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WO2009014941A1 - 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinone derivatives as multi-target protein kinase inhibitors and histone deacetylase inhibitors - Google Patents

3-(4-amidopyrrol-2-ylmethlidene)-2-indolinone derivatives as multi-target protein kinase inhibitors and histone deacetylase inhibitors Download PDF

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WO2009014941A1
WO2009014941A1 PCT/US2008/070075 US2008070075W WO2009014941A1 WO 2009014941 A1 WO2009014941 A1 WO 2009014941A1 US 2008070075 W US2008070075 W US 2008070075W WO 2009014941 A1 WO2009014941 A1 WO 2009014941A1
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pyrrole
mmol
amino
indol
oxo
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PCT/US2008/070075
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French (fr)
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Xian-Ping Lu
Zhibin Li
Zhuqiang Ning
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Shenzen Chipscreen Bioscience, Ltd.
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Publication of WO2009014941A1 publication Critical patent/WO2009014941A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

  • the present invention relates to certain 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinone derivatives which are capable of inhibiting protein kinases and histone deacetylases.
  • the compounds of this invention are therefore useful in treating diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities.
  • Pharmaceutical compositions comprising these compounds, methods of treating diseases utilizing pharmaceutical compositions comprising these compounds, and methods of preparing these compounds are also disclosed.
  • Protein kinases are a family of enzymes that catalyze the phosphorylation of proteins, in particular the hydroxy group of specific tyrosine, serine and threonine residues in proteins. Protein kinases play a critical role in the regulation of a wide variety of cellular processes, including metabolism, cell proliferation, cell differentiation, cell survival, environment-host reaction, immune response, and angiogenesis. Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include inflammatory diseases, autoimmune diseases, cancer, neurological and neurodegenerative diseases, cardiovascular diseases, allergies and asthma or hormone-related disease (Tan, S-L.,2006, J. Immunol., 176: 2872-2879; Healy, A.
  • the protein kinases can be conventionally divided into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases (STKs).
  • PTKs protein tyrosine kinases
  • STKs serine-threonine kinases
  • the protein tyrosine kinases are divided into two classes: the non-transmembrane tyrosine kinases and transmembrane growth factor receptor tyrosine kinases (RTKs).
  • RTKs transmembrane growth factor receptor tyrosine kinases
  • EGFR epidermal growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • PDGFR platelet derived growth factor receptor growth factor receptor
  • FGFR fibroblast growth factor receptor
  • the epidermal growth factor receptor (EGFR) family comprises four transmembrane tyrosine kinase growth factor receptors: HER1 , HER2, HER3 and HER4. Binding of a specific set of ligands to the receptor promotes EGFR dimerization and results in the receptors autophosphorylation on tyrosine residues (Arteaga, C-L.,2001, Curr. Opin. Oncol., 6: 491-498). Upon autophosphorylation of the receptor several signal transduction pathways downstream of EGFR become activated.
  • the EGFR signal transduction pathways have been implicated in the regulation of various neoplastic processes, including cell cycle progression, inhibition of apoptosis, tumor cell motility, invasion and metastasis.
  • EGFR activation also stimulates vascular endothelial growth factor (VEGF), which is the primary inducer of angiogenesis (Petit, A-M. et al.,1997, Am. J. Pathol., 151 : 1523-1530).
  • VEGF vascular endothelial growth factor
  • deregulation of the EGFR-mediated signal transduction pathways is associated with oncogenesis (Wikstrand, C-J. et al.,1998, J Natl Cancer Inst, 90: 799-800).
  • Mutations leading to continuous activation of amplification and over expression of EGFR proteins are seen in many human tumors, including tumors of breast, lung, ovaries and kidney. These mutations are a determinant of tumor aggressiveness (Wikstrand, C-J.
  • EGFR over expression is frequently seen in non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • Activity of EGFR can be inhibited either by blocking the extracellular ligand binding domain with the use of anti-EGFR antibodies or by the use of small molecules that inhibit the EGFR tyrosine kinase, thus resulting in inhibition of downstream components of the EGFR pathway (Mendelsohn, J., 1997, Clin. Can. Res., 3: 2707-2707).
  • VEGF vascular endothelial growth factor
  • VEGF-A The binding of VEGF-A to VEGFR-1 induces endothelial cell migration. Binging to VEGFR-2 induces endothelial cell proliferation, permeability and survival. VEGFR-3 is thought to mediate lymphangiogenesis.
  • the binding of VEGF to VEGFR-2 receptors results in activation and autophosphorylation of intracellular tyrosine kinase domains which further triggers other intracellular signaling cascades (Parikh, A-A., 2004, Hematol. Oncol. CHn. N. Am., 18:951-971).
  • STKs serine-threonine kinases
  • STKs are predominantly intracellular although there are a few receptor kinases of the STK type.
  • STKs are the most common forms of the cytosolic kinases that perform their function in the part of the cytoplasm other than the cytoplasmic organelles and cytoskelton.
  • Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase comprised of ⁇ and ⁇ isoforms that are each encoded by distinct genes. GSK-3 has been found to phosphorylate and modulate the activity of a number of regulatory proteins. GSK-3 has been implicated in various diseases including diabetes, Alzheimer's disease, CNS disorders such as manic depressive disorder and neurodegenerative diseases, and cardiomyocete hypertrophy (Haq, et al., 2000, J. Cell Biol., 151: 117). Aurora-2 is a serine-threonine protein kinase that has been implicated in human cancer, such as colon, breast, and other solid tumors.
  • Aurora-2 may play a role in controlling the accurate segregation of chromosomes during mitosis. Misregulation of the cell cycle can lead to cellular proliferation and other abnormalities.
  • the Aurora-2 protein has been found to be over expressed (Schumacher, et a!., 1998, J. Cell Biol., 143: 1635-1646; Kimura et al., 1997, J. Biol. Chem., 272: 13766-13771).
  • CDKs The cyclin-dependent kinases
  • CDKs are serine-threonine protein kinase that regulates mammalian cell division. CDKs play a key role in regulating cell machinery. To date, nine kinase subunits (CDK 1-9) have been identified. Each kinase associates with a specific regulatory partner and together makes up the active catalytic moiety. Uncontrolled proliferation is a hallmark of cancer cells, and misregulation of CDK function occurs with high frequency in many important solid tumors. CDK2 and CDK4 are of particular interest because their activities are frequently misregulated in a wide variety of human cancers.
  • Raf kinase a downstream effector of ras oncoprotein, is a key mediator of signal-transduction pathways from cell surface to the cell nucleus. Inhibition of raf kinase has been correlated in vitro and in vivo with inhibition of the growth of variety of human tumor types (Monia et al., 1996, Nat. Med., 2: 668-675).
  • serine-threonine protein kinases include the protein kinase A, B and C. These kinases, known as PKA 1 PKB and PKC, play key roles in signal transduction pathways.
  • cyclic compounds U.S. Pat. No. 7,151 ,096
  • bicyclic compounds U.S. Pat. No. 7,189,721
  • tricyclic compounds U.S. Pat. No. 7,132,533
  • (2-oxindol-3-ylidenyl) acetic acid derivatives U.S. Pat. No. 7,214,700
  • 3-(4-amidopyrroi-2-ylmethlidene)-2-indolinone derivatives U.S. Pat. No. 7,179,910
  • fused pyrazole derivatives U.S. Pat. No.
  • the present invention is directed to certain 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinone derivatives which are capable of selectively inhibiting protein kinases and histone deacetylases and are therefore useful in treating diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities.
  • they are highly effective against hematological malignancy and solid carcinoma.
