituted by identical or different hydroxy or the group -OR ⁵ , mean and their isomers, diastereomers, enantiomers and / or salts. Compounds of general formula I, according to claim 1, in which A and D are each independently halogen, hydroxy, cyano or the group -OR ⁵ or optionally mono- or polysubstituted, identical or different, with halogen or hydroxy or with the group -OR ^{5 is} substituted C ₁ -C ₄ -alkyl or C ₃ -C ₆ -cycloalkyl, where the alkyl radical may optionally be branched, X is -NH-, -N (C ₁ -C ₃ -alkyl) - or - O-, wherein the alkyl radical may optionally be branched, R ^{1 is} halogen or cyano, R ^{2 is} optionally mono- or polysubstituted by identical or different C ₁ -C ₃ -alkoxy-substituted hydroxy-C ₁ -C ₈ - alkyl, wherein the alkyl radical may optionally be branched, R ³ and R ^{4 are} each independently hydrogen and R ^{5 is} -CF ₃ or C ₁ -C ₄ alkyl, wherein the alkyl radical may optionally be branched, mean , as well as their isomers, diastereomers, enantiomers and / or salts. Compounds of general formula I, according to claim 1 or 2, wherein A and D are each independently halogen, hydroxy, cyano or the group -OR ⁵ or optionally mono- or polysubstituted by identical or different halogen-substituted C ₁ -C ₄ -alkyl or C ₃ -C ₆ -cycloalkyl, X is -NH- or -O-, R ^{1 is} halogen, R ^{2 is} optionally substituted by the group -OR ⁵ substituted hydroxy-C ₁ -C ₈ alkyl R ³ and R ^{4 are} hydrogen and R ^{5 is} C ₁ -C ₄ alkyl, and their isomers, diastereomers, enantiomers and / or salts. Compounds of general formula I, according to one of claims 1 to 3, in which A and D are each independently of the other C ₁ -C ₄ -alkyl, X is -NH- or -O-, R ^{1 is} halogen, R ^{2 represents} optionally substituted by the group -OR ⁵ substituted hydroxy-C ₁ -C ₈ -alkyl, R ³ or R ^{4 are} hydrogen and are, and their isomers, diastereomers, enantiomers and / or salts. Use of the compound of general formula IIa or IIb

in the A and D, and R ³ and R ^{4 have} the meanings given in the general formula (I), and their isomers, diastereomers, enantiomers and / or salts as intermediates for the preparation of the compound of general formula I. Use of the compound of general formula IIa, according to claim 5, characterized in that A or D is halogen, cyano, hydroxy, methoxy, methyl, CF ₃ , ethyl, isopropyl, isobutyl or cyclopropyl, and their isomers, diastereomers, enantiomers and / or salts. Use of the compound of general formula IIb, according to claim 5, characterized in that A or D is halogen, cyano, hydroxy, methoxy, methyl, CF ₃ , ethyl, isopropyl, isobutyl or cyclopropyl and R ³ or R ^{4 are} hydrogen , as well as their isomers, diastereomers, enantiomers and / or salts. Use of the compound of general formula IIa, according to claim 5 or 6 or of general formula (IIb), according to claim 5 or 7, characterized in that A or D are methyl and R ³ or R ^{4 are} hydrogen, and their isomers , Diastereomers, enantiomers and / or salts. Pharmaceutical agents comprising a compound of the general formula I according to at least one of the claims 1 to 4. Use of the compounds of the general formula I, according to claims 1 to 4, for the manufacture of a medicament for the treatment of cancer, Angiofribroma, arthritis, eye diseases, autoimmune diseases, Chemotherapeutic-induced alopecia and mucositis, Crohn's disease, Endometriosis, fibrotic diseases, hemangioma, cardiovascular diseases, infectious Diseases, nephrological diseases, chronic and acute neurodegenerative diseases, as well as injuries of the nervous tissue, viral infections, to inhibit reocclusion of vessels Balloon catheter treatment, in vascular prosthetics or after insertion of mechanical devices for holding vessels open, such as z. Stents, as immunosuppressive agents, to aid scar-free wound healing, for age spots and contact dermatitis. Use according to claim 10, characterized in that cancer includes solid tumors, tumor or metastasis growth, Kaposis sarcoma, Hodgkin's disease and leukemia, arthritis, rheumatoid arthritis, eye diseases, diabetic retinopathy, neovascular glaucoma, autoimmune psoriasis, alopecia and multiple sclerosis , fibrotic diseases, cirrhosis of the liver, mesangial cell proliferative diseases, arteriosclerosis, diseases caused by