WO2007057397A1

WO2007057397A1 - Treatment of cancer

Info

Publication number: WO2007057397A1
Application number: PCT/EP2006/068459
Authority: WO
Inventors: Wolfgang Sommergruber; Norbert Kraut; Norbert Schweifer; Wolfgang Rettig; Frank Hilberg; Flavio Solca; Martin Steegmaier
Original assignee: Boehringer Ingelheim International Gmbh; Boehringer Ingelheim Pharma Gmbh & Co. Kg
Priority date: 2005-11-15
Filing date: 2006-11-14
Publication date: 2007-05-24

Abstract

The invention is based on the finding that indolinones are useful for the therapy of diseases which result from aberrant activity of certain tyrosine kinases selected from the group comprising ABL, FGFR3, FLT3, and RET.

Description

TREATMENT OF CANCER

TECHNICAL FIELD OF THE INVENTION

The invention relates to the field of cancer therapy, particularly to the use of compounds for the treatment of cancers associated with certain tyrosine kinases.

BACKGROUND OF THE INVENTION

Tyrosine kinases are a class of enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate to tyrosine residues in protein substrates. Tyrosine kinases play critical roles in signal transduction for a number of cell functions via substrate phosphorylation.

Tyrosine kinases fall within the categories of receptor type or non-receptor type kinases. Receptor type tyrosine kinases have an extracellular, a transmembrane, and an intracellular portion, while non-receptor type tyrosine kinases are intracellular.

The protein kinase family includes kinases such as, for example, the Abelson tyrosine kinase (ABL), fibroblast growth factor receptor 3 (FGFR3), FMS-related tyrosine kinase 1 and 3 (FLTl and FLT3) and receptor tyrosine kinase RET, and the like.

The receptor type tyrosine kinases are transmembrane receptors with diverse biological activity. Members of the HER subfamily are EGFR, HER2, HER3, and HER4. Ligands of this subfamily of receptors include epithelial growth factor, TGF-α, amphiregulin, HB- EGF, betacellulin and heregulin. Another subfamily of these receptor-type tyrosine kinases is the insulin subfamily, which includes INS-R, IGF-IR, and IR-R. The PDGF subfamily includes the PDGF-α and β receptors, CSFlR, c-KIT and FLK-II. The FLK family is comprised of the kinase insert domain receptor (KDR), fetal liver kinase- 1 (FLK-I), fetal liver kinase-4 (FLK-4) and the fins-like tyrosine kinase- 1 (FLT-I). Other receptor tyrosine kinase families are RET and AXL (AXL, MER, Tyro3). The non-receptor type of tyrosine kinases also comprises numerous subfamilies, including SRC, FRK, BTK, CSK, ABL, ZAP70, FES/FPS, FAK, JAK, ACK, and LIMK. Each of these subfamilies is further sub-divided into varying receptors. For example, the SRC subfamily is one of the largest and includes SRC, YES, FYN, LYN, LCK, BLK, HCK, FGR, and YRK. The SRC subfamily of enzymes has been linked to oncogenesis.

Certain mutations in the genes of receptor protein tyrosine kinases result in activation of the receptor in a manner that is independent of the presence of a ligand. Such ligand- independent, or constitutive, receptor protein tyrosine kinase activation results in increased receptor activity. The clinical manifestations of certain mutations are skeletal and proliferative disorders and diseases, including achondroplasia and various cancers.

Though the exact mechanisms of signal transduction have not been fully elucidated, tyrosine kinases have been shown to be important contributing factors in cell proliferation, carcinogenesis and cell differentiation.

Compounds that regulate and/or modulate tyrosine kinase signal transduction and compositions containing such compounds are useful for treating tyrosine kinase-dependent diseases and conditions, such as angiogenesis and cancer and inflammatory diseases.

The ABL (ABLl) protooncogene encodes a cytoplasmic and nuclear protein tyrosine kinase that is involved in a variety of different biological processes such as cell differentiation, cell division, cell adhesion, and stress response. Alterations of ABL by chromosomal rearrangement or viral transduction lead to malignant transformation, as in CML. Activity of c-ABL protein is negatively regulated by its SH3 domain, and deletion of the SH3 domain turns ABLl into an oncogene. The t(9;22) translocation occurs in more than 90% of CML, 25 to 30% of adult and 2 to 10% of childhood acute lymphoblastic leukemia (ALL), and rare cases of AML. The underlying cause of CML is the BCR-ABL oncogene, which results from a reciprocal t(9;22) chromosome translocation in a hematopoietic stem cell (Deininger et al., 2000, Blood 96: 3343-3356; Chissoe et al., 1995, Genomics 27: 67-82). This fusion gene encodes a chimeric BCR-ABL protein, in which the tyrosine kinase activity of ABL is constitutively activated. CML patients express the 210 kDa BCR-ABL, whereas patients with Ph+ ALL usually express a 190 kDa BCR- ABL protein arising from a different breakpoint in the BCR gene (MeIo et al., 1994, Leukemia 8:208-211; Ravandi et al., 1999, Br. J. Haematol. 107).

The ubiquitously expressed ABL tyrosine kinase is localized to the nucleus and binds to DNA. The DNA-binding activity is regulated by CDC2-mediated phosphorylation. Welch and Wang showed that the tyrosine kinase activity of nuclear ABL is regulated in the cell cycle through a specific interaction with the retinoblastoma (RBl) protein (Welch and Wang, 1993, Cell 75: 779-790). Alternative splicing leads also to a 145-kD ABL protein classified as a "nonreceptor tyrosine kinase" (Chissoe et al., 1995, Genomics 27: 67-82).

A domain in the C-terminus of RB binds to the ATP-binding lobe of the ABL tyrosine kinase, resulting in kinase inhibition. Hyperphosphorylation of RB correlates with release of ABL and activation of the tyrosine kinase in S phase cells. The nuclear ABL tyrosine kinase can enhance transcription, and this activity is inhibited by RB. Thus, nuclear ABL is an S phase-activated tyrosine kinase that might participate directly in the regulation of transcription. Fellner et al. (1994, Trends Biochem. Sci. 19: 453-458) described the SRC homology domains SH2 and SH3 as molecular adhesives on many proteins involved in signal transduction including the interaction of ABL and CRK (SH2 and SH3 interaction). Barila and Superti-Furga (1998, Nature Genet. 18: 280-282) presented evidence for an intramolecular inhibitory interaction of the SH3 domain with the catalytic domain. Site- directed mutations in each of these elements activated c-ABL. Barila and Superti-Furga proposed that regulators of c-ABL will have opposite effects on its activity depending on their ability to favor or disrupt these intramolecular interactions.

Imatinib mesylate (Gleevec, STI571; Novartis Pharma AG) is a drug targeting the tyrosine kinase activity of BCR-ABL (Buchdunger et al., 2001, Biochim. Biophys. Acta 1551: Ml 1-M18) and is an effective therapy for CML. However, in ALL or in CML patients who have progressed to either the accelerated or blastic phases of the disease, response rates to imatinib therapy are significantly decreased and, of those who initially respond to treatment, many relapse within 12 months. Relapse is often associated with point mutations in BCR-ABL that reduce the binding affinity of imatinib, or occasionally with amplification of the BCR-ABL gene (for details see Weisberg et al., 2005, Cancer Cell 7: 129-141 and references therein). Thus, there is a need for additional BCR-ABL tyrosine kinase inhibitors that are more potent and active against imatinib-resistant BCR-ABL mutants. Several second-generation inhibitors that are effective against imatinib-resistant mutant ABL kinases have been developed, showing improved potency and activity against most ABL mutations (most notably AMN107 and BMS-354825/dasatinib). However, neither of these new inhibitors is highly effective against the imatinib-resistance mutant ABL(T315I) (Shah et al., 2004, Science, 305: 399- 401; Weisberg et al., 2005, Cancer Cell 7: 129-141; O'Hare et al., 2005, Cancer Res. 65: 4500- 4505).

