USE OF INDIGOID BISINDOLE DERIVATIVES FOR THE MANUFACTURE OF A MEDICAMENT TO INHIBIT CYCLIN DEPENDENT KINASES

Abstract: The present invention relates to the use of indigoid bisindole derivatives for the manufacture of a medicament for inhibiting cyclin dependent kinases, particularly CDK 1, CDK 2, CDK 4 and CDK 5, more particularly ATP:Proteinphosphotransferase p34<cdc2> (CDK1).

Description: "Use of indigoid bisindole derivatives for the manufacture of a medicament to inhibit cyclin dependent kinases" Description The present invention relates to the use of indigoid bisindole derivatives for the manufacture of a medicament for inhibiting cyclin dependent kinases, particularly CDK 1 , CDK2, CDK 4 and CDK 5, more particularly ATP:Proteinphosphotransfer- ase p34 cdc2 (CDK1 ) . Indigoid bisindoles comprise a spectrum of natural dye stuffs. Many of these can be obtained from plants. Accordingly, indirubine, indigo and isoindigo are natural products which can be obtained from different plants: namely, Baphicacanthus cusia (Acanthaceae), Indigofera suffruticosa (Fabaceae), Isatis indigotica (Brassi- caceae) and others. Indican, a glycoside which is found in plants, gives glucose and 3-hydroxyindole due to acidic or enzymatic hydrolysis. 3-Hydroxyindole is converted by air-oxidation into indigo and its isomers. Indigo naturalis (Chinese: quingdai) is the natural blue dye obtained from plant material, e.g. Isatis indigotica (Brassicaceae) . Indirubine, an isomer of indigo, can be found in Indigo natura- Ms in an amount of up to 60% (Falbe J . & Regitz M., Rόmpp Chemie Lexikon (1 992), 9. Aufl., Stuttgart, Georg Thieme Veriag) . It occurs also in Isatis tincto- ria in an amount of up to 5% which is indigenous to Central Europe (Gelius R., Z. Chem., 20, (1 980), 340-341 ) . Indigo naturalis is reported to be used in traditional Chinese medicine εs a hemostatic, antipyretic, anti-inflammatory and sedative agent in the treatment of bacterial and viral infections. Antileukemic effects of Indigo naturalis have also been reported, with indirubine being the effective principle (Ji X. et al., Acta Pharm. Sin., 1 6, ( 1 981 ), 146-1 48; Gan W. J . et al., J . Hematol., 6, (1 985), 61 1 -61 3) . Furthermore, derivatives of indirubine are known for a long time as dyes of low persistence. Few, structurally quite different types of p34 cdc2 inhibitors have been described up to now. They are either of natural origin or derived from natural compounds and show varying degrees of inhibitory activities. Examples are Staurosporine, an alkaloid from Streptomyces sp, Butyrolactone-I from Aspergillus terreus var., Flavopiridol, a novel promising anti-tumour agent derived by partial synthesis from a parent structure found in the indian plant Dysoxylum binectariferum, 9- Hydroxyellipticin from the plants Ochrosia elliptica and Ochrosia acuminata, the purine derivatives olomoucine, roscovitine and isopentenyladenine and some peptides. The mechanism of these compounds is based on competitive inhibition of ATP binding (Meijer , Trends in Cell Biology, 6, (1 996), 393-397) . For determination of the IC 50-vaiues: p34 cdc2 /cyclin B was purified from M phase starfish (Marthasterias glacialis) oocytes by affinity chromatogaphy on p9 CKShs1 -Sepharose beads, from which it was eluted by free p 9 CKShs1 as described (Meijer et al., Eur. J. Biochem., 243, (1 997), 527-536) . It was assayed with 1 mg histone H 1 (Sigma type lll-S)/ml, in the presence of 1 5 μM |>- 32 P] ATP (3,000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 10 min. incubation at 30°C, 25 μl aliquots of supernatant were spotted onto 2.5 x 3 cm pieces of Whatman P81 phosphocellulose paper, and after 20 sec, the filters v^re washed five times (for at least 5 min. each time) in a solution of 10 ml phosphoric acid/liter of water. The wet filters were transferred into 6 ml plastic scintillation vials, 5 ml ACS (Amersham) scintillation fluid was added and the radioactivity measured in a Packard scintillation counter. The kinase activity was expressed in pmoles phosphate