  • Histone deacetylase (HDAC) proteins play a critical role in regulating gene expression in vivo by altering the accessibility of genomic DNA to transcription factors. Specifically, HDAC proteins remove the acetyl group of acetyl-lysine residues on histones, which can result in nucleosomal remodelling (Grunstein, M., 1997, Nature, 389: 349-352). Due to their governing role in gene expression, HDAC proteins are associated with a variety of cellular events, including cell cycle regulation, cell proliferation, differentiation, reprogramming of gene expression, and cancer development (Ruijter, A-J-M., 2003, Biochem.
  • HDAC inhibitors have been demonstrated to reduce tumor growth in various human tissues and in animal studies, including lung, stomach, breast, and prostrate (Dokmanovic, M. ,2005, J. Cell Biochenm., 96: 293-304).
  • Mammalian HDACs can be divided into three classes according to sequence homology.
  • Class I consists of the yeast Rpd3-like proteins (HDAC 1, 2, 3, 8 and 11).
  • Class Il consists of the yeast HDA1-like proteins (HDAC 4, 5, 6, 7, 9 and 10).
  • Class III consists of the yeast SIR2-like proteins (SIRT 1, 2, 3, 4, 5, 6 and 7).
  • HDAC1 has been linked to cell proliferation, a hallmark of cancer.
  • mammalian cells with knock down of HDAC1 expression using siRNA were antiproliferative (Glaser, K-B., 2003, Biochem. Biophys. Res. Comm., 310: 529-536). While the knock out mouse of HDAC1 was embryonic lethal, the resulting stem cells displayed altered cell growth (Lagger, G, 2002, EMBO J., 21 : 2672-2681). Mouse cells overexpressing HDAC1 demonstrated a lengthening of G 2 and M phases and reduced growth rate (Bartl. S., 1997, MoI. Cell Biol., 17: 5033-5043). Therefore, the reported data implicate HDAC1 in cell cycle regulation and cell proliferation.
  • HDAC2 regulates expression of many fetal cardiac isoforms. HDAC2 deficiency or chemical inhibition of histone deacetylase prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inpp ⁇ f) resulting in constitutive activation of glycogen synthase kinase 3 ⁇ (Gsk3 ⁇ ) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1).
  • Inpp ⁇ f inositol polyphosphate-5-phosphatase f
  • Gsk3 ⁇ glycogen synthase kinase 3 ⁇
  • Akt thymoma viral proto-oncogene
  • Pdk1 3-phosphoinositide-dependent protein kina
  • HDAC2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3 ⁇ .
  • Chemical inhibition of activated Gsk3 ⁇ allowed HDAC2-def ⁇ cient adults to become sensitive to hypertrophic stimulation.
  • HDAC3 are maximally expressed in proliferating crypt cells in normal intestine. Silencing of HDAC3 expression in colon cancer cell lines resulted in growth inhibition, a decrease in cell survival, and increased apoptosis. Similar effects were observed for HDAC2 and, to a lesser extent, for HDAC1. HDAC3 gene silencing also selectively induced expression of alkaline phosphatase, a marker of colon cell maturation. Concurrent with its effect on cell growth, overexpression of HDAC3 inhibited basal and butyrate-induced p21 transcription in a Sp1/Sp3-dependent manner, whereas silencing of HDAC3 stimulated p21 promoter activity and expression. These findings identify HDAC3 as a gene deregulated in human colon cancer and as a novel regulator of colon cell maturation and p21 expression (Wilson, A-J., 2006, J. Biol. Chem., 281: 13548-13558).
  • HDAC6 is a subtype of the HDAC family that deacetylates alpha-tubulin and increases cell motility.
  • OSCC oral squamous cell carcinoma
  • NOKs normal oral keratinocytes
  • HDAC6 mRNA and protein expression were commonly up-regulated in all cell lines compared with the NOKs.
  • Immunofluorescence analysis detected HDAC6 protein in the cytoplasm of OSCC cell lines. Similar to OSCC cell lines, high frequencies of HDAC6 up-regulation were evident in both mRNA (74%) and protein (51%) levels of primary human OSCC tumors.
  • the clinical tumor stage was found to be associated with the HDAC6 expression states.
  • the analysis indicated a significant difference in the HDAC6 expression level between the early stage (stage I and II) and advanced-stage (stage III and IV) tumors (P O.014).
  • HDAC epigenetic silencing of functional chromosomes by HDAC is one of major mechanisms occurred in many pathological processes, in which functionally critical genes are repressed or reprogrammed by HDAC activities leading to the loss of phenotypes in terminal differentiation, maturation and growth control, and the loss of functionality of tissues.
  • tumor suppressor genes are often silenced during development of cancer and chemical inhibitor of HDAC can derepressed the expression of these tumor suppressor genes, leading to growth arrest and differentiation (Glaros S et al., 2007, Oncogene June 4 Epub ahead of print; Mai, A, et al., 2007, lnt J. Biochem Cell Bio., April 4, Epub ahead of print; Vincent A.
  • HDAC inhibitors include (1) short-chain fatty acids, e.g. butyrate and phenylbutyrate; (2) organic hydroxamic acids, e.g. suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA); (3) cyclic tetrapeptides containing a 2-amino-8-oxo 9,10-expoxydecanoyl (AOE) moiety, e.g. trapoxin and HC-toxin; (4) cyclic peptides without the AOE moiety, e.g. apicidin and FK228; and (5) benzamides, e.g.
  • SAHA suberoylanilide hydroxamic acid
  • TSA trichostatin A
  • AOE 2-amino-8-oxo 9,10-expoxydecanoyl
  • benzamides e.g.
  • Sorafenib developed by Bayer Pharmaceuticals, is the first drug targeting both the RAF/MEK/ERK pathway (involved in cell proliferation) and the VEGFR2/PDGFR ⁇ signaling cascade (involved in angiogenesis).
  • This drug was first approved in December 2005 for advanced kidney cancer, a disease that is believed to be highly dependent on angiogenesis.
  • these target therapies although are effective against some solid tumors, but far from satisfaction in terms of reaching a better efficacy as a single agent against other solid tumors while tolerable sides associated with treatment can be maintained.
  • PROVIDED HEREIN are new chemical compounds that combine anti-angiogenesis and anti-proliferation activities of RTK's together with differentiation-inducing, immune modulation, cell cycle arrest and apoptosis activities of more selective HDACi, to reach a better efficacy against solid tumors while overcoming side effects such as hypertension, QT prolongation, thyroid gland regression, skin rash and discoloration, and pains associated with currently marketed RTK inhibitors.
  • the present invention provides a compound having the structure represented by formula (I), or its stereoisomer, enantiomer, diastereomer, hydrate, or pharmaceutically acceptable salts thereof:
  • X is a valence bond or -C(O)-N H-(CH 2 ) m -;
  • R 1 , R 2 , R 3 and R 4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
  • R 5 is -NHOH or
  • R 6 , R 7 , R 8 and R 9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; n is an integer ranging from 1 to 5; m is an integer ranging from 1 to 5.
  • halo as used herein means fluorine, chlorine, bromine or iodine.
  • alkyl as used herein includes methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl and the like.
  • alkoxy as used herein includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and the like.
  • the condensation reaction (a) and (d) are conducted by using a peptide condensing agent such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbo- diimide (DCC), N,N'-carbonyldiimidazole (CDI), etc.
  • EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • DCC dicyclohexylcarbo- diimide
  • CDI N,N'-carbonyldiimidazole
  • the reaction may be conducted at O to 80 0 C for 4 to 72 hours.
  • Solvents which may be used are normal solvents such as benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, N, N-dimethylformamide, etc.
  • a base such as sodium hydroxide, tri
  • the condensation reaction (c) is conducted by using CICOOEt as a condensing agent.