Specific germline activating point mutations in the gene encoding the tyrosine kinase receptor FGFR3 (fibroblast growth factor receptor 3) result in autosomal dominant human skeletal dysplasias. The identification in multiple myeloma and in two epithelial cancers - bladder and cervical carcinomas - of somatic FGFR3 mutations identical to the germinal activating mutations found in skeletal dysplasias, together with functional studies, have suggested an oncogenic role for this receptor. Activating point mutations in the FGFR3 gene occur most frequently in low-grade and low-stage bladder carcinomas, whereas they are rare in high-grade carcinomas (van Rhijn et al., 2002, J. Pathol. 198: 245-251).

The FMS-related tyrosine kinase 3 (FLT3) was originally identified as a stem cell tyrosine kinase 1 (STKl). FLT3 is a member of the type III receptor tyrosine kinase family that includes KIT, FMS, and PDGFR. FLT3 expression is restricted in human blood and marrow to CD34⁺ cells, a population greatly enriched by stem/progenitor cells. FLT3 functions as a growth factor receptor on hematopoietic stem and/or progenitor cells and plays an essential role in acute myeloid leukemia (AML). Internal tandem duplication (ITD; mutations within the juxtamembrane domain leads to sustained dimerization) mutations causing constitutive activation of the receptor. FLT3 becomes phosphorylated independently of the ligand (Abu-Duhier et al., 2000, Brit. J. Haemat. I l l: 190-195). FLT3 mutations found to be the strongest prognostic factor for overall survival in patients under the age of 60 years. Kelly et al. (2002, Proc. Nat. Acad. Sci. 99: 8283-8288) noted that acute promyelocytic leukemia (APL) cells express aberrant fusion proteins involving the retinoic acid receptor alpha (RARA). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARA in the balanced reciprocal chromosomal translocation, t(15;17)(q22;ql 1). Expression of PML/RARA in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3 include e. g. ITD. FLT3(ITD) expression transforms hematopoietic cell lines to factor independent growth and also induces a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML; for this a cooperation of FLT3(ITD) and PML/RARA is necessary.

Activation of the FLT3 due to ITD is found in 20 to 25% of AML patients (Kottaridis et al., 2001, Blood 98: 1752-1759; Meshinchi et al., 2001, Blood 97: 89-94). The observed length mutation is usually an internal duplication. Point mutations and deletions of codons 835-836 of FLT3, which are located in the activation loop of the protein tyrosine kinase domain, are detected in -7% of all AML cases (Yamamoto et al., 2001, Blood 97: 2434- 2439; Abu-Duhier et al., 2001, Brit. J. Haemat. 113: 983-988). Single point mutations mainly found in the activation loop domain (hotspot region for activating) e. g. D835 (similar to position D816 in the KIT oncogene). The D835 mutation occurs independently of the FLT3 ITD mutation. The FLT3 ITD mutation is found in 20% of patients with AML and is strongly associated with leukocytosis and a poor prognosis. Yamamoto et al. (2001, Blood 97: 2434-2439) found missense mutations in 7% of AML, in -3% of myelodysplastic syndrome cases, and in -3% of ALL patients. Activating mutations of the FLT3 receptor tyrosine kinase are common in AML but are rare in adult ALL. Armstrong et al. (Blood, 2004, 103: 3544-3546) found that ~ 25% of hyperdiploid ALL samples possess FLT3 mutations. In samples from patients whose disease would relapse, FLT3 mutations were identified. These data suggested that patients with hyperdiploid or relapsed ALL in childhood might be considered candidates for therapy with small-molecule inhibitors of FLT3. FLT3 ITD mutations were also detected in myeloid sarcoma that typically occurs in the setting of AML or myeloproliferative disorders (Ansari-Lari et al, 2004, Brit. J. Haemat. 126: 785-791).

There are several agents in clinical development that are capable of inhibiting the kinase activity of this activated enzyme (Stone, Medscape 2003), one of them being MLN518 that was used for treatment of patients with advanced AML, with either wild type or mutant FLT3 (De Angelo et al., 2003, Blood 102: 65a. Abstract 219). Another FLT3 inhibitor, CEP701, inhibits FLT3 with an IC₉0 of 10-20 nM. It is highly protein bound, and, similar to MLN518, also inhibits other kinases. PKC 412, a third FLT3 inhibitor, which also inhibits c-kit, vascular endothelial growth factor receptor (VEGFR), and protein kinase C, was associated with responses in mutant FLT3 patients and a more modest level of response in AML patients with wild-type FLT3 (Estey et al., 2003, Blood 102: 614a. Abstract 2270). Since clinical resistance has been reported for the kinase inhibitor PKC412 in AML by mutation of Asn-676 in the FLT3 tyrosine kinase domain (Heidel et al., 2005, Blood; doi:10.1182/blood-2005-06-2469), there exists a high medical need for a specific FLT3 inhibitor targeting also its mutated forms (Levis and Small, 2005, Int. J. Hematol. 82: 100-107).

GTP14564, another tyrosine kinase inhibitor active against FLT3, was shown to inhibit not only mutated FLT3 in RS4;11 cells but also FLT3 amplified wild-type cells such as SEMK2-M1. Responses to GTP14564 in all cell types were directly related to the level of STAT5 phosphorylation in the cells (Yao et al., 2005, Leukemia 19: 1605-1612).

The RET receptor tyrosine kinase protooncogene undergoes oncogenic activation in vivo and in vitro by cytogenetic rearrangement (Grieco et al., 1990, Cell 60: 557-563). Mutations in the RET gene are associated with multiple endocrine neoplasia, type HA, multiple endocrine neoplasia, type HB, Hirschsprung disease (HSCR), aganglionic megacolon, and medullary thyroid carcinoma (MTC). Salvatore et al. (2000, J. Clin. Endocr. Metab. 85: 3898-3907) noted that oncogenic mutations cause constitutive activation of the kinase function of RET, which in turn results in the autophosphorylation of RET tyrosine residues critical for signaling. Regardless of the nature of the underlying activating mutation, the concomitant phosphorylation of Y1015 and Y 1062 cause multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC).

FMTC-associated RET mutations, Y791F and S891A, respectively, affect the tyrosine kinase domain of the receptor; FMTC mutants are monomeric receptors which are autophosphorylated and activated independently of glial cell line-derived neurotrophic factor (GDNF; ligand of RET, normally expressed during lung development). The mutations lead to constitutive activation of signal transducers and activators of transcription 3 (STAT3). Furthermore, it was shown that STAT3 activation is mediated by a signaling pathway involving SRC, JAKl, and JAK2, differing from STAT3 activation promoted by REJ^C634R which was previously found to be independent of SRC and JAKs (Menacho et al, 2005, Cancer Res. 65: 1729-1737).