incorporated into histone H 1 /10 min incubation or in % of maximal activity. The strongest inhibitors of p34 are derivatives of staurosporine (IC 50 0,003-0,03 μM) . The selectivity of these inhibitors is, however, rather poor. They also show more or less potent inhibitory activity to quite a wide range of cellular kinases. Table 1 shows the specifity of known chemical inhibitors of cyclin-dependent kinases (IC 50 -values given in μM) (cf. Meijer L, Trends in Cell Biology, 6, ( 1 996), 393-397) . Tab. 1 : Enzyme StauroUCN- Butyro- Flavopi- Olomo- Rosco- 9-Hydro- sporine 01 lactone- ridol ucin vitine xy-ellip- ticine CDK1 0.003- 0.031 0.60 0.40 7 0.65 a ca. 1 0.009 CDK2 0.007 0.030 1 .50 0.40 7 0.70 ND CDK4 < 0.032 no 0.40 > 1 000 > 100 ND 1 0.000 effect MAPK 0.020 0.910 94 ND 30 30 ND PKA 0.008 ND 260 145 > 2000 > 1000 ND PKG 0.009 ND ND 6 > 2000 > 1000 ND PKC 0.005 0.007 1 60 ND > 1 000 > 100 ND Tyrosine- 0.006 ND > 590 25 440 70 ND kinase (EGF-R) (EGF-R) (EGF-R) (EGF-R) (l-R) 0.006 (Src) CDK: cyclin-dependent kinase; EGF-R: epidermal growth factor receptor tyrosine kinase; l-R: insulin receptor-tyrosine kinase; MAPK: mitogene-activated protein kinase; ND: not determined; PKA: cAMP-dependent protein kinase; PKG: cGMP-dependent protein kinase. a IC 50 -value for racemic mixture; IC 50 for (R)-roscovitin is 0.45 μM. Cyclins and cyclin dependent kinases (CDK) have an essential role for driving the cell through the cell cycle (cell division cycle, cdc) . During the cell division cycle, oscillations in concentrations and activities of cyclins are observed. This applies, e.g. to cyclins D and E in the so-called G 1 -phase of the cell cycle and to cyclinA (S- and M-Phase) and cycIinB (G2- and M-Phase) . The cyclin dependent kinases are activated by association with a member of the cyclin-family. Up to now, eight human CDKs have been described: CDK1 ( = p34 cdc2 ), CDK2 to CDK8. CDK-proteins consist of a catalytic subunit and a regulatory subunit, the cyclins (Meijer et al., Eur. J. Biochem., 243, ( 1 997), 527-536) . Every step of the cell division cycle is regulated by specific CDK/cyc- lin complexes which ascertain a strict control. Important checkpoints are at the transition from G 1 phase to S phase and from G2 phase to M phase (Pines J., Cancer Biology, 6, (1 995), 63-72). The p34 cdc2 /cyclinB complexes are important components at the G 2 -M-checkpoint. Fig.1 shows the points of action of the cyclin CDK complex cdc2/cyclinB in the cell divsion cycle (M = mitosis, cell division; G = gap; S = synthesis; interphase = G^ S + G^. The cdc2/cyclinB complexes are the primary active protein kinases in mitosis. They accumulate in an inactive state in the cytoplasm in interphase cells, and are then rapidly activated by cdc25 phosphatase and translocated into the nucleus at the beginning of mitosis (Pines J., Hunter J., Cell Biol., 1 1 5(1 ) , ( 1 991 ), 1 -1 7) . CyclinB is degraded at the metaphase-anaphase transition, inactivating cdc2, which is necessary for exit from mitosis (Glotzer et al., Nature, 349(6305). (1 991 ), 132-138; Murray A.W., Nature, 339(6222). (1 989), 280-286; Surana U. et al., Cell, 65(1). (1 991 ), 145-1 61 ) . Multiple changes of CDK proteins and their regulators are associated with the development of human tumours (Cordon-Cardo C, Am. J. Pathol., 147(3), (1 995), 545-560) . Thus, the technical problem underlying the present invention is to provide new inhibitors for cyclin dependent kinases, particularly CDK 1 , CDK2, CDK 4 and CDK 5, more particularly ATP:Proteinphosphotransferase p34 cdc2 (CDK1 ), which exhibit a high selectivity as well as high efficiency compared to the inhibitors known in the art. The solution to the above technical problem is achieved by the embodiments characterized in the claims. In particular, the present invention relates to the use of indigoid bisindole derivatives for the manufacture of a medicament for inhibiting cyclin dependent kinases, particularly CDK 1 , CDK2, CDK 4 and CDK 5, more particularly ATP:Proteinphosphotransferase p34 cdc2 (CDK1 ), in mammals, preferably in man. Preferably, the indigoid bisindole derivatives are selected from indigo derivatives, isoindigo derivatives or indirubine derivatives. In a preferred embodiment of the present invention, the indirubine derivate is a compound having the general formula (I) (I) wherein the groups R 1 and R 6 can be the same or different and represent a hydrogen atom; a halogen atom; a hydroxy group; a methylenehydroxy group; a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms; a straight-chain or branched-chain alkyloxy group having 1 to 1 8 carbon atoms; a straight-chain or branched-chain methylenealkoxy group having 1 to 18 carbon atoms; a cycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a substituted or unsubstituted aryl group which can comprise one or more heteroatoms; a substituted or unsubstituted aralkyl group which can comprise one or more heteroatoms; a substituted or unsubstituted aryloxy group which can comprise one or more heteroatoms; a mono-, di- or trialkylsilyl group having 1 to 6 carbon atoms independently of each other in each instance in the straight-chain or branched-chain alkyl group; a mono-, di- or triarylsilyl group with substituted or unsubstituted aryl groups independently of each other in each instance; a trifluoromethyl group; a -COM group; a -COOM group; a -CH 2 COOM group, wherein M is hydrogen, a straight- chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, or an aryl group which can comprise one or more heteroatoms and can be substituted with one or more halogen atoms, one or more alkyl groups or one or more alkoxy groups; a -NR 11 R 12 group, wherein R 1 1 and R 12 can be the same or different and represent a hydrogen atom, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, a substituted or unsubstituted aryl group which can comprise one or more heteroatoms; or an acyl group; a methylene-amino group -CH 2 -NR 11 R 12 , wherein R 1 1 and R 12 have the above definitions; a benzyl group, wherein the benzene nucleus can comprise one or more heteroatoms; a methylenecycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a physiological amino acid residue bound to the nitrogen as an amide; an O-glycoside or a N-glycoside, wherein the glycoside is selected from monosaccharides or disaccharides; or a methylenesulfonate group; R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 and R 10 can be the same or different and represent a hydrogen atom; a halogen atom; a hydroxy group; a nitroso group; a nitro group; an alkoxy group; a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups; a substituted or unsubstituted aryl group which can comprise one or more heteroatoms; a substituted or unsubstituted aralkyl group which can comprise one or more heteroatoms; a substituted or unsubstituted aryloxy group which can comprise one or more heteroatoms; a substituted or unsubstituted methylenearyloxy group which can comprise one or more heteroatoms; a cycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a methylenecycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a trifluoromethyl group; a -COM group; a -COOM group; a -CH 2 COOM group, wherein M is hydrogen, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, or an aryl group which can comprise one or more heteroatoms and can be substituted with one or more halogen atoms, one or more alkyl groups or one or more alkoxy groups; a -NR 1 R 12 group, wherein R 1 1 and R 12 can be the same or different and represent a hydrogen atom, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, a substituted or unsubstituted aryl group which can comprise one or more heteroatoms, or an acyl group, or wherein the nitrogen atom is part of a cycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatom(s); a -CONR 1 1 R 12 group, wherein R 1 and R 12 have the above definitions; a hydroxylamino group; a phosphate group; a phosphonate group; a sulfate group; a sulfonate group; a sulfonamide group; a -S0 2 NR 11 R 12 group, wherein R 11 and R 12 have the above definitions; an azo group -N = N-R 13 , in