  • the reaction may be conducted at 0 to 80 0 C for 1 to 24 hours.
  • Solvents which may be used are normal solvents such as benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, N, N-dimethylformamide, etc. If necessary, a base such as sodium hydroxide, triethylamine and pyridine may be added to the reaction system.
  • the hydrolysis reaction (b) is conducted by using a hydrolysis agent such as lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.
  • a hydrolysis agent such as lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.
  • the reaction may be conducted at 0 to 80 0 C for 2 to 72 hours.
  • Solvents which may be used are normal solvents such as water, methanol, ethanol, tetrahydrofuran, dioxane, N, N-dimethylformamide, etc.
  • the compounds represented by formula (I) and the intermediate (3) and (4) may be purified or isolated by the conventional separation method such as extraction, recrystallization, column chromatography and the like.
  • This invention also provides a compound having the structure represented by formula (II), or its stereoisomer, enantiomer, diastereomer, hydrate, or pharmaceutically acceptable salts thereof:
  • Y is a moiety having -CH 2 -, -CO-, -CS-, -SO- or -SO 2 -, which is linear with a length of 4.0 to 12.0 A and links A and B;
  • R 1 , R 2 , R 3 and R 4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
  • R 6 , R 7 , R 8 and R 9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl;
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are independently hydrogen or alkyl.
  • the compounds represented by formula (I) or formula (II) are capable of inhibiting protein kinases and histone deacetylases and are therefore useful in treating diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities. In particular, they are highly effective against hematological malignancy and solid carcinoma.
  • the compounds represented by formula (I) or formula (II) useful as a drug may be used in the form of a general pharmaceutical composition.
  • the pharmaceutical composition may be in the forms normally employed, such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, may contain flavourants, sweeteners etc. in suitable solids or liquid carriers or diluents, or in suitable sterile media to form injectable solutions or suspensions.
  • Such composition typically contains from 0.5 to 70%, preferably 1 to 20% by weight of active compound, the remainder of the composition being pharmaceutically acceptable carriers, diluents or solvents or salt solutions.
  • the compounds represented by formula (I) or formula (II) are clinically administered to mammals, including man and animals, via oral, nasal, transdermal, pulmonary, or parenteral routes. Administration by the oral route is preferred, being more convenient and avoiding the possible pain and irritation of injection. By either route, the dosage is in the range of about 0.0001 to 200 mg/kg body weight per day administered singly or as a divided dose. However, the optimal dosage for the individual subject being treated will be determined by the person responsible for treatment, generally smaller dose being administered initially and thereafter increments made to determine the most suitable dosage.
  • Example 1 Representative compounds of the present invention are shown in Table 1 below.
  • the compound numbers correspond to the "Example numbers” in the Examples section. That is, the synthesis of compound 1 as shown in the Table 1 is described in “Example 1” and the synthesis of compound 52 as shown in the Table 1 is described in “Example 52".
  • the compounds presented in the Table 1 are exemplary only and are not to be construed as limiting the scope of this invention in any manner.
  • This assay is used to measure in vitro kinase activity of PDGFR in an ELISA assay.
  • Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) (Cell Signaling)
  • Wash Buffer 1 X PBS, 0.05% Tween-20 (PBS/T)
  • BSA Bovine Serum Albumin
  • Stop Buffer 50 mM EDTA, pH 8.
  • ECL Enhanced chemiluminescence
  • P-Tyr-100 Phospho-Tyrosine Monoclonal Antibody
  • This assay is used to measure in vitro kinase activity of VEGFR2 in an ELISA assay.
  • Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) (Cell Signaling)
  • VEGF Receptor 2 Kinase (recombinant, human) (Cell Signaling)
  • Wash Buffer 1 X PBS, 0.05% Tween-20 (PBS/T)
  • BSA Bovine Serum Albumin
  • ECL Enhanced chemiluminescence
  • P-Tyr-100 Phospho-Tyrosine Monoclonal Antibody
  • This assay is used to measure in vitro kinase activity of c-KIT in an ELISA assay.
  • Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) (Cell Signaling)
  • wash Buffer 1 X PBS, 0.05% Tween-20 (PBSAT)
  • BSA Bovine Serum Albumin
  • ECL Enhanced chemiluminescence
  • P-Tyr-100 Phospho-Tyrosine Monoclonal Antibody
  • NIH-3T3 mouse fibroblasts cell line engineered to stably express human PDGFRC was constructed and used to evaluate PDGF dependent cell proliferation.
  • PDGFRDNIH-3T3 cells were plated into 96-well plates at 5,000 per well and incubated with serum-free medium for 24 hours.
  • Compounds and PDGF BB (50ng/ml) were added and incubated for 72 hours in serum-free medium.
  • the effects on proliferation were determined by addition of MTS reagent (Promega) according to the instruction, incubation for 2 hours at 37°C in CO 2 incubator, and record the absorbance at 490nm using an ELISA plate reader.
  • HUVEC cells were plated into 96-well plates at 6,000 per well and incubated with serum-free medium for 2 hours. Compounds and VEGF 165 (50ng/ml) were added and incubated for 72 hours in serum-free medium. The effects on proliferation were determined by addition of MTS reagent (Promega) according to the instruction, incubation for 2 hours at 37°C in CO 2 incubator, and record the absorbance at 490nm using an ELISA plate reader.
  • MTS reagent Promega
  • HHDDAACC (SSRREE ((GGDDFF1111 ( ⁇ -tubulin
  • HDAC Fluorimetric Assay/Drug Discovery Kit (BIOMOL) according to manufacture's instruction. 1. Add Assay buffer, diluted trichostatin A or test inhibitor to appropriate wells of the microtiter plate. Following table lists examples of various assay types and the additions required for each test.
  • HDAC subtype selectivity inhibition assay of tested compounds was carried out by several reporter gene assays experiments. Briefly, HeLa cells were seeded in 96-well plates the day before transfection to give a confluence of 50-80%. Cells were transfected with one of reporter gene plasmids containing a promoter sequences or response elements upstream of a luciferase gene construct using FuGene ⁇ transfection reagent according to the manufacturer's instructions (Roche). The promoter or response elements including p21-promoter, gdfH -promoter, serum response element (SRE), MEF-binding element were fused upstream to the luciferase gene reporter construct.
  • SRE serum response element
  • a GFP expression plasmid was cotransfected.
  • Cells were allowed to express protein for 24 hours followed by addition of individual compounds or the vehicle (DMSO). 24 hours later the cells were harvested, and the luciferase assays were performed using the luciferase assay kit according to the manufacturer's instructions (Promega).
  • ⁇ -galactosidase activity from transfected cells was measured using a kit (Promega) as instructed by the manufacturer.
  • This assay is used to measure in vivo inhibition of HDAC6 in a cytoblot assay.
  • Fixation solution 95% ethanol, 5% acetic acid
  • TBS 0.15M NaCI, 0.02M Tris-CI pH7.4
  • Procedure for performing the assay in 96-well plate 1. A549 cells were seeded at a density of 20000 cells/200 ⁇ l/well in 96-well white plate and incubated at 37°C for 24 hours.
  • This assay is used to measure the in vivo inhibition of total HDAC activity in a cytoblot assay.
  • Fixation solution 95% ethanol, 5% acetic acid
  • TBS 0.15M NaCI, 0.02M Tris-CI pH7.4
  • ADB TBS + 2%BSA + 0.1% Triton X-100
  • ECL Enhanced chemiluminescence
  • A549 cells were seeded at a density of 20000 cells/200 ⁇ l/well in 96-well white plate and incubated at 37°C for 24 hours.