Giordano et al. (2005, Oncogene 24: 6646-6656) performed transcriptional profiling for more than 50 papillary thyroid carcinomas. The tumors were genotyped for their common activating mutations: BRAF V600E point mutation, RET/PTC 1 and 3 rearrangement and point mutations of KRAS, HRAS and NRAS. By combining expression profiles with mutational status, they defined distinct expression profiles for the BRAF, RET/PTC and RAS mutation groups. By this one tumor without an obvious detectable mutation was predicted by the classifier to have a RET/PTC rearrangement and indeed was shown to contain one by fluorescence in situ hybridization analysis. These data demonstrate that the mutational status is the primary determinant of gene expression variation within these tumors.

RET oncoproteins expressed in thyroid carcinomas have been considered as targets for therapeutic intervention. Oncogenic activation of the receptor tyrosine kinase encoding RET gene occurs, in addition to gene rearrangement in papillary thyroid carcinomas (PTC), by missense mutation in medullary thyroid carcinomas (MTC). These genetic alterations lead to the expression of deregulated products characterized by ligand- independent activation of the intrinsic tyrosine kinase of RET. The cellular effects of the arylidene 2-indolinone RET inhibitor RPI-I on the human PTC cell line TPC-I which spontaneously harbors the RET/PTC 1 oncogene, have been described by Lanzi et al. (2003, Tumori. 89: 520-522). The reported results provide evidence that RPI-I is able to inhibit cell growth and to interfere with RET/ptcl -driven signaling. Carlomagno et al.

(2002, Cancer Res. 62: 7284-7290) showed that ZD6474, an anilinoquinazoline, blocked in vivo phosphorylation and signaling of oncogenic RET kinases. ZD6474 blocked in vivo phosphorylation and signaling of the RET/PTC3 and RET/MEN2B oncoproteins and of an epidermal growth factor (EGF)-activated EGF-receptor/RET chimeric receptor. RET/PTC3-transformed cells treated with ZD6474 lost proliferative autonomy and showed morphological reversion. In addition, ZD6474 prevented the growth of two human PTC cell lines that carry spontaneous RET/PTC 1 rearrangements, FB2 and TPCl (papillary carcinomas harboring the RET/PTC 1 rearrangement) whereas ARO harboring a wtRET did not respond. Finally, ZD6474 blocked anchorage-independent growth of RET/PTC3- transformed NIH3T3 fibroblasts and the formation of tumors after injection of NIH- RET/PTC3 cells into nude mice.

The alteration of the RET proto-oncogene has also been reported in several neuroendocrine tumors, among them small cell lung cancer (SCLC) which is considered to be a neuroendocrine tumor with high medical need (Jafri and Salgia, 2004, J. Biol. Regul. Homeost. Agents 18: 275-290).

Based on the knowledge about the dependence/addictance of tumors on ABL, FGFR3, FLT3, RET and their respective mutants in resistant tumors (see above), there is a need for new compounds, formulations, treatments and therapies to treat diseases and disorders associated with ABL, FGFR3, FLT3, and RET.

BRIEF SUMMARY OF THE INVENTION

It is, therefore, an object of this invention to provide compounds for the manufacture of a medicament useful in the treatment or prevention or amelioration of such diseases and disorders comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition comprising a compound of formula (I).

The invention is based on the finding that indolinones are useful for the therapy of diseases which result from aberrant activity of certain tyrosine kinases.

Indolinones, their preparation as well as the pharmacological activity of these compounds based on inhibition of kinases, e. g. VEGFR-2, suitable for therapy of cancer, are disclosed in WO 01/27081 and WO2004/13099. The cited documents are herewith incorporated by reference with respect to any aspects relating to these specific compounds.

DESCRIPTION OF THE DRAWINGS

TABLE 1

Kinase inhibition profile of 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)-methylcarbonyl)-N- methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2-indolinone.

IC50 values are based on the Upstate Kinase Selectivity Screening Service. A detailed description of substrates used, of buffer composition and assay conditions for each of the analysed kinases is given in Kinase Profiler™ Assay Protocols, Upstate Group, Inc. Oct.

2003. (h) refers to the human homologue of the respective kinase.

TABLE 2

Copy Number analysis of selected genes in 45 tumor cell lines for the detection of amplified chromosomal loci. The Assays are performed using Affymetrix GeneChip Human Mapping 5OK Xba arrays (Affymetrix Inc., Santa Clara, CA, USA). This subset of Mapping Arrays provides a high genomic coverage with a mean marker distance of 50 kb. The data are analyzed on the GeneChip DNA Analysis Software (GDAS) and subsequently copy numbers are calculated using the GeneChip Chromosome Copy Number Analysis Tool (CNAT) Version 2.0. (Huang, et al, 2004, Human Genomics 1 : 287-299). Numbers indicate the calculated numbers of chromosomal copies of the corresponding gene.

TABLE 3 TABLE 3A

Indicated cell lines have been selected according to their FLT3 status. BT 549 and COLO

205 contain an amplified FLT3 locus (13ql2-27.5) 3-fold and 4-fold, respectively, whereas

HL 60 and THPl exhibit a normal chromosomal status (listed as 2-fold) n.a. indicates that no chromosomal data are yet available for these cell lines. wtFLT3 : wilde-type FLT3

FLT3-ITD: FLT3 internal tandem duplications

IC50 values are listed in the nM concentration range. Doxorubicin is used as a positive control for the proliferation assay (Alamar Blue).

TABLE 3B Cell culture medium compositions and cell culture conditions are listed for the cell lines utilized. Additionally, the optimized cell number and the most appropriate developing time for each cell line in the 96-well plate Alamar Blue setting is listed.

FIGURE 1 Cluster analysis of transcription levels of selected genes versus 49 cell lines. Expression data were extracted from the BioExpress database (GeneLogic) and hierachical clustered using the Spotfire DecisionSite 8.1™. Expression levels: Black boxes indicate high, bright grey low or no expression. For methodologic details see FIGURE 2 and 4.

FIGURE 2

ABL expression profile (GeneLogic BioExpress data base) in various human normal and tumor tissues.

For expression analysis, box-and-whisker plots were generated as described (Shen-Ong et al, 2003, Cancer Res. 63: 3296 - 3301; Dolznig et al, 2005, Cancer Immun. 5: 10). The center line indicates the median, the box itself represents the interquartile range (IQR) between the first and third quartiles. Whiskers extend to 1.5 times the IQR. The human sample collection has been described by the originator of the BioExpress database (Shen- Ong et al., 2003, Cancer Res. 63: 3296 - 3301). The respective hybridizations were performed on Affymetrix HG-Ul 33A/B oligonucleotide chips (Affymetrix Inc., Santa Clara, CA, USA). These chips are based on 25-mer oligonucleotides and allow the detection of more than 39,000 human transcripts, with probe sets of 11 oligonucleotides used per transcript. Chip data were normalized with the statistical algorithm implemented in the Microarray Suite version 5.0 (Affymetrix Inc.). Briefly, the raw expression intensity for a given chip experiment is multiplied by a global scaling factor to allow comparisons between chips. This factor is calculated by removing the highest 2% and the lowest 2% of the values of the non-normalized expression values, and calculating the mean for the remaining values, as trimmed mean. One hundred divided by the trimmed mean gives the scaling factor, where 100 is the standard value used by GeneLogic. Numbers in brackets indicate the sample numbers. The Affymetrix identification code for ABL is 202123_s_at_HG-U133A. Ki N: normal kidney, Ki RCC: renal cell carcinoma, Ki CCC: kidney clear cell carcinoma, Ki_WT: Kidney Wilm's tumor, LN_N: normal lymph node, LN_ACMet: lymph node adeno carcinoma metastases, LN SCCMet: lymph node squamous cell carcinoma metastases, Br N: normal breast, LN IDCMet: metastatic infiltrating ductal carcinoma in lymph node, So N: normal soft tissue, So Os: soft tissue osteosarcoma, So_S: soft tissue sarcoma, So_MFH: soft tissue malignant fibrous histocytoma, CC_N: normal cerebral cortex, Bn GMF: glioblastoma multiforme.