infectious diseases by unicellular parasites, cardiovascular diseases, stenoses, such as, for example, Stent-induced restenosis, arteriosclerosis and Restenosis, nephrological disorders glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndromes, transplantation rejections and glomerulopathy, chronic neurodegenerative diseases Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, AIDS dementia and Alzheimer's disease, brain ischemia in acute neurodegenerative diseases Neurotrauma, and viral infections include cytomegalovirus infections, herpes, hepatitis B or C, and HIV disorders. Medicinal products containing at least one compound according to at least one of the claims 1 to 4 included. Medicament according to claim 12, in addition contain suitable formulation and / or carriers. Medicament according to claim 12 or 13, for the treatment of cancer, angiofibribroma, arthritis, eye diseases, Autoimmune diseases, chemotherapeutic-induced alopecia and Mucositis, Crohn's disease, endometriosis, fibrotic diseases, hemangioma, cardiovascular Diseases, infectious Diseases, nephrological diseases, chronic and acute neurodegenerative diseases, as well as injuries of the nervous tissue, and viral infections and to inhibit reocclusion of vessels Balloon catheter treatment, in vascular prosthetics or after insertion of mechanical devices for holding vessels open, such as z. As stents, and as immunosuppressants, and in support of scar-free wound healing, and in age spots and contact dermatitis. Medicament for use according to claim 14, wherein Solid cancer tumors, tumor or metastasis growth, Kaposis sarcoma, Hodgkin's disease and leukemia, under Arthritis, rheumatoid arthritis, eye diseases, diabetic Retinopathy, neovascular glaucoma, under autoimmune diseases Psoriasis, alopecia and multiple sclerosis, under fibrotic Diseases, liver cirrhosis, mesangial cell proliferative diseases, Arteriosclerosis, infectious diseases caused by unicellular parasites caused diseases, under cardiovascular diseases Stenoses, such. B. stent-induced Restenosis, atherosclerosis and restenosis, under nephrological Diseases glomerulonephritis, diabetic nephropathy, malignant Nephrosclerosis, thrombotic microangiopathic syndromes, transplantation rejections and glomerulopathy under chronic neurodegenerative diseases Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, AIDS dementia and Alzheimer's Illness, of acute neurodegenerative diseases ischemia of the Brain and neurotraumata, and among viral infections cytomegalovirus infections, Herpes, Hepatitis B or C, and HIV disorders are understood. Use of the compounds of the general formula I according to at least one of the claims 1 to 4 and / or the pharmaceutical agent according to claim 9 as inhibitors of cyclin-dependent Kinases. Use according to claim 16, characterized in that the kinases CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 or CDK9. Use of the compounds of the general formula I according to at least one of the claims 1 to 4 and / or the pharmaceutical agent according to any one of claims 9 as Inhibitors of glycogen synthase kinase (GSK-3R). Use of the compounds of the general formula I according to at least one of the claims 1 to 4 and / or the pharmaceutical agent according to any one of claims 9 as Inhibitors of VEGF receptor tyrosine kinases. Use of the compounds of the general formula I according to at least one of the claims 1 to 4 and / or the pharmaceutical agent according to any one of claims 9 as Inhibitors of cyclin-dependent kinases and the VEGF receptor tyrosine kinases. Use of the compounds of general formula I, according to at least one of claims 1 to 4, in the form of a pharmaceutical preparation for enteral, parenteral and oral administration. Compounds of general formula I, according to at least one of the claims 1 to 4, and drugs according to at least one of the claims 12 to 15 with suitable formulation and excipients. Use of the compounds of the general formula I, according to at least one of the claims 1 to 4 in the form of a pharmaceutical preparation for the enteral, parenteral and oral administration. Use of the pharmaceutical agent according to claim 9 in the form of a preparation for the enteral, parenteral and oral administration.