  • Tumor cells were trypsinized and plated into 96-well plates at 3,000 per well and incubated in complete medium with 10% FBS for 24 hours. Compounds were added over a final concentration range of 100 ⁇ mol/L to 100 nmol/L in 0.1% DMSO and incubated for 72 hours in complete medium. The effects on proliferation were determined by addition of MTS reagent (Promega) according to the instruction, incubation for 2 hours at 37°C in CO 2 incubator, and record the absorbance at 490nm using an ELISA plate reader.
  • MTS reagent Promega
  • HeLa cells were cultured in each 10-cm-diameter tissue culture plate in DMEM medium supplemented with 10% calf serum. After cultured for 24 h, the cells were treated with CS055 and SAHA as a positive control and the same volume of DMSO as a negative control respectively for 24 h. The treated cells were harvested and histones of cultured cells were extracted according to standard procedure. Protein was quantitated using a protein assay kit (plusOne 2-D quant kit, Amersham Biosciences). About 10 ⁇ g of extracted cellular histones were separated by SDS-PAGE on a 12% separating gel and electrophoretically transferred to PVDF membrane.
  • the total histone H3 proteins were identified by using anti-histone H3 antibody (Upstate,1:1000) as primary antibody and the goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Upstate, 1:2000) as secondary antibody.
  • the bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak).
  • the acetylated histone H3 proteins were detected by using anti-acetyl-histone H3 antibody (Upstate, 1 :50000) as primary antibody and the goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (Upstate, 1 :2000) as secondary antibody.
  • the bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak).
  • HeLa cells were cultured in each well of 6-well plate in DMEM medium supplemented with 10% calf serum. After cultured for 24 h, the cells were treated with CS055 and SAHA as a positive control and the same volume of DMSO as a negative control respectively for 24 h. The treated cells were washed once with PBS and treated with 100 ⁇ l of M-PER mammalian protein extraction reagent (PIERCE). The lysate was collected and the protein was quantitated using a protein assay kit (plusOne 2-D quant kit, Amersham Biosciences). About 10 ⁇ g of the lysate was separated by SDS-PAGE on a 10% separating gel and electrophoretically transferred to PVDF membrane.
  • PIERCE M-PER mammalian protein extraction reagent
  • the total ⁇ -Tubulin proteins were identified by using anti- ⁇ -Tubulin antibody (Santa Cruz, 1:500) as primary antibody and the goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Upstate, 1 :2000) as secondary antibody.
  • the bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak).
  • the acetylated ⁇ -Tubulin were detected by using anti-acetyl- ⁇ -Tubulin antibody (Santa Cruz, 1:1000) as primary antibody and the goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Upstate, 1:2000) as secondary antibody.
  • the bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak).
  • HDAC Fluorimetric Assay/Drug Discovery Kit (BIOMOL) according to manufacture's instruction.
  • HCT-8 Human Colon Tumor Xenograft Experiment on Female NCr-nu/nu Mice Treated with Compound 13, 14 and 4. Effect of HDACi on HCT-8 tumor growth(Vt-V0)/V0*100
  • mice Female NCr-nu/nu mice were used for all studies. Tumors of HCT-8 model were established and maintained through in vivo passage of s.c. fragments (3x3 mm) implanted in the flank using a 12-gauge trocar. A new generation of the passage was initiated every three weeks, and studies were conducted between generations 3 and 12 of this line. Tumor bearing animal was randomized for grouping and treatment was initiated when tumor size exceeds 6 mm. The mice were divided into 4 groups and each contains 8. Treatment for all but the compound 13 was administered orally once daily at 80mg/kg body weight for the duration indicated in each experiment. The compound 13 was given by IP once daily at 40mg/kg body weight for the duration indicated in each experiment.
  • mice Female NCr-nu/nu mice were used for all studies Tumors of MCF-7 model were established and maintained through in vivo passage of s.c. fragments (3x3 mm) implanted in the flank using a 12-gauge trocar. A new generation of the passage was initiated every three weeks, and studies were conducted between generations 3 and 12 of this line. Tumor bearing animal was randomized for grouping and treatment was initiated when tumor size exceeds 6 mm. The mice were divided into 5 groups and each contains 8. The compound 14 was administered orally once daily at 120mg/kg (14-120) or 240mg/kg (14-240) body weight for the duration indicated in each experiment. The compound 13 was given by IP once daily at 30mg/kg (13-30) or 60mg/kg (13-60) body weight for the duration indicated in each experiment. Tumor weight was weighted at the day after the last treatment.

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Abstract

Isolated compounds of formula (I): and stereoisomers, enantiomers, diastereomers, and pharmaceutically acceptable salts thereof are described, as well as processes for production, and methods of use of these compounds and compositions thereof for the treatment of diseases associated with abnormal protein kinase activities and/or abnormal histone deacetylase activities including, for example, inflammatory diseases, autoimmune diseases, cancer, amelioration of abnormal cell proliferation, neurological and neurodegenerative diseases, cardiovascular diseases, allergies and asthma and/or hormone-related diseases.

Description

3-(4-AMIDOPYRROL-2-YLMETHLIDENE)-2-INDOLINONE DERIVATIVES AS MULTI-TARGET PROTEIN KINASE INHIBITORS AND HISTONE DEACETYLASE
INHIBITORS
Priority is derived from US 60/935,041 , filed July 24, 2007.
FIELD OF THE INVENTION
The present invention relates to certain 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinone derivatives which are capable of inhibiting protein kinases and histone deacetylases. The compounds of this invention are therefore useful in treating diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities. Pharmaceutical compositions comprising these compounds, methods of treating diseases utilizing pharmaceutical compositions comprising these compounds, and methods of preparing these compounds are also disclosed.
BACKGROUND OF THE INVENTION
Protein kinases are a family of enzymes that catalyze the phosphorylation of proteins, in particular the hydroxy group of specific tyrosine, serine and threonine residues in proteins. Protein kinases play a critical role in the regulation of a wide variety of cellular processes, including metabolism, cell proliferation, cell differentiation, cell survival, environment-host reaction, immune response, and angiogenesis. Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include inflammatory diseases, autoimmune diseases, cancer, neurological and neurodegenerative diseases, cardiovascular diseases, allergies and asthma or hormone-related disease (Tan, S-L.,2006, J. Immunol., 176: 2872-2879; Healy, A. ea al.,2006, J. Immunol., 177: 1886-1893; Salek-Ardakani, S. et al.,2005, J. Immunol., 175: 7635-7641; Kim, J. et al.,2004, J. Clin. Invest., 114: 823-827). Therefore, considerable effort has been made to identify protein kinase inhibitors that are effective as therapeutic agents against these diseases.
The protein kinases can be conventionally divided into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases (STKs).
The protein tyrosine kinases (PTKs) are divided into two classes: the non-transmembrane tyrosine kinases and transmembrane growth factor receptor tyrosine kinases (RTKs). At present, at least nineteen distinct subfamilies of RTKs have been identified, such as the epidermal growth factor receptor (EGFR), the vascular endothelial growth factor receptor (VEGFR), the platelet derived growth factor receptor growth factor receptor (PDGFR), and the fibroblast growth factor receptor (FGFR).