FIGURE 3

ABL expression in various cell lines (GeneLogic BioExpress data base). The Affymetrix identification code for ABL is 202123_s_at_HG-U133A. Methodologic description see FIGURE 2 and 4.

FIGURE 4

FLT3 expression in various cell lines. Expression data are derived from the BioExpress database (GeneLogic) and have been generated using the Affymetrix GeneChip Human Genome U133 (HG-U133) Set (Affymetrix Inc., Santa Clara, CA, USA) as described in Figure 6. The Affymetrix identification code for FLT3 is 206674_at_HG133A. The bold vertical lines indicate present (informative) calls as defined by Affymetrix Microarray Suit 5.0 (MAS5.0) the faint vertical lines indicate absent (non-informative) calls. Methodologic description see FIGURE 2. Cell lines with strong FLT3 expression signals:

HL60: promyelocytic leukemia, THP-I: acute monocytic leukemia

FIGURE 5

RET expression in various cell lines. Expression data are derived from the BioExpress database (GeneLogic) as described in Figure 2 and Figure 4. The Affymetrix identification code for RET is 205879_x_at_HG-U133A

Cell lines with strong RET expression signals:

BT-549: breast adenocarcinoma, COLO 205: colon adenocarcinoma, HL-60: acute promyelocytic leukaemia, MCF7: breast adenocarcinoma, MDA-MB-231 : breast adenocarcinoma, NCI-H522: lung large cell carcinoma, SR: anaplastic large cell lymphoma, THP-I: acute monocytic leukaemia.

FIGURE 6

RET expression profile (GeneLogic BioExpress data base) in human normal and tumor tissues. Methodologic description see FIGURE 2 and 4.

The Affymetrix identification code for RET is 205879_x_at_HG-U133A. Br N: normal breast, Br IDC: breast infiltrating ductal carcinoma, Co N: normal colon, Co AC: colon adenocarcinoma, Lu N: normal lung, Lu AC: lung adenocarcinoma, Lu SCC: lung squamous cell carcinoma, Pr N: normal prostate, Pr AC: prostate adenocarcinoma.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the finding that indolinones are useful for the therapy of diseases which result from aberrant activity of a tyrosine kinase selected from the group comprising ABL, FGFR3, FLT3, and RET. The present invention relates therefore to the use of a compound of formula (I)

wherein

R¹ is hydrogen, amino, methyl or ethyl;

R² is -(CH₂)_PNR^CR^C, substituted or unsubstituted piperidinyl-lylmethyl, substituted or unsubstituted imidazolyl, and

R^c is independently from each other hydrogen, methyl, ethyl, -C(O)R^a, -(CH₂)_pNR^aR^a, -

C(O)(CH₂)_pNR^aR^a, -(CH₂)_pC(O)NR^aR^a or S(O)₂R^a,

R^a is hydrogen, methyl or ethyl and p is 0, 1 or 2 or its tautomers, enantiomers, diastereomers, mixtures and salts thereof for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated with at least one tyrosine kinase selected from the group comprising

ABL, FGFR3, FLT3, and RET.

Another aspect of the invention is the use of a compound selected from the group consisting of

(a) 3-Z-[I -(4-(piperidin- 1 -yl-methyl)-anilino)- 1 -phenyl-methylene] -6-ethoxycarbonyl-2- indolinone,

(b) 3-Z-[( 1 -(4-(piperidin- 1 -yl-methyl)-anilino)- 1 -phenyl-methylene]-6-carbamoyl-2- indolinone,

(c) 3-Z-[I -(4-(piperidin- 1 -yl-methyl)-anilino)- 1 -phenyl-methylene] -6-methoxycarbonyl-2- indolinone,

(d) 3-Z-[ 1 -(4-(dimethylaminomethyl)-anilino)- 1 -phenyl-methylene]-6-ethoxycarbonyl-2- indolinone,

(e) 3-Z-[l-(4-((2,6-dimethyl-piperidin-l-yl)-methyl)-anilino)-l-phenyl-methylene]-6- ethoxycarbonyl-2-indolinone,

(f) 3-Z-[I -(4-(N-(2-dimethylamino-ethyl)-N-acetyl-amino)-anilino)- 1 -phenyl-methylene] - 6-ethoxycarbonyl-2-indolinone,

(g) 3-Z-[l-(4-(N-(3-dimethylamino-propyl)-N-acetyl-amino)-anilino)-l-phenyl- methylene]-6-ethoxycarbonyl-2-indolinone,

(h) 3-Z-[l-(4-(N-(2-dimethylamino-ethyl)-N-methylsulphonyl-amino)-anilino)-l-phenyl- methylene]-6-ethoxycarbonyl-2-indolinone, (i) 3-Z-[ 1 -(4-(dimethylaminomethyl)-anilino)- 1 -phenyl-methylene] -6-methoxycarbonyl-2- indolinone,

(j) 3-Z-[I -(4-(N-acetyl-N-dimethylaminocarbonylmethyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone,

(k) 3-Z-[l-(4-ethylaminomethyl-anilino)-l-phenyl-methylene]-6-methoxycarbonyl-2- indolinone,

(1) 3-Z-[I -(4-(I -methyl-imidazol-2-yl)-anilino)-l -phenyl-methylene] -6-methoxycarbonyl-

2-indolinone,

(m) 3 -Z- [ 1 -(4-(N-dimethylaminomethylcarbonyl-N-methyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone, (n) 3-Z-[l-(4-(N-(2-dimethylamino-ethyl)-N-methylsulphonyl-amino)-anilino)-l-phenyl- methylene]-6-methoxycarbonyl-2-indolinone,

(o) 3-Z-[ 1 -(4-(N-(3-dimethylamino-propyl)-N-methylsulphonyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone,

(p) 3-Z-[ 1 -(4-(N-dimethylaminocarbonylmethyl-N-methylsulphonyl-amino)-anilino)- 1 - phenyl-methylene] -6-methoxycarbonyl-2-indolinone,

(q) 3-Z-[ 1 -(4-(N-((2-dimethylamino-ethyl)-carbonyl)-N-methyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone,

(r) 3 -Z- [ 1 -(4-(N-(2-dimethylamino-ethyl)-N-acetyl-amino)-anilino)- 1 -phenyl-methylene] -

6-methoxycarbonyl-2-indolinone, (s) 3 -Z- [ 1 -(4-methylaminomethyl-anilino)- 1 -phenyl-methylene] -6-methoxycarbonyl-2- indolinone and (t) 3 -Z- [ 1 -(4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 - phenyl-methylene]-6-methoxycarbonyl-2-indolinone, or a tautomer or salt thereof for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated with at least one tyrosine kinase selected from the group comprising ABL, FGFR3, FLT3, and RET.

A further aspect of the invention is the use 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylene]-6-methoxycarbonyl-2- indolinone or a salt thereof for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated with at least one tyrosine kinase selected from the group comprising ABL, FGFR3, FLT3, and RET.

A further aspect of the invention is the use 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylene]-6-methoxycarbonyl-2- indolinone monoethanesulfonate for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated with at least one tyrosine kinase selected from the group comprising ABL, FGFR3, FLT3, and RET.