which R 13 represents an aromatic system which can be substituted by one or more carboxyl groups, phosphoryl groups or sulfonate groups; or a O-glycoside or a N-glycoside, wherein the glycoside is selected from monosaccharides or disaccharides; or R 1 and R 5 , and R 6 and R 10 , respectively, form independently from each other a ring together having 1 to 4, optionally substituted, CH 2 groups; and X and Y can be the same or different and represent an oxygen atom; a sulfur atom; a selenium atom; a tellurium atom; a NR 14 group in which the group R 14 represents a hydrogen atom, a straight-chain or branched-chain alkyl group having 1 to 18 carbon atoms which can be substituted by one or more carboxyl groups, phosphoryl groups or sulfonate groups, a substituted or unsubstituted aryl group which can comprise one or more heteroatoms, an aralkyl group, or a sulfonate group; or a NOR 14 group, wherein the group R 14 has the above definitions. With respect to the benzene nuclei constituting the indirubine derivates of the the above general formula (I) one or more ring atoms can be replaced by nitrogen atoms. Further, one or more aromatic or non-aromatic ring systems which can comprise one or more heteroatoms independently of each other, can be condensed to the indirubine system. Furthermore, the indirubine derivatives having the above general formula (I) can also be bound to a polyethyleneglycolester or a polyethyleneglycolether by ester bondings or ether bondings, respectively. In another embodiment of the present invention, the isoindigo derivate is a compound having the general formula (II) wherein R 1 to R 14 and X and Y have the above definitions. With respect to the benzene nuclei constituting the isoindigo derivates of the above general formula (II) one or more ring atoms can be replaced by nitrogen atoms. Further, one or more aromatic or non-aromatic ring systems which can comprise one or more heteroatoms independently of each other, can be condensed to the isoindigo system. Furthermore, the isoindigo derivatives having the above general formula (II) can be bound to a polyethyleneglycolester or a polyethyleneglycolether by ester bondings or ether bondings, respectively. In a further embodiment of the present invention, the indigo derivate is a compound having the general formula (III) (III) wherein R 1 to R 14 and X and Y have the above definitions. With respect to the benzene nuclei constituting the indigo derivates of the above general formula (III) one or more ring atoms can be replaced by nitrogen atoms. Further, one or more aromatic or non-aromatic ring systems which can comprise one or more heteroatoms independently of each other, can be condensed to the indigo system. Furthermore, the indigo derivatives having the above general formula (III) can be bound to a polyethyleneglycolester or a polyethyleneglycolether by ester bondings or ether bondings, respectively. The above indigoid bisindole derivatives having the general formulas (l), (II) or (III) can also be in the form of their physiologically acceptable salts. In the search for new selective inhibitors of cellular signalling pathways, it has surprisingly been found that indigoid bisindole derivatives are highly selective inhibitors of the enzyme complex p34 cdc2 /cyclinB. An inhibition of p34 cdc2 -kinase by indigoid bisindole derivatives is not described in the prior art. Surprisingly, it has turned out that indigoid bisindole derivatives are both highly selective and highly effective inhibitors of cdc2-kinase and other cyclin dependent kinases (CDK's), showing IC 50 -values with the isolated enzyme down to submicromolar range. The inhibition of CDK-activities also is observed in cell culture, using human tumor cell lines. As an example, indirubin-3'-monoxime was found to inhibit histone H 1 phosphorylation as a measure for CDK1 /cyclin B activity after 24 h incubation of MCF-7 mammary carcinoma cells. Moreover, the content of cyclin B complex was significantly reduced. Cells arrested by serum deprivation after treatment for 24 h