The epidermal growth factor receptor (EGFR) family comprises four transmembrane tyrosine kinase growth factor receptors: HER1 , HER2, HER3 and HER4. Binding of a specific set of ligands to the receptor promotes EGFR dimerization and results in the receptors autophosphorylation on tyrosine residues (Arteaga, C-L.,2001, Curr. Opin. Oncol., 6: 491-498). Upon autophosphorylation of the receptor several signal transduction pathways downstream of EGFR become activated. The EGFR signal transduction pathways have been implicated in the regulation of various neoplastic processes, including cell cycle progression, inhibition of apoptosis, tumor cell motility, invasion and metastasis. EGFR activation also stimulates vascular endothelial growth factor (VEGF), which is the primary inducer of angiogenesis (Petit, A-M. et al.,1997, Am. J. Pathol., 151 : 1523-1530). In experimental models, deregulation of the EGFR-mediated signal transduction pathways is associated with oncogenesis (Wikstrand, C-J. et al.,1998, J Natl Cancer Inst, 90: 799-800). Mutations leading to continuous activation of amplification and over expression of EGFR proteins are seen in many human tumors, including tumors of breast, lung, ovaries and kidney. These mutations are a determinant of tumor aggressiveness (Wikstrand, C-J. et al.,1998, J Natl Cancer Inst., 90: 799-800). EGFR over expression is frequently seen in non-small cell lung cancer (NSCLC). Activity of EGFR can be inhibited either by blocking the extracellular ligand binding domain with the use of anti-EGFR antibodies or by the use of small molecules that inhibit the EGFR tyrosine kinase, thus resulting in inhibition of downstream components of the EGFR pathway (Mendelsohn, J., 1997, Clin. Can. Res., 3: 2707-2707).
The vascular endothelial growth factor (VEGF) is secreted by almost all solid tumors and tumor associated stroma in response to hypoxia. It is highly specific for vascular endothelium and regulates both vascular proliferation and permeability. Excessive expression of VEGF levels correlate with increased microvascular density, cancer recurrence and decreased survival (Parikh, A-A., 2004;, Hematol. Oncol. Clin. N. Am., 18:951-971). There are 6 different ligands for the VEGF receptor, VEGF-A through -E and placenta growth factor. Ligands bind to specific receptors on endothelial cells, mostly VEGFR-2. The binding of VEGF-A to VEGFR-1 induces endothelial cell migration. Binging to VEGFR-2 induces endothelial cell proliferation, permeability and survival. VEGFR-3 is thought to mediate lymphangiogenesis. The binding of VEGF to VEGFR-2 receptors results in activation and autophosphorylation of intracellular tyrosine kinase domains which further triggers other intracellular signaling cascades (Parikh, A-A., 2004, Hematol. Oncol. CHn. N. Am., 18:951-971).
The serine-threonine kinases (STKs) are predominantly intracellular although there are a few receptor kinases of the STK type. STKs are the most common forms of the cytosolic kinases that perform their function in the part of the cytoplasm other than the cytoplasmic organelles and cytoskelton.
Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase comprised of α and β isoforms that are each encoded by distinct genes. GSK-3 has been found to phosphorylate and modulate the activity of a number of regulatory proteins. GSK-3 has been implicated in various diseases including diabetes, Alzheimer's disease, CNS disorders such as manic depressive disorder and neurodegenerative diseases, and cardiomyocete hypertrophy (Haq, et al., 2000, J. Cell Biol., 151: 117). Aurora-2 is a serine-threonine protein kinase that has been implicated in human cancer, such as colon, breast, and other solid tumors. This kinase is believed to be involved in protein phosphorylation events that regulate cell cycle. Specifically, Aurora-2 may play a role in controlling the accurate segregation of chromosomes during mitosis. Misregulation of the cell cycle can lead to cellular proliferation and other abnormalities. In human colon cancer tissue, the Aurora-2 protein has been found to be over expressed (Schumacher, et a!., 1998, J. Cell Biol., 143: 1635-1646; Kimura et al., 1997, J. Biol. Chem., 272: 13766-13771).
The cyclin-dependent kinases (CDKs) are serine-threonine protein kinase that regulates mammalian cell division. CDKs play a key role in regulating cell machinery. To date, nine kinase subunits (CDK 1-9) have been identified. Each kinase associates with a specific regulatory partner and together makes up the active catalytic moiety. Uncontrolled proliferation is a hallmark of cancer cells, and misregulation of CDK function occurs with high frequency in many important solid tumors. CDK2 and CDK4 are of particular interest because their activities are frequently misregulated in a wide variety of human cancers.
Raf kinase, a downstream effector of ras oncoprotein, is a key mediator of signal-transduction pathways from cell surface to the cell nucleus. Inhibition of raf kinase has been correlated in vitro and in vivo with inhibition of the growth of variety of human tumor types (Monia et al., 1996, Nat. Med., 2: 668-675).
Other serine-threonine protein kinases include the protein kinase A, B and C. These kinases, known as PKA1 PKB and PKC, play key roles in signal transduction pathways.
Many attempts have been made to identify small molecules which act as protein kinase inhibitors useful in the treatment of diseases associated with abnormal protein kinases activities. For example, cyclic compounds (U.S. Pat. No. 7,151 ,096), bicyclic compounds (U.S. Pat. No. 7,189,721), tricyclic compounds (U.S. Pat. No. 7,132,533), (2-oxindol-3-ylidenyl) acetic acid derivatives (U.S. Pat. No. 7,214,700), 3-(4-amidopyrroi-2-ylmethlidene)-2-indolinone derivatives (U.S. Pat. No. 7,179,910), fused pyrazole derivatives (U.S. Pat. No. 7,166,597), aminofurazan compounds (U.S. Pat. No. 7,157,476), pyrrole substituted 2-indolinone compounds (U.S. Pat. No. 7,125,905), triazole compounds (U.S. Pat. No. 7,115,739), pyrazolylamine substituted quinazoline compounds (U.S. Pat. No. 7,098,330) and indazole compounds (U.S. Pat. No. 7,041 ,687) have all been described as protein kinase inhibitors. Several protein kinase inhibitors such as Glivec, Suten, and Sorafenib have been successfully approved by FDA as anti-cancer therapy. Their clinic uses demonstrated clear advantages over existing chemotherapeutical treatments, fueling continuing interests in innovation of mechanism-based treatments and improvement of chemical scaffolds to discover new compounds with excellent oral bioavailability, significant anti-tumor activity, and lower toxicity at well-tolerated dose. SUMMARY OF THE INVENTION
The present invention is directed to certain 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinone derivatives which are capable of selectively inhibiting protein kinases and histone deacetylases and are therefore useful in treating diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities. In particular, for example, they are highly effective against hematological malignancy and solid carcinoma.
DETAILED DESCRIPTION OF THE INVENTION
Various publications are cited throughout the present application. The contents of these publications and contents of documents cited in these publications are herein incorporated by reference.
Histone deacetylase (HDAC) proteins play a critical role in regulating gene expression in vivo by altering the accessibility of genomic DNA to transcription factors. Specifically, HDAC proteins remove the acetyl group of acetyl-lysine residues on histones, which can result in nucleosomal remodelling (Grunstein, M., 1997, Nature, 389: 349-352). Due to their governing role in gene expression, HDAC proteins are associated with a variety of cellular events, including cell cycle regulation, cell proliferation, differentiation, reprogramming of gene expression, and cancer development (Ruijter, A-J-M., 2003, Biochem. J., 370: 737-749; Grignani, F., 1998, Nature, 391: 815-818; Lin, R-J., 1998, 391 : 811-814; Marks, P-A., 2001 , Nature Reviews Cancer, 1 : 194). In fact, HDAC inhibitors have been demonstrated to reduce tumor growth in various human tissues and in animal studies, including lung, stomach, breast, and prostrate (Dokmanovic, M. ,2005, J. Cell Biochenm., 96: 293-304).
Mammalian HDACs can be divided into three classes according to sequence homology. Class I consists of the yeast Rpd3-like proteins (HDAC 1, 2, 3, 8 and 11). Class Il consists of the yeast HDA1-like proteins (HDAC 4, 5, 6, 7, 9 and 10). Class III consists of the yeast SIR2-like proteins (SIRT 1, 2, 3, 4, 5, 6 and 7).