A further aspect of the invention is the use of a compound of formula (I) for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated the tyrosine kinase ABL.

A further aspect of the invention is the use of a compound of formula (I) for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated the tyrosine kinase FGFR3.

A further aspect of the invention is the use of a compound of formula (I) for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated the tyrosine kinase FLT3. A further aspect of the invention is the use of a compound of formula (I) for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated the tyrosine kinase RET.

Such diseases are characterized by aberrant activity of the above-mentioned tyrosine kinases that results from aberrant expression, e. g. overexpression of the wildtype protein, or from expression of a mutated version leading to a constitutive signaling.

Compounds of formula (I) are described in WO 01/27081. The compounds are useful to regulate and/or modulate signal transduction of tyrosine kinase selected from ABL, FGFR3, FLT3, and RET.

ABL is found overexpressed in Kidney WiIm 's tumor, soft tissue osteosarcoma, and glioblastoma multiforme. Mutated versions of ABL are predominantly detected in Ph+ leukemias such as chronic myelogeneous leukemia (CML) or acute lymphocytic leukemia (ALL).

Recent publications (LΗote and Knowles, 2005, Exp. Cell Res. 304: 417-431 and references therein; Oiu et al, 2005, World J. Gastroenterol. 11: 5266-5272) on the constitutively activated FGFR3 in a large proportion of two common epithelial cancers, bladder and cervix, as well as in multiple myeloma and hepatocellular carcinoma, underlines the oncogenic role for FGFR3 in carcinomas.

Besides cancer, FGFR3 has been shown to be also involved in skeletal abnormalities, including achondroplasia and hypochondraplasia (WO02/102972).

Diseases involving deregulated FLT3 receptor tyrosine kinase activity include, but are not limited to, leukemias including acute myeloid leukemia (AML), AML with trilineage myelodysplasia (AML/TMDS), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS). This term also, specifically includes diseases resulting from FLT3 receptor mutation. Modulation of c-RET activity may also be useful in treating cancers of the nerve tissue, such as neuroblastoma, even if an abnormality is not found in the signaling pathway. Hereditary and spontaneous mutations that activate the RET kinase lead to several types of cancers, including multiple endocrine neoplasias type 2A and 2B (MEN2A and MEN2B), familial medullary thyroid carcinomas (FMTC), and papillary thyroid carcinomas (PTC). Subsets of mutations associate with each of these cancer types. Missense mutations in one of five cysteines of the RET extracellular domain are present in nearly all cases of MEN2A and FMTC, and presumably constitutively activate RET's tyrosine kinase activity by mimicking the effects of ligand binding to the extracellular domain. Most patients with MEN2B harbor mutations in codon 918 (Met-> Thr) in the ATP binding pocket of intracellular tyrosine kinase domain. This mutation presumably activates the kinase and alters its substrate specificity.

It is of interest that besides endocrine neoplasia, HSCR, and medullary thyroid carcinoma, breast cancer cell lines show an upregulation of RET (see FIGURE 5). The importance of this observation is supported by the upregulation of RET detected in infiltrating ductal breast cancer samples (see FIGURE 6).

The compounds can be used for the prevention or short-term or long-term treatment of the above mentioned diseases including, where appropriate, in combination with other state-of- the-art compounds such as other anti-tumour substances, cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids or antibodies.

The compounds of the general formula (I) can be used on their own or in combination with other active compounds according to the invention and, where appropriate, in combination with other pharmacologically active compounds as well. Chemotherapeutic agents which can be administered in combination with the compounds according to the invention include, without being restricted thereto, hormones, hormone analogs and antihormones (e. g. tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, aminoglutethimide, cyproterone acetate, finasteride, buserelin acetate, fludrocortisone, fluoxymesterone, medroxyprogesterone and octreotide), aromatase inhibitors (e. g. anastrozole, letrozole, liarozole, vorozole, exemestane and atamestane), LHRH agonists and antagonists (e. g. goserelin acetate and luprolide), inhibitors of growth factors (growth factors such as platelet-derived growth factor and hepatocyte growth factor, examples of inhibitors are growth factor antibodies, growth factor receptor antibodies and tyrosine kinase inhibitors, such as gefitinib, imatinib, lapatinib and trastuzumab); antimetabolites (e. g. antifolates such as methotrexate and raltitrexed, pyrimidine analogs such as 5-fluorouracil, capecitabine and gemcitabine, purine and adenosine analogs such as mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine and fludarabine); antitumor antibiotics (e. g. anthracyclines, such as doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin C, bleomycin, dactinomycin, plicamycin and streptozocin); platinum derivatives (e. g. cisplatin, oxaliplatin and carboplatin); alkylating agents (e. g. estramustine, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazine, cyclophosphamide, ifosfamide and temozolomide, nitrosoureas such as carmustine and lomustine and thiotepa); antimitotic agents (e. g. vinca alkaloids such as vinblastine, vindesine, vinorelbine and vincristine; and taxans such as paclitaxel and docetaxel); topoisomerase inhibitors (e. g. epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan and mitoxantrone) and various other chemotherapeutic agents such as amifostin, anagrelide, clodronate, filgrastin, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotan, pamidronate and porfimer.

The compounds may be administered by oral, transdermal or parenteral route or by inhalation. The compounds are present as active ingredients in conventional preparations, e. g. in compositions consisting essentially of an inert pharmaceutical carrier and an effective dose of the active substance, such as for example plain or coated tablets, capsules, lozenges, powders, solutions, suspensions, emulsions, syrups, suppositories, transdermal systems, etc. An effective dose of the compounds according to the invention is between 1 and 100, preferably between 1 and 50, most preferably between 5-30 mg/dose, for oral administration, and between 0.001 and 50, preferably between 0.1 and 10 mg/dose for intravenous or intramuscular administration. For inhalation, solutions containing 0.01 to 1, preferably 0.1 to 0.5 % of active substance are suitable according to the invention. For inhalation, the use of powders is preferred. It is also possible to use the compounds according to the invention as a solution for infusion, preferably in physiological saline or nutrient salt solution.

The compounds may be used on their own or in conjunction with other active substances, optionally also in conjunction with other pharmacologically active substances. Suitable preparations include for example tablets, capsules, suppositories, solutions, elixirs, emulsions or dispersible powders. Suitable tablets may be obtained, for example, by mixing the active substance(s) with known excipients, for example inert diluents such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatine, lubricants such as magnesium stearate or talc and/or agents for delaying release, such as carboxymethyl cellulose, cellulose acetate phthalate, or polyvinyl acetate. The tablets may also comprise several layers.

Coated tablets may be prepared accordingly by coating cores produced analogously to the tablets with substances normally used for tablet coatings, for example collidone or shellac, gum arabic, talc, titanium dioxide or sugar. To achieve delayed release or prevent incompatibilities the core may also consist of a number of layers. Similarly the tablet coating may consist of a number of layers to achieve delayed release, possibly using the excipients mentioned above for the tablets.

Syrups or elixirs containing the active substances or combinations thereof may additionally contain a sweetener such as saccharine, cyclamate, glycerol or sugar and a flavour enhancer, e. g. such as vanillin or orange extract. They may also contain suspension adjuvants or thickeners such as sodium carboxymethyl cellulose, wetting agents such as, for example, condensation products of fatty alcohols with ethylene oxide, or preservatives such as p-hydroxybenzoates.