with indirubin-3'-monoxime in serum containing medium exhibited an arrest in G 1 -phase of the cell cycle at low micromolar concentrations of the substance. In concentrations > 5 μM, an additional arrest at G2/M-phase of the cell cycle became apparent. Cells arrested in G2/m by Nocodazole treatment after release of the block exhibited a significant increase of G2/M arrested cells under treatment with > 5 μM indirubin-3'-monoxime, resulting in a massive accumulation of cells in G2/M. Concomitantly with the observed intracellular effects, growth inhibition was induced in the same concentration range, resulting in an IC 50 -value of 3.3 ± 0.7 μM after 3 days incubation (IC 50 = concentration that induces 50% growth inhibition as compared to vehicle control) . In addition, induction of apoptotic cell death was observed. Based on the above mentioned selective inhibitory potency down to the nanomolar range, indirubine derivatives can be used for analytical biochemistry, especially for the study of cell cycle effects. Furthermore, these compounds can be used for the treatment of diseases in patients, which are connected to the loss of proliferation control without any restriction to these potential areas of application. These include cancers, psoriasis, cardiovascular diseases (stenosis, restenosis) (Brooks et al., J. Biol. Chem., 272, ( 1 997), 29207-2921 - 1 ), infectious diseases (unicellular parasites (Trypanosoma, Toxoplasma, Plasmodium, etc.), fungi, etc.), nephrology (glomerulonephritis: Pippin et al., J. Gin. Invest., 100, ( 1 997), 251 2-2520), neurodegenerative disorders such as Alzheimer disease (Imahori, K., Uchida T., J. Biochem., 1 21 , ( 1 997), 1 79-1 88), viral infections such as cytomegalovirus (Bresnahan W. A. et al., Virology 231,(1 997), 239-247) and HIV (Mancebo H.S.Y. et al., Genes & Dev., H, (1 997), 2633-2644) . The present invention is explained further by the following examples: Example 1 : Synthesis of indirubine To a solution of 0.42 g (2.4 mmol) of indoxyl acetate in 20 ml methanol under argon 0.35 g (2.4 mmol) of isatin and 0.55 g (5.2 mmol) of sodium carbonate are added. The mixture is stirred for 30 min at ambient temperature. After 24 h standing at ambient temperature, the reaction mixture is filtered off. The precipitate is washed with little methanol and water until the filtrate shows a neutral pH. Residual water is removed by storage in an evacuated exsiccator over potassium hydroxide. Recrystallisation from ethanol or pyridine gives deep purple crystals (Russell G.A., Kaupp G. ( 1 969), J. Am. Chem. Soc, 9JL, 3851 - 9, modified) . Yield: 0.51 g (81 %), fine, deep-purple needles, Fp: 341 -343 ° C CHN-analysis: (C 16 H 10 N 2 O 2 ); MW: 262,26 g/mol; calc: 73.3% C, 3.8% H, 10.7% N; found: 73.2% C, 4.0% H, 10.6% N mass spectrum: m/z = 262: (M + , 100%), 234: (43%), 205 (25%), 1 58 (3%), 1 31 (4%), 1 03 (7%), 76 (3%) 1 H-NMR and 13 C-NMR-spectrum are in accordance with the proposed structure. IR-spectrum: 3340 cm "1 : v (N-H), 1 710 cm "1 : v (3 ' -C = O), 1 650 cm "1 : v (2- C = 0), 1 590 cm- 1 : v (C = C, aryl), 1450 cm "1 : v (C = C, aryl), 745 crτr 1 : v (aryl with four neighbouring H-atoms) . UV/Vis-spectrum (DMSO): 290 nm, 363 nm, 383 nm (shoulder), 551 nm Essentially the same synthetic procedere was applied for the following Examples 2 to 9 and 1 1 and 1 2: Example 2: 5-lodoindirubine Yield: 80%, fine, deep-purple needles, Fp: 334-335 ° C (decomposition); CHN-analysis (C 16 H 9 IN 2 0 2 ); MG = 388.1 6 g/mol; calc : 49.5% C, 2.3% H, 7.2% N; found. : 49.7% C, 2.5% H, 7.1 % N; Mass spectrum: 388 (M + , 1 00%), 360 (3%), 269 (9%), 261 (6%), 233 ( 1 6%), 205 ( 1 6%), 1 28 (1 %); 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. UV/Vis-spectrum (DMSO): 370 nm, 386 nm (shoulder), 555 nm. Example 3: 5-Bromoindirubine Yield: 70%, fine, deep-purple needles; CHN-analysis (C 16 H 9 BrN 2 O 2 ); MG = 341 .1 6 g/mol, calc: 56.3% C, 2.7% H, 8.2% N; found 56.4% C, 2.7% H, 8.2% N; Mass spectrum: 342(M + , 100%), 340 (M + , 99%), 314 (18%), 262 (64%), 233 (34%), 205 (81 %), 1 77 (10%); 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Example 4: 5-Chloroindirubine Yield: 95%, fine, deep-purple needles; CHN-analysis (C 16 H 9 CIN 2 0 2 ); MG = 296.70 g/mol; calc: 49.5% C, 2.3% H, 7.2% N; found: 49.7% C, 2.5% H, 7.1 % N; Mass spectrum: m/z = 296 (M + , 1 00%), 268 (39%), 239 (8%), 233 (35%), 205 (50%), 1 77 (7%), 1 53 (6%), 1 37 (7%), 77 (7%), 1 20 (4%), 1 02 (6%), 77 (7%) . 