The activity of HDAC1 has been linked to cell proliferation, a hallmark of cancer. Particularly, mammalian cells with knock down of HDAC1 expression using siRNA were antiproliferative (Glaser, K-B., 2003, Biochem. Biophys. Res. Comm., 310: 529-536). While the knock out mouse of HDAC1 was embryonic lethal, the resulting stem cells displayed altered cell growth (Lagger, G, 2002, EMBO J., 21 : 2672-2681). Mouse cells overexpressing HDAC1 demonstrated a lengthening of G2 and M phases and reduced growth rate (Bartl. S., 1997, MoI. Cell Biol., 17: 5033-5043). Therefore, the reported data implicate HDAC1 in cell cycle regulation and cell proliferation. HDAC2 regulates expression of many fetal cardiac isoforms. HDAC2 deficiency or chemical inhibition of histone deacetylase prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inppδf) resulting in constitutive activation of glycogen synthase kinase 3β (Gsk3β) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1). In contrast, HDAC2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3β. Chemical inhibition of activated Gsk3β allowed HDAC2-defιcient adults to become sensitive to hypertrophic stimulation. These results suggest that HDAC2 is an important molecular target of HDAC inhibitors in the heart and that HDAC2 and Gsk3β are components of a regulatory pathway providing an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure (Trivedi, C-M., 2007, Nat. Med,. 13: 324-331).
HDAC3 are maximally expressed in proliferating crypt cells in normal intestine. Silencing of HDAC3 expression in colon cancer cell lines resulted in growth inhibition, a decrease in cell survival, and increased apoptosis. Similar effects were observed for HDAC2 and, to a lesser extent, for HDAC1. HDAC3 gene silencing also selectively induced expression of alkaline phosphatase, a marker of colon cell maturation. Concurrent with its effect on cell growth, overexpression of HDAC3 inhibited basal and butyrate-induced p21 transcription in a Sp1/Sp3-dependent manner, whereas silencing of HDAC3 stimulated p21 promoter activity and expression. These findings identify HDAC3 as a gene deregulated in human colon cancer and as a novel regulator of colon cell maturation and p21 expression (Wilson, A-J., 2006, J. Biol. Chem., 281: 13548-13558).
HDAC6 is a subtype of the HDAC family that deacetylates alpha-tubulin and increases cell motility. Using quantitative real-time reverse transcription polymerase chain reaction and Western blots on nine oral squamous cell carcinoma (OSCC)-derived cell lines and normal oral keratinocytes (NOKs), HDAC6 mRNA and protein expression were commonly up-regulated in all cell lines compared with the NOKs. Immunofluorescence analysis detected HDAC6 protein in the cytoplasm of OSCC cell lines. Similar to OSCC cell lines, high frequencies of HDAC6 up-regulation were evident in both mRNA (74%) and protein (51%) levels of primary human OSCC tumors. Among the clinical variables analyzed, the clinical tumor stage was found to be associated with the HDAC6 expression states. The analysis indicated a significant difference in the HDAC6 expression level between the early stage (stage I and II) and advanced-stage (stage III and IV) tumors (P=O.014). These results suggest that HDAC6 expression may be correlated with tumor aggressiveness and offer clues to the planning of new treatments (Sakuma, T, 2006, Int. J. Oncol., 29: 117-124).
Epigenetic silencing of functional chromosomes by HDAC is one of major mechanisms occurred in many pathological processes, in which functionally critical genes are repressed or reprogrammed by HDAC activities leading to the loss of phenotypes in terminal differentiation, maturation and growth control, and the loss of functionality of tissues. For example, tumor suppressor genes are often silenced during development of cancer and chemical inhibitor of HDAC can derepressed the expression of these tumor suppressor genes, leading to growth arrest and differentiation (Glaros S et al., 2007, Oncogene June 4 Epub ahead of print; Mai, A, et al., 2007, lnt J. Biochem Cell Bio., April 4, Epub ahead of print; Vincent A. et al., 2007, Oncogene, April 30, Epub ahead of print; our unpublished results); and repression of structural genes such as FXN in Friedreich's ataxia and SMN in spinal muscular atrophy can be reversed by HDAC inhibitors that lead to re-expression of FXN and SMN genes and resume the functions in the tissues (Herman D et al., 2006, Nature Chemical Biology, 2(10):551-8; Avila AM et al., 2007, J Clinic Investigation, 117(3)659-71; de Bore J, 2006, Tissue Eng. 12(10):2927-37); Induction of entire MHC Il family gene expression through reprogramming of HDAC "hot spot" in chromosome 6p21-22 by HDAC inhibitor further extend epigenetic modulation of immune recognition and immune response (Gialitakis M et al., 2007, Nucleic Acids Res., 34(1);765-72).
Several classes of HDAC inhibitors have been identified, including (1) short-chain fatty acids, e.g. butyrate and phenylbutyrate; (2) organic hydroxamic acids, e.g. suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA); (3) cyclic tetrapeptides containing a 2-amino-8-oxo 9,10-expoxydecanoyl (AOE) moiety, e.g. trapoxin and HC-toxin; (4) cyclic peptides without the AOE moiety, e.g. apicidin and FK228; and (5) benzamides, e.g. MS-275 (EP0847992A1, US2002/0103192A1 , WO02/26696A1 , WO01/70675A2, WO01/18171A2). Although, HDAC inherited very promising biological roles as a drug target, the success of SAHA from Merck is currently only limited to the treatment of cutaneous T cell lymphoma whereas no major solid tumors yet been reported to be highly effective by this treatment. Therefore, there is still a need to discover new compounds with improved profiles, such as stronger HDAC inhibitory activity and anti-cancer activity, more selective inhibition on different subtype of HDAC, and lower toxicity.
The favorite metaphor for cancer drug developers has long been the target therapy. One hoped to design a drug that could hit tumor cells in one specific target, knocking out tumor cells while leaving normal cells undamaged. Cancer cells, however, can use multiple biological triggers and pathways to grow and spread throughout the body. Hitting them in one target will also render them to regroup and redeploy along new growth paths. That realization has led to the development of combination target therapies, which are becoming the new paradigm for cancer treatment. Several multi-target kinase inhibitors are now in development, two, Sorafenib and Suten, are already approved in the United States. For example, Sorafenib, developed by Bayer Pharmaceuticals, is the first drug targeting both the RAF/MEK/ERK pathway (involved in cell proliferation) and the VEGFR2/PDGFRβ signaling cascade (involved in angiogenesis). This drug was first approved in December 2005 for advanced kidney cancer, a disease that is believed to be highly dependent on angiogenesis. However, these target therapies, although are effective against some solid tumors, but far from satisfaction in terms of reaching a better efficacy as a single agent against other solid tumors while tolerable sides associated with treatment can be maintained.
PROVIDED HEREIN are new chemical compounds that combine anti-angiogenesis and anti-proliferation activities of RTK's together with differentiation-inducing, immune modulation, cell cycle arrest and apoptosis activities of more selective HDACi, to reach a better efficacy against solid tumors while overcoming side effects such as hypertension, QT prolongation, thyroid gland regression, skin rash and discoloration, and pains associated with currently marketed RTK inhibitors.
Particularly, the present invention provides a compound having the structure represented by formula (I), or its stereoisomer, enantiomer, diastereomer, hydrate, or pharmaceutically acceptable salts thereof:
Figure imgf000008_0001
(I) wherein
X is a valence bond or -C(O)-N H-(CH2)m-;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
R5 is -NHOH or
Figure imgf000008_0002
R6, R7, R8 and R9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; n is an integer ranging from 1 to 5; m is an integer ranging from 1 to 5.
In the above structural formula (I) and throughout the present specification, the following terms have the indicated meaning:
The term "halo" as used herein means fluorine, chlorine, bromine or iodine.
The term "alkyl" as used herein includes methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl and the like.
The term "alkoxy" as used herein includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and the like.
The compounds of this invention are prepared as described below: (a) Compound 1 is condensed with compound 2 to give compound 3;
Figure imgf000009_0001
(b) Compound 3 is hydrolyzed to give compound 4;
Figure imgf000009_0002
(c) Compound 4 is condensed with hydroxylamine to give compound 5a;
Figure imgf000009_0003
(d) Compound 4 is condensed with compound 6 to give compound 5b.
Figure imgf000009_0004
The condensation reaction (a) and (d) are conducted by using a peptide condensing agent such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbo- diimide (DCC), N,N'-carbonyldiimidazole (CDI), etc. The reaction may be conducted at O to 80 0C for 4 to 72 hours. Solvents which may be used are normal solvents such as benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, N, N-dimethylformamide, etc. If necessary, a base such as sodium hydroxide, triethylamine and pyridine may be added to the reaction system.
The condensation reaction (c) is conducted by using CICOOEt as a condensing agent. The reaction may be conducted at 0 to 80 0C for 1 to 24 hours. Solvents which may be used are normal solvents such as benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, N, N-dimethylformamide, etc. If necessary, a base such as sodium hydroxide, triethylamine and pyridine may be added to the reaction system.
The hydrolysis reaction (b) is conducted by using a hydrolysis agent such as lithium hydroxide, sodium hydroxide, potassium hydroxide, etc. The reaction may be conducted at 0 to 80 0C for 2 to 72 hours. Solvents which may be used are normal solvents such as water, methanol, ethanol, tetrahydrofuran, dioxane, N, N-dimethylformamide, etc.
The compounds represented by formula (I) and the intermediate (3) and (4) may be purified or isolated by the conventional separation method such as extraction, recrystallization, column chromatography and the like.
This invention also provides a compound having the structure represented by formula (II), or its stereoisomer, enantiomer, diastereomer, hydrate, or pharmaceutically acceptable salts thereof:
A-Y-B (N)
wherein A is
Figure imgf000010_0001
B is
Figure imgf000011_0001
Y is a moiety having -CH2-, -CO-, -CS-, -SO- or -SO2-, which is linear with a length of 4.0 to 12.0 A and links A and B;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
R6, R7, R8 and R9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl;
R10, R11, R12, R13, R14 and R15 are independently hydrogen or alkyl.
The compounds represented by formula (I) or formula (II) are capable of inhibiting protein kinases and histone deacetylases and are therefore useful in treating diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities. In particular, they are highly effective against hematological malignancy and solid carcinoma.
The compounds represented by formula (I) or formula (II) useful as a drug may be used in the form of a general pharmaceutical composition. The pharmaceutical composition may be in the forms normally employed, such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, may contain flavourants, sweeteners etc. in suitable solids or liquid carriers or diluents, or in suitable sterile media to form injectable solutions or suspensions. Such composition typically contains from 0.5 to 70%, preferably 1 to 20% by weight of active compound, the remainder of the composition being pharmaceutically acceptable carriers, diluents or solvents or salt solutions.
The compounds represented by formula (I) or formula (II) are clinically administered to mammals, including man and animals, via oral, nasal, transdermal, pulmonary, or parenteral routes. Administration by the oral route is preferred, being more convenient and avoiding the possible pain and irritation of injection. By either route, the dosage is in the range of about 0.0001 to 200 mg/kg body weight per day administered singly or as a divided dose. However, the optimal dosage for the individual subject being treated will be determined by the person responsible for treatment, generally smaller dose being administered initially and thereafter increments made to determine the most suitable dosage.
Representative compounds of the present invention are shown in Table 1 below. The compound numbers correspond to the "Example numbers" in the Examples section. That is, the synthesis of compound 1 as shown in the Table 1 is described in "Example 1" and the synthesis of compound 52 as shown in the Table 1 is described in "Example 52". The compounds presented in the Table 1 are exemplary only and are not to be construed as limiting the scope of this invention in any manner. TABLE 1
Exampl
Structure Name e
N-hydroxy-2-{5-[5-fluoro-2-oxo- 1 ,2-dihydro-indol-<3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole- 3-carbonyl}amino-acetamide
N-(2-aminophenyl)-2-{5-[5- fluoro-2-oxo-1 ,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl- 1 H-pyrrole-3-carbonyl}amino- acetamide
N-(2-amino-4-fluorophenyl)-2- {5-[5-fluoro-2-oxo-1 ,2-dihydro- indol -(3Z)-ylidene-methyl]- 2,4-dimethyl-1 H-pyrrole-3- carbonyl}amino-acetamide
N-hydroxy-3-{5-[5-fluoro-2-oxo- 1 ,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4- dimethyl-1 H-pyrrole- 3-carbonyl}amino-propanamide
N-(2-aminophenyl)-3-{5-[5- fluoro-2-oxo-1 ,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl- 1 H-pyrrole-3-carbonyl}amino- propanamide
N-(2-amino-4-fluorophenyl)- 3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-
10 indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amino-propanamide
Figure imgf000012_0001
N-hydroxy-6-{5-[5-fluoro-2-oxo-
1 ,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4- dimethyl-1 H-pyrrole- 3-carbonyl}amino-hexanamide
N-(2-aminophenyl)-6-{5-[5- fluoro-2-oxo-1 ,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl- 1 H-pyrrole-3-carbonyl}amino- hexanamide
N-(2-amino-4-fluorophenyl)-6- {5-[5-fluoro-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amiπo-hexanamide
Figure imgf000013_0001
H π . OH N-hydroxy-2-{2-{5-[5-fluoro-2-
U Il H oxo-1 ,2-dihydro-indol-(3Z)-
18 ylidene- methyl]-2,4-dimethyl- 1 H-pyrrole-3-carbonyQamino}
Figure imgf000013_0002
acetylamino-acetamide
N-(2-aminophenyl)-2-{2-{5-[5- fluoro-2-oxo-1 ,2-dihydro-indol-
19 (3Z)-ylidenemethyl]-2,4-dimethyl- 1 H-pyrrole-3-carbonyl}amino}- acetylamino-acetamide
N-(2-amino-4-fluoropheπyl)-2- {2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-
20 indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amino}acetylamino-acetamide
N-hydroxy-2-{3-{5-[5-fluoro-2- oxo-1 ,2-dihydro-indol-(3Z)-
23 ylidenemethyl]-2,4-dimethyl-1 H- pyrrole-3-carbonyl}amino}- propionylamino-acetamide
Figure imgf000013_0003
N-(2-aminophenyl)-2-{3-{5- [5-fluoro-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amino}propionylamino-acetamide
N-(2-amino-4-fluorophenyl)-2- {3-{5-[5-fluoro-2-oxo-1 ,2-di- hydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3- carbonyl}amino}propionylamino- acetamide
N-hydroxy-6-{2-{5-[5-fluoro-2- oxo-1 ,2-dihydro-indol-(3Z)- ylidenemethyl]-2,4-dimethyl-1 H- pyrrole-3-carbonyl}amino}- acetylamino-hexanamide
N-(2-aminophenyl)-6-{2-{5-[5- fluoro-2-oxo-1 ,2-dihydro-indol- (3Z)-ylidenemethyl]-2,4-dimethyl- 1 H-pyrrole-3-carbonyl}amino}- acetylamino-hexanamide
N-(2-amino- 4-fluorophenyl)-6- {2-{5-[5-fluoro-2-oxo-1 ,2-di- hydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3- carbonyl}amino}acetylamino- hexanamide
N-(2-amino-4-chlorophenyl)-6- {5- [5-fluoro-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amino-hexanamide
N-(2-amino-4-methylphenyl)-6- {5- [5-fiuoro-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amino-hexanamide
Figure imgf000014_0001
N-(2-amino-4-methoxyphenyl)-6- {5-[5-fluoro-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amino-hexanamide
N-(2-amino-4-trifluoromethyl- phenyl)-6-{5-[5-fluoro-2-oxo-1 ,2- dihydro-indol-(3Z)-ylidenemethyl] -2,4-dimethyl-1 H-pyrrole-3- carbonyl}amino-hexanamide
N-(2-aminophenyl)-6-{5-[2-oxo- 1 ,2-dihydro-indol-(3Z)- ylidenetnethyl]-2,4-dimethyl-1 H- pyrrole-3-carbonyl}amino- hexanamide
N-(2-aminophenyl)-6-{5-[5- chloro-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}- amino-hexanamide
N-hydroxy-6-{5-[4-methyl-2-oxo-
1 ,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4- dimethyl-1 H-pyrrole- 3-carbonyl}amino-hexanamide
N-(2-aminophenyl)-6-{5-[5-nitro- 2-0X0-1 ,2-dihydro-indol-(3Z)- ylidenemethyl]-2,4-dimethyl-1 H- pyrrole-3-carbonyl}amino- hexanamide
N-(2-aminophenyl)-6-{5-[6- methoxy-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1 H-pyrrole-3-carbonyl}-
Figure imgf000015_0001
amino-hexanamide N-(2-aminophenyl)-6-{5-[6- trifluoromethyl-2-oxo-1 ,2-
52 dihydro-indol-(3Z)-ylidene- methyl]-2,4-dimethyl-1 H-pyrrole- 3-carbonyl}amino-hexanamide
Figure imgf000016_0001
Further, all parts and percentages in the examples, as well as in the remainder of the specification, are by weight unless otherwise specified. Any range of numbers recited in the specification or paragraphs hereinafter describing or claiming various aspects of the invention, such as that representing a particular set of properties, units of measure, conditions, physical states or percentages, is intended to literally incorporate expressly herein by reference or otherwise, any number falling within such range, including any subset of numbers or ranges subsumed within any range so recited. The term "about" when used as a modifier for, or in conjunction with, a variable, is intended to convey that the numbers and ranges disclosed herein are flexible and that practice of the present invention by those skilled in the art using temperatures, concentrations, amounts, contents, carbon numbers, and properties that are outside of the range or different from a single value, will achieve the desired result.
Example 1
Preparation of 2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-acetic acid methyl ester
Figure imgf000016_0002
δ-Iδ-fluoro^-oxo-i ^-dihydro-indol^SZJ-ylidenemethyll^^-dimethyl-IH-pyrrole-S-carbox- ylic acid (300 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and glycine methyl ester hydrochloride (151.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-acetic acid methyl ester (325 mg, 88% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.44 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.66 (s, 3H, COOCH3), 3.98 (d, J= 8.0 Hz, 2H, CH2CO), 6.84 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.72 (s, 1 H, vinyl-H), 7.76 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 8.01 (t, J= 8.0Hz, 1H, CONH), 10.91 (s, 1H, indolinone-NH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 372 (M+1). Example 2
Preparation of 2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-yiidenemethyl]- 2,4-dimethyl-1 H-pyrrole-S-carbonylJamino-acetic acid
Figure imgf000017_0001
2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyljamino-acetic acid methyl ester (371 mg, 1 mmol) and 300ml Of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 2-{5-[5-fluoro-2-oxo- 1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car-bonyl}amino-acetic acid (342 mg, 96% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.44 (s, 3H, pyrrole-CH3), 2.49 (S, 3H, pyrrole-CH3), 3.88 (d, J= 8.0 Hz, 2H, CH2CO), 6.84 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 7.72 (s, 1H, vinyl-H), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.87 (t, J= 8.0Hz, 1H, CONH), 10.91 (s, 1H, indolinone-NH), 12.57 (s, 1H, COOH), 13.70 (s, 1H, pyrrole-NH). LC-MS (m/z) 358 (M+1).
Example 3
Preparation of N-hydroxy-2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-acetamide
Figure imgf000017_0002
2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-acetic acid (357 mg, 1 mmol), triethylamine (151 mg, 1.5 mmol) and 20 ml of DMF were stirred at O0C while ethyl chloroformate (163 mg, 1.5 mmol) was added. The mixture was stirred at O0C for 2 hours, and then 50% aqueous solution of hydroxylamine (1.32 g, 20 mmol) was added. The mixture was stirred for 3 hours at room temperature, and then diluted with 1000 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-hydroxy-2-{5-[5-fluoro-2-oxo-1 ,2 -dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-acetamide (327 mg, 88%) as a brown solid. 1H NMR (DMSO-d6)δ2.46 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrroie-CHs), 3.75 (d, J= 4.0 Hz, 2H, CH2CO), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.91 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.71 (s, 1H, vinyl-H), 7.74 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.76 (t, J= 4.0Hz, 1H, CONH), 8.83 (br s, 1H, N-OH), 10.86 (br s, 2H, NH-O and indolinone-NH), 13.69 (s, 1 H, pyrrole-NH). LC-MS (m/z) 373 (M+1).
Example 4 Preparation of
N-(2-aminophenyl)-2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-acetamide
Figure imgf000018_0001
2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-acetic acid (357 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol) and o-phenylenediamine (864 mg, 8 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-2-{5-[5-fluoro-2- oxo-i ^-dihydro-indol^SZJ-ylidenemethyll^^-dimethyl-IH-pyrrole-S-carbonylJamino-ace- tamide (371 mg, 83% yield) as a brown solid. 1H NMR (DMSO-d6)δ2.45 (s, 3H, pyrrole-CHs), 2.47 (s, 3H, pyrrole-CH3), 4.0 (d, J= 4.0 Hz, 2H, CH2CO), 4.83 (s, 2H, benzene-NH2), 6.53 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz1 1H, Ar-H), 6.82 (m, 3H, Ar-H), 7.14 (d, J= 8.0 Hz, 1H, Ar-H), 7.72 (s, 1H, vinyl-H), 7.67 (t, J= 4.0Hz, 1H, CONH), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 9.18 (s, 1 H, benzene-NH), 10.91 (s, 1H, indolinone-NH), 13.72 (s, 1H, pyrrole-NH). LC-MS (m/z) 448 (M+1).
Example 5 Preparation of
N-(2-amino-4-fluorophenyl)-2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-acetamide
Figure imgf000018_0002
2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-acetic acid (357 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol) and 4-fluoro-o-phenylenediamine (1.02 g, 8 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and ethanol, and then dried under vacuum to give N-(2-amino-4-fluorophenyl)-2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidene-methyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyI}amino-acetamide (367 mg, 79% yield) as a brown solid.1H NMR (DMSO-d6)δ2.46 (s, 3H, pyrrole-CH3), 2.48 (s, 3H, pyrrole-CH3), 4.02 (d, J= 4.0 Hz, 2H, CH2CO), 5.26 (s, 2H, benzene-NH2), 6.29 (m, 1H, Ar-H), 6.46 (d, J= 8.0 Hz, 1H, Ar-H), 6.84 (m, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.05 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.72 (s, 1H, vinyl-H), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.84 (t, J= 4.0Hz, 1H, CONH), 9.18 (s, 1H, benzene-NH), 10.91 (s, 1H, indolinone-NH), 13.72 (s, 1H, pyrrole-NH). LC-MS (m/z) 466 (M+1).
Example 6
Preparation of 3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-propionic acid methyl ester