Solutions for injection and infusion are prepared in the usual way, e. g. with the addition of preservatives such as p-hydroxybenzoates, or stabilisers such as alkali metal salts of ethylenediamine tetra-acetic acid, and transferred into injection vials or ampoules. Capsules containing one or more active substances or combinations of active substances may for example be prepared by mixing the active substances with inert carriers such as lactose or sorbitol and packing them into gelatine capsules.

Suitable suppositories may be made for example by mixing with carriers provided for this purpose, such as neutral fats or polyethyleneglycol or the derivatives thereof.

A therapeutically effective daily dose is between 1 and 800 mg, preferably 10 - 300 mg, in adults.

The following examples are presented in order to more fully illustrate the embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

Example 1

Inhibition of ABL and mutants thereof by 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2- indolinone.

3-Z-[ 1 -(4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 - phenyl-methylen]-6-methoxycarbonyl-2-indolinone exhibits not only an efficient inhibitory activity towards wtABL, but also towards ABL (T315I) mutant (see TABLE 1). The compounds are analyzed in conventional proliferation studies such as Alamar Blue assays or in apoptosis assays such as the Annexin-V-Fluos Staining kit (Boehringer Mannheim, Indianapolis, IN). In such assays, cell lines overexpressing wild type and mutant ABL as described (Shah et al, 2004, Science, 305: 399- 401; Weisberg et al, 2005, Cancer Cell 7: 129-141; O'Hare et al., 2005, Cancer Res. 65: 4500- 4505) are used. Also included are cell lines which are identified to overexpress ABL such as NCI-H520 (see FIGURE 1). Additional cell lines are selected from the list in FIGURE 3 (preferentially cell lines derived from leukemias such as JURKAT and K-562). Lysates of treated cell lines are subjected to Western Blot analysis for the inhibition of the autophosphorylation of BCR-ABL and BCR-ABL mutants antibodies useful in such assays have been described Weisberg et al., 2005, Cancer Cell 7: 129-141 (supplemental data) and O'Hare et al, 2005, Cancer Res. 65: 4500- 4505).

The tumor- inhibiting activity of the compounds of formula (I) can also be demonstrated in vivo. For the in vivo studies, a systemic 320 BCR-ABL leukemia model in mice and a bioluminescent BCR-ABL model of CML as described (Weisberg et al., 2005, Cancer Cell 7: 129-141; Armstrong et al., 2003, Cancer Cell, 3: 173-183) can be used.

The tumor-inhibiting activity of the compound is determined using female Balb/c nude or NMRI nude mice in which cell lines, such as of human adeno or squamous cell carcinoma origin, or human tumor tissue are transplanted. On day 0, a ca. 25 mg piece of solid tumor is transplanted subcutaneously under inhaled isofiuorane narcosis on the left flank and the small incision wound is closed with a suture clip. Alternatively human tumor cells grown in tissue culture can be injected subcutaneously into one flank of the animal at a concentration of 1-2 x 10 per ml in PBS containing 5% FCS. On day 6 after the tumor transplantation or when the tumor has reached a median size of 100-200 mm³, the mice are randomized in groups of 10 animals and treatment is commenced. The treatment is carded out for 15 days or until the control treated tumors have reached a median size of 1250 mm³ by administering a compound of formula (I), e. g. suspension in 0.5% Natrosol or dimethyl sulfoxide/Tween 80/sodium chloride solution in the different doses perorally or intraperitoneally once daily. The tumors are measured twice weekly with a sliding caliper and the tumor volume determined. In this assay, the peroral or intraperitoneal administration of a compound of formula (I) effects a marked reduction in the average tumor volume compared with the untreated controls.

Osteopontin (OPN) expression has been shown to be involved in the progression and increased aggression and invasiveness of many solid tumors. It recently has been demonstrated that OPN expression is also induced in CML. The specific signaling pathway required for the induction of OPN expression by p210 BCR-ABL is driven via the sequential activation of RAS, phosphatidylinositol-3 kinase, atypical protein kinase C, RAF-I, and mitogen-activated protein kinase kinase. The data strongly suggest that these molecules represent a single pathway and also that there is no redundancy in this pathway, as inhibition of any individual component results in a block in the induction of OPN (Hickey et al, 2005, J. Leukoc. Biol. 78: 289-300). Based on these data, Western Blot analyses on the phosphorylation status of the reported signaling molecules in the BCR- ABL driven cascade utilizing phospho-specific antisera such as for p-RAFl (phospho- Ser338, GeneTex) or for phospho-aPKC (Roche Molecular Biochemicals) are conducted.

In addition, as seen in FIGURE 2 ABL is also found overexpressed in Kidney WiIm 's tumor, soft tissue osteosarcoma, and glioblastoma multiforme. According to their expression profile these tumors may be regarded as indications for ABL targeting as well.

Example 2

Inhibition of FGFR3 -dependent tumors by 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2- indolinone.

As described in Example 1 , the compounds are further analyzed in conventional proliferation studies such as Alamar Blue assays or in apoptosis assays such as the Annexin-V-Fluos Staining kit (Boehringer Mannheim, Indianapolis, IN).

Specific germline activating point mutations in the gene encoding the tyrosine kinase receptor FGFR3 (fibroblast growth factor receptor 3) result in autosomal dominant human skeletal dysplasias. The identification in multiple myeloma and in two epithelial cancer' s- bladder and cervical carcinomas-of somatic FGFR3 mutations identical to the germinal activating mutations found in skeletal dysplasias, together with functional studies, have suggested an oncogenic role for this receptor. Activating point mutations in the FGFR3 gene occur most frequently in low-grade and low-stage bladder carcinomas, whereas they are rare in high-grade carcinomas (van Rhijn et al., 2002, J. Pathol. 198: 245-251). The response to inhibition by 3-Z-[l-(4-(Λ^(4-methyl-piperazin-l-yl)-methylcarbonyl)-iV- methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2-indolinone is determined in tumors and tumor cell lines carrying these activating FGFR3 mutations. It will be determined if tumors and tumor cell lines carrying these activating FGFR3 mutations will respond to inhibition by 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2- indolinone.

Based on the in vitro data on the pronounced inhibition of FGFR3 by 3-Z-[l-(4-(iV-((4- methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl-methylen]-6- methoxycarbonyl-2-indolinone (see TABLE 1) and on published data (Fiedler et al., 2003, Blood, 102: 2763-2767; Mesters et al., 2001, Blood 98: 241-243; O'Farrell et al., 2003, Clin. Cancer Res. 9: 5465-5476) suggesting that receptor tyrosine kinase inhibitors with different target specificities show clinical activity in AML, leading to complete and partial remissions in some patients, 3-Z-[l-(4-(Λ^(4-methyl-piperazin-l-yl)-methylcarbonyl)-iV- methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2-indolinone is tested in myeloid cell lines such as HL-60, Kasumi-1, K-562, and Mono-Mac- 1. 3-Z-[l-(4-(iV-((4- methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl-methylen]-6- methoxycarbonyl-2-indolinone is tested for the inhibition of colony formation in semisolid media as described (Topp et al., 1995, Cancer Res. 55: 2173-2176) in the presence and absence of VEGF, bFGF (ligand for FGFRl), and PDGF and analyzed in a flow cytometry apoptosis assay as described above. Furthermore, Western blotting is performed according to standard procedures using an enhanced chemiluminescence system (Amersham, Little Chalfont, United Kingdom) analyzing the inhibitory effect of 3-Z-[l-(4-(N-((4-methyl- piperazin-l-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylen]-6- methoxycarbonyl-2-indolinone on the down-stream signaling. Therefore, the following antibodies are used as described (Demiroglu et al., 2001, Blood 98: 3778-3783): 4G10 (phosphotyrosine; Upstate Biotechnology, Dundee, United Kingdom), FGFRl , CREB- 1 , ERK-I, phospho ERK (Santa Cruz Biotechnology, Santa Cruz, CA) and MEKl /2, AKT, phospho AKT, and phospho-CREB (New England Biolabs, Hitchin, United Kingdom). Example 3

Inhibition of receptor tyrosine kinase FLT3 and mutants thereof by 3-Z-[l-(4-(iV-((4- methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl-methylen]-6- methoxycarbonyl-2-indolinone. 3-Z-[ 1 -(4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 - phenyl-methylen]-6-methoxycarbonyl-2-indolinone has a strong inhibitory effect (see TABLE 1) on FLT3 (wt and mutation D835) when tested in vitro.