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Example 5: 5-Fluoroindirubine Yield: 92%, fine, deep-purple needles; CHN-analysis (C 16 H 9 FN 2 0 2 ), MG = 280.25 g/mol, calc: 68.6% C, 3.2% H, 9.9% N; found: 68.0%C, 3.2% H, 9.9% N; Mass spectrum: m/z = 281 (M + + H + , 19%), 280 (M + , 100%), 252 (73%), 223 (32%), 176 (6%), 140 (7%), 121 (13%), 94 (4%), 76 (12%), 77 (7%), 57 (4%), 44(15%). 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Example 6: 5-Methylindirubine: Yield: 92%, fine, deep-purple needles; CHN-analysis (C 17 H l2 N 2 0 2 ), MG = 276.28 g/mol, calc: 73.9% C, 4.4% H, 10.1% N; found: 73.8%C, 4.3% H, 10.2% N; Mass spectrum: m/z = 276 (M + , 100%), 261 (10%), 248 (47%), 247 (53%), 220 (6%), 219 (18%), 205 (7%), 171 (4%), 165 (10%), 138 (4%), 133 (15%), 104 (7%), 77 (7%); 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Example 7: 5-Nitroindirubine Yield: 88%, fine, deep-purple needles; CHN-analysis (C 16 H 9 N 3 0 4 ), MG = 307.26 g/mol; calc: 62.5% C, 3.0% H, 13.7% N; found: 62.4%C, 3.0% H, 13.3% N; Mass spectrum: m/z = 307 (M + , 5%), 276 (10%), 262 (100%), 234 (23%), 205 (22%), 158 (6%), 131 (10), 104 (19%), 76 (12%), 50 (6%). 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Example 8: 5'-Bromoindirubine Yield: 92%, fine, deep-purple needles; CHN-analysis (C 16 H 9 BrN 2 0 2 ), MG = 341.16 g/mol, calc: 56.3% C, 2.7% H, 8.2% N; found: 55.7%C, 2.5% H, 8.0% N. 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Example 9: 5,5'-Dibromoindirubine Yield: 94%, fine, deep-purple needles; CHN-analysis (C 16 H 8 Br 2 N 2 0 2 ), MG = 420.06 g/mol; calc: 45.7% C, 1.9% H, 6.7% N; found: 45.8% C, 2.0% H, 6.4% N. 1 H-NMR-spectrum is in accordance with the proposed structure. Example 10: lndirubine-3'-oxime lndirubine-3'-oxime was synthesized by reaction of indirubine with hydroxylamine hydrochloride in a pyridine solution (Farbwerke vorm. Meister Lucius & Brϋning in Hoechst a.M., Patentschrift des Reichspatentamtes Nr. 283726 (1913)). 13 C-NMR-spectroscopy revealed the location of the hydroxyimino residue in 3'-Position (<J(C2) = 171.05 ppm; c5(C3') = 145.42 ppm; DMSO-d 6 , RT) Yield: 90 %, red crystals; CHN-analysis (C 16 H l1 N 3 O 2 ), MG = 277.30g/mol; calc: 69.3% C, 4.0% H, 15.2 % N; found: 69.0% C, 4.0% H, 14.9% N; 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Example 11 : lndirubine-5-sulfonic acid Yield: 76%, crystalline, deep-purple substance; Mass spectrum: 388 (M + , 100%), 360 (3%), 269 (9%), 261 (6%), 233 (16%), 205 (16%), 128 (1%). 1 H-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Table 2 shows the structures of the compunds of Examples 1 to 11. Tab. 2: Table 3 shows the specificity of the compounds of Examples 1 to 1 1 (IC 50 . values given in μM) to inhibit cdc2 kinase in comparison to other cellular kinases. Tab. 3: ND: not dedected; PKA: cAMP-dependent protein kinase; PKC: Ca 2 + - dependent protein kinase Example 1 2: Isoindigo Isoindigo was synthesized by reaction of oxindole with isatin in acetic acid with addition af hydrochloric acid (Wahl A., Bayard P., Comptes Rendues Hebdomadaires des Seances de L'Academie des Sciences, 148, ( 1 909), 71 6- 71 9) . Yield: 84%, crystalline, brown substance; CHN-analysis (C 16 H 10 N 2 0 2 ), MG = 262.26 g/mol; calc: 73,3% C, 3.8% H, 10,7%o N; found: 73.0% C, 3.8% H, 1 0.9% N; Mass spectrum: m/z = 262 (M + , 1 00%), 234 (85%), 220 (5%), 205 (1 8%), 1 90 (4%), 1 77 (5%), 1 51 (5%), 1 32 ( 1 7%), 103 (6%), 76 (4%), 32 (26%) . ^-NMR- and 13 C-NMR-spectrum are in accordance with the proposed structure. Isoindigo shows an IC 50 -value of 80μM for the p34 cdc2 /cyclinB complex. Table 4 shows the kinase inhibition selectivity of indigo, indirubin, 5-chloroindi- rubine, indirubine-3'-monoxim and indirubine-5 sulfonic acid . The indicated IC 50 values were calculated from respective dose response curves and are presented in μM. Tab. 4:

Claims: Claims 1 . Use of indigoid bisindole derivatives for the manufacture of a medicament for inhibiting cyclin dependent kinases. 2. Use according to claim 1 , wherein the cyclin dependent kinases are selected from CDK 1 , CDK 2, CDK 4 or CDK5. 3. Use according to claim 1 or 2, wherein the indogoid bisindole derivatives are selected from indigo derivatives, isoindigo derivatives or indirubine derivatives. 4. Use according to claim 3, wherein the indirubine derivate is a compound having the general formula (I) (I) wherein the groups R 1 and R 6 can be the same or different and represent a hydrogen atom; a halogen atom; a hydroxy group; a methylenehydroxy group; a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms; a straight-chain or branched-chain alkyloxy group having 1 to 1 8 carbon atoms; a straight-chain or branched-chain methylenealkoxy group having 1 to 1 8 carbon atoms; a cycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a substituted or unsubstituted aryl group which can comprise one or more heteroatoms; a substituted or unsubstituted aralkyl group which can comprise one or more heteroatoms; a substituted or unsubstituted aryloxy group which can comprise one or more heteroatoms; a mono-, di- or trialkylsilyl group having 1 to 6 carbon atoms independently of each other in each instance in the straight-chain or branched-chain alkyl group; a mono-, di- or triarylsilyl g roup with substituted or unsubstituted aryl groups independently of each other in each instance; a trifluoromethyl group; a -COM group; a -COOM group; a -CH 2 COOM group, wherein M is hydro- gen, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, or an aryl group which can comprise one or more heteroatoms and can be substituted with one or more halogen atoms, one or more alkyl groups or one or more alkoxy groups; a -NR 1 1 R 12 group, wherein R 11 and R 12 can be the same or different and represent a hydrogen atom, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, a substituted or unsubstituted aryl group which can comprise one or more heteroatoms; or an acyl group; a methyleneamino group -CH 2 -NR 11 R 12 , wherein R 1 1 and R 12 have the above definitions; a benzyl group, wherein the benzene nucleus can comprise one or more heteroatoms; a methylenecycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a physiological amino acid residue bound to the nitrogen as an amide; an O-glycoside or a N-glycoside, wherein the glycoside is selected from monosaccharides or disaccharides; or a methylenesulfonate group; R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 and R 10 can be the same or different and represent a hydrogen atom; a halogen atom; a hydroxy group; a nitroso group; a nitro group; an alkoxy group; a straight- chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups; a substituted or unsubstituted aryl group which can comprise one or more heteroatoms; a substituted or unsubstituted aralkyl group which can comprise one or more heteroatoms; a substituted or unsubstituted aryloxy group which can comprise one or more heteroatoms; a substituted or unsubstituted methylenearyloxy group which can comprise one or more heteroatoms; a cycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a methylenecycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatoms; a trifluoromethyl group; a -COM group; a -COOM group; a -CH 2 COOM group, wherein M is hydrogen, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, or an aryl group which can comprise one or more heteroatoms and can be substituted with one or more halogen atoms, one or more alkyl groups or one or more alkoxy groups; a -NR 1 1 R 12 group, wherein R 11 and R 12 can be the same or different and represent a hydrogen atom, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can additionally carry one or more hydroxy