Based on SNP Chip analyses cell lines which exhibit amplification of the FLT3 gene locus (PC-3, SW 480, COLO 205, BT 549, and MDA-MB 453) are identified and used in the proliferation studies (TABLE 2). According to the inhibitory profile 3-Z-[l-(4-(iV-((4- methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl-methylen]-6- methoxycarbonyl-2-indolinone is a substance potently inhibiting the activity of FLT3 mutants such as FLT3(D835Y) in vitro (see TABLE 1). Further cell lines expressing either wtFLT3 such as RS4;11 (B cell precursor leukemia) or FLT3 ITD such as MV4;11 (AML; both alleles are mutated) or MOLM- 13 (AML) with one wt and one mutated allele (Lopes de Menezes et al, 2005, Clin. Cancer Res. 11: 5281-5291; Quentmeier et al, 2003, 17: 120-124) are also tested. Expression of FLT3 seems to be highly restricted to leukemia cell lines as shown in FIGURE 4; e.g. cell lines HL60 and THP-I show the most prominent present (informative) calls for the FLT3 transcript.

The efficacy of 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)-methylcarbonyl)-N-methyl- amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2-indolinone is monitored on Western Blots with phospho-specific antisera towards p-FLT3 (inhibition of auto- phosphorylation). As STAT-5 phosphorylation is also part of the FLT3 signaling chain and STAT-5 molecules are shown to be constitutive Iy phosphorylated in FLT3 ITD-positive cells (Lopes de Menezes et al., 2005, Clin. Cancer Res. 11: 5281-5291; Yao et al., Leukemia, 2005, 19:1605-1612). 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2- indolinone is further tested on the decrease of the phospho-specific STAT-5 signal in treated cell lines as well.

Furthermore, based on the findings that FLT3-ligand and FLT3 facilitate migration/homing and mobilization of normal and transformed hematopoietic cells by regulating the CXCL12/CXCR4 signaling pathways, 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2- indolinone is tested in an in vitro migration assay as described (Fukuda et al., 2005, Blood, 105: 3117-3126).

In order to analyze whether wtFLT3 alone or an amplification of wtFLT3 or an (activating) mutation is the driving "oncogenic" force, the inhibitory effect of 3-Z-[l-(4-(iV-((4-methyl- piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl-methylen]-6- methoxycarbonyl-2-indolinone is tested in cell lines expressing either wtFLT3 such as in RS4;11 (B cell precursor leukemia) or a mutated version of FLT3 such as FLT3 ITD in MV4;11 (AML; both alleles are mutated) or MOLM- 13 (AML) with one wt and one mutated allele (Lopes de Menezes et al., 2005, Clin. Cancer Res. 11: 5281-5291; Quentmeier et al., 2003, 17: 120-124).

For this purpose selected cell lines (see TABLE 3) are taken into culture following the protocol listed in TABLE 3 and analyzed in an Alamar Blue proliferation assay. As a control doxorubicin is used in parallel. In most cases two independent assay runs are performed.

Cell lines are treated as summarized in TABLE 3. Appropriate stock solutions of 3-Z-[I- (4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl- methylen]-6-methoxycarbonyl-2-indolinone or doxorubicin, respectively are prepared in DMSO. Further dilutions are performed in IxPBS + 5% DMSO (highest concentration used: 10 μM). Cells are seeded into 96-well plates and incubated for 3 days in the presence of various concentrations of 3 -Z- [ 1 -(4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-iV- methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2-indolinone or doxorubicin, respectively. After this period 20 μl of Alamar Blue dye (Biosource DALlOO) are added to each well (96-well plate) and exposed for the indicated time (TABLE 3). The 96-well plate is shaken carefully after each hour. Fluorescence is measured at Ex: 544 nm / Em: 590 nm.

In TABLE 3 the IC50 values are listed together with a short description of the cell lines according to their origin and FLT3 status. The data clearly indicate that only those cell lines harboring a mutated FLT3 (FLT3-ITD) are sensitive towards 3-Z-[l-(4-(iV-((4- methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl-methylen]-6- methoxycarbonyl-2-indolinone. Interestingly, amplification of the FLT3-locus alone as shown for cell line BT 549 and COLO 205 do not lead to sensitive tumors cells. Altogether, from these data it can be concluded that wtFLT3 or amplification of FLT3 alone is not sufficient for the essential contribution ("oncogenic" driving force) to tumorigenesis.

In summary, 3-Z-[ 1 -(4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)- anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2-indolinone is an efficient compound in targeting tumors expressing mutated FLT3.

Example 4

Inhibition of receptor tyrosine kinase RET by 3-Z-[l-(4-(N-((4-methyl-piperazin-l-yl)- methylcarbonyl)-N-methyl-amino)-anilino)-l-phenyl-methylen]-6-methoxycarbonyl-2- indolinone.

3-Z-[ 1 -(4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 - phenyl-methylen]-6-methoxycarbonyl-2-indolinone has a strong inhibitory effect (see TABLE 1) on RET when tested in vitro. The compound is tested in proliferation and apoptosis assays (see Examples 1 and 2) on cell lines overexpressing RET, such as BT- 549, COLO 205, HL-60, MCF7, MDA-MB-231, NCI-H522, SR, and THP-I (see FIGURE 5). Further cell lines with mutated RET are identified by exon sequencing for correlation with the mutational status. Besides endocrine neoplasia, HSCR, and medullary thyroid carcinoma, breast cancer cell lines show an upregulation of RET (see FIGURE 5). Furthermore, two thyroid carcinoma cell lines, HTB- 107 (thyroid squamous cell carcinoma) and TT (thyroid medulla carcinoma) are also included. The TT cell line was used in a xenograft model and shown to be sensitive for Irinotecan treatment resulting in complete remission in 100% of xenografts treated. The duration of remission is further enhanced by combination with the kinase inhibitor, CEP-751 (Strock et al., 2005, J. Clin. Endocrinol. Metab; doi:10.1210/jc.2005-1882). Therefore, the cell line TT is used as an excellent xenograft model to test the efficacy of 3-Z-[l-(4-(iV-((4-methyl-piperazin-l-yl)- methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl-methylen]-6-methoxycarbonyl-2- indolinone in a thyroid medulla carcinoma preclinical model.

The Examples that follow illustrate pharmaceutical formulations of the indolinones for use according to the present invention.

A) Tablets per tablet active substance 100 mg lactose 140 mg corn starch 240 mg polyvinylpyrrolidone 15 mg magnesium stearate 5 mg

500 mg

The finely ground active substance, lactose and some of the corn starch are mixed together. The mixture is screened, then moistened with a solution of polyvinylpyrrolidone in water, kneaded, wet-granulated and dried. The granules, the remaining corn starch and the magnesium stearate are screened and mixed together. The mixture is compressed to produce tablets of suitable shape and size.

B) Tablets per tablet active substance 80 mg lactose 55 mg corn starch 190 mg microcrystalline cellulose 35 mg polyvinylpyrrolidone 15 mg sodium-carboxymethyl starch 23 mg magnesium stearate 2 mg

400 mg

The finely ground active substance, some of the corn starch, lactose, microcrystalline cellulose and polyvinylpyrrolidone are mixed together, the mixture is screened and worked with the remaining corn starch and water to form a granulate which is dried and screened. The sodiumcarboxymethyl starch and the magnesium stearate are added and mixed in and the mixture is compressed to form tablets of a suitable size.

C) Coated tablets per coated tablet Active substance 5 mg

Corn starch 41.5 mg

Lactose 30 mg Polyvinylpyrrolidone 3 mg

Magnesium stearate 0.5 mg 80 mg

The active substance, corn starch, lactose and polyvinylpyrrolidone are thoroughly mixed and moistened with water. The moist mass is pushed through a screen with a 1 mm mesh size, dried at about 45 ⁰C and the granules are then passed through the same screen. After the magnesium stearate has been mixed in, convex tablet cores with a diameter of 6 mm are compressed in a tablet-making machine. The tablet cores thus produced are coated in known manner with a covering consisting essentially of sugar and talc. The finished coated tablets are polished with wax. D) Capsules per capsule Active substance 50.0 mg Corn starch 268.5 mg Magnesium stearate 1.5 mg 320.0 mg

The substance and corn starch are mixed and moistened with water. The moist mass is screened and dried. The dry granules are screened and mixed with magnesium stearate. The finished mixture is packed into size 1 hard gelatine capsules.

E) Ampoule solution active substance 50 mg sodium chloride 50 mg water for inj. 5 ml

The active substance is dissolved in water at its own pH or optionally at pH 5.5 to 6.5 and sodium chloride is added to make it isotonic. The solution obtained is filtered free from pyrogens and the filtrate is transferred under aseptic conditions into ampoules which are then sterilised and sealed by fusion. The ampoules contain 5 mg, 25 mg and 50 mg of active substance.

F) Suppositories

Active substance 50 mg

Solid fat 1650 mg 1700 mg

The hard fat is melted. At 40 ⁰C the ground active substance is homogeneously dispersed. It is cooled to 38 ⁰C and poured into slightly chilled suppository moulds.

Claims

Claims:

1. Use of a compound of formula (I)

wherein

R¹ is hydrogen, amino, methyl or ethyl;

R^c is independently from each other hydrogen, methyl, ethyl, -C(O)R^a, -(CH₂)_pNR^aR^a, -C(O)(CH₂)_pNR^aR^a, -(CH₂)_pC(O)NR^aR^a or S(O)₂R^a, R^a is hydrogen, methyl or ethyl and p is 0, 1 or 2 or its tautomers, enantiomers, diastereomers, mixtures and salts thereof for the manufacture of a medicament for the treatment or prevention or amelioration of diseases or disorders associated with at least one tyrosine kinase selected from the group comprising ABL, FGFR3, FLT3, and RET.

2. Use of a compound according to claim 1, wherein the compound is selected from the group consisting of (a) 3-Z-[ 1 -(4-(piperidin- 1 -yl-methyl)-anilino)- 1 -phenyl-methylene] -6- ethoxycarbonyl-2-indolinone,

(b) 3-Z-[( 1 -(4-(piperidin- 1 -yl-methyl)-anilino)- 1 -phenyl-methylene]-6-carbamoyl- 2-indolinone, (c) 3-Z-[ 1 -(4-(piperidin- 1 -yl-methyl)-anilino)- 1 -phenyl-methylene] -6- methoxycarbonyl-2-indolinone,

(d) 3-Z-[ 1 -(4-(dimethylaminomethyl)-anilino)- 1 -phenyl-methylene] -6- ethoxycarbonyl-2-indolinone, (e) 3-Z-[l-(4-((2,6-dimethyl-piperidin-l-yl)-methyl)-anilino)-l-phenyl-methylene]-

6-ethoxycarbonyl-2-indolinone,

(f) 3-Z-[I -(4-(N-(2-dimethylamino-ethyl)-N-acetyl-amino)-anilino)- 1 -phenyl- methylene]-6-ethoxycarbonyl-2-indolinone,

(h) 3-Z-[l-(4-(N-(2-dimethylamino-ethyl)-N-methylsulphonyl-amino)-anilino)-l- phenyl-methylene]-6-ethoxycarbonyl-2-indolinone,

(i) 3-Z-[l-(4-(dimethylammomethyl)-anilmo)-l-phenyl-methylene]-6- methoxycarbonyl-2-indolinone, (j) 3-Z-[I -(4-(N-acetyl-N-dimethylaminocarbonylmethyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone,

(k) 3 -Z-[ 1 -(4-ethylaminomethyl-anilino)- 1 -phenyl-methylene] -6-methoxycarbonyl-

2-indolinone,

(1) 3-Z-[I -(4-(I -methyl-imidazol-2-yl)-anilino)- 1 -phenyl-methylene] -6- methoxycarbonyl-2-indolinone,

(m) 3 -Z- [ 1 -(4-(N-dimethylaminomethylcarbonyl-N-methyl-amino)-anilino)- 1 - phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(n) 3-Z-[l-(4-(N-(2-dimethylamino-ethyl)-N-methylsulphonyl-amino)-anilino)-l- phenyl-methylene]-6-methoxycarbonyl-2-indolinone, (o) 3-Z-[ 1 -(4-(N-(3-dimethylamino-propyl)-N-methylsulphonyl-amino)-anilino)- 1 - phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(p) 3-Z-[ 1 -(4-(N-dimethylaminocarbonylmethyl-N-methylsulphonyl-amino)- anilino)- 1 -phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(q) 3-Z-[l-(4-(N-((2-dimethylamino-ethyl)-carbonyl)-N-methyl-amino)-anilino)-l- phenyl-methylene] -6-methoxycarbonyl-2-indolinone,

(r) 3 -Z- [ 1 -(4-(N-(2-dimethylamino-ethyl)-N-acetyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone, (s) 3 -Z- [l-(4-methylaminomethyl-anilino)-l -phenyl-methylene] -6- methoxycarbonyl-2-indolinone and

(t) 3 -Z- [ 1 -(4-(iV-((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-iV-methyl-amino)- anilino)- 1 -phenyl-methylene]-6-methoxycarbonyl-2-indolinone, or a tautomer or salt thereof.

3. Use of a compound according to claim 1, wherein the compound is 3-Z-[I -(4-(N- ((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone or a salt thereof.

4. Use of a compound according to claim 3, wherein the compound is 3-Z-[I -(4-(N- ((4-methyl-piperazin- 1 -yl)-methylcarbonyl)-Λf-methyl-amino)-anilino)- 1 -phenyl- methylene]-6-methoxycarbonyl-2-indolinone monoethanesulfonate.

5. Use of a compound according to claim 1 - 4, wherein the tyrosine kinase is ABL.

6. Use of a compound according to claim 1 - 4, wherein the tyrosine kinase is FGFR3.

7. Use of a compound according to claim 1 - 4, wherein the tyrosine kinase is FLT3.

8. Use of a compound according to claim 1 - 4, wherein the tyrosine kinase is RET.