and/or amino groups, a substituted or unsubstituted aryl group which can comprise one or more heteroatoms, or an acyl group, or wherein the nitrogen atom is part of a cycloalkyl group having 3 to 7 carbon atoms which can comprise one or more heteroatom(s); a -CONR 1 1 R 12 group, wherein R 1 and R 2 have the above definitions; a hydroxylamino group; a phosphate group; a phosphonate group; a sulfate group; a sulfonate group; a sulfonamide group; a -S0 2 NR 11 R 12 group, wherein R 1 1 and R 12 have the above definitions; an azo group -N = N-R 13 , in which R 13 represents an aromatic system which can be substituted by one or more carboxyl groups, phosphoryl groups or sulfonate groups; or a O-glycoside or a N-glycoside, wherein the glycoside is selected from monosaccharides or disaccharides; or R 1 and R 5 , and R 6 and R 10 , respectively, form independently from each other a ring together having 1 to 4, optionally substituted, CH 2 groups; and X and Y can be the same or different and represent an oxygen atom; a sulfur atom; a selenium atom; a tellurium atom; a NR 14 group in which the group R 14 represents a hydrogen atom, a straight-chain or branched-chain alkyl group having 1 to 1 8 carbon atoms which can be substituted by one or more carboxyl groups, phosphoryl groups or sulfonate groups, a substituted or unsubstituted aryl group which can comprise one or more heteroatoms, an aralkyl group, or a sulfonate group; or a NOR 14 group, wherein the group R 14 has the above definitions. 5. Use according to claim 4, wherein one or more ring atoms of the benzene nuclei of the compound having the general formula (I) are replaced by nitrogen atoms. 6. Use according to claim 4 or 5, wherein one or more aromatic or non- aromatic ring systems which can comprise one or more heteroatoms independently of each other, are condensed to the indirubine system. 7. Use according to anyone of claims 4 to 6, wherein the compound having the general formula (I) is bound to a polyethyleneglycolester or a polyethyleneglycolether. 8. Use according to claim 3, wherein the isoindigo derivate is a compound having the general formula (II) (ID wherein R to R and X and Y have the meanings as defined in claim 4. 9. Use according to claim 8, wherein one or more ring atoms of the benzene nuclei of the compound having the general formula (II) are replaced by nitrogen atoms. 0. Use according to claim 8 or 9, wherein one or more aromatic or non- aromatic ring systems which can comprise one or more heteroatoms independently of each other, are condensed to the isoindigo system. 1 . Use according to anyone of claims 8 to 1 0, wherein the compound having the general formula (II) is bound to a polyethyleneglycolester or a polyethyleneglycolether. 2. Use according to claim 3, wherein the indigo derivate is a compound having the general formula (III) (III) wherein R 1 to R 14 and X and Y have the meanings as defined in claim 4. 3. Use according to claim 1 2, wherein one or more ring atoms of the benzene nuclei of the compound having the general formula (111) are replaced by nitrogen atoms. 4. Use according to claim 1 2 or 1 3, wherein one or more aromatic or non- aromatic ring systems which can comprise one or more heteroatoms independently of each other, are condensed to the indigo system. 5. Use according to anyone of claims 1 2 to 1 4, wherein the compound having the general formula (III) is bound to a polyethyleneglycolester or a polyethyleneglycolether. 6. Use according to anyone of claims 1 to 1 5, wherein the indigoid bisindole derivative is in the form of a physiologically acceptable salt. 7. Compound which is 5-fluoroindirubine. 8. Compound which is 5-nitroindirubine. 9. Compound which is 5,5'-dibromoindirubine.

Inventors: EISENBRAND GERHARD, EISENBRAND, GERHARD
Applicants: CNRS CENTRE NATIONAL DE RECH S, EISENBRAND GERHARD, CNRS (CENTRE NATIONAL DE RECHERCHE SCIENTIFIQUE) FRANCE INNOVATION SCIENTIFIQUE ET TRANSFERT, EISENBRAND, GERHARD
Application year: 1999
Main classification: A61K31
IPC codes: A61K31, A61K47, A61P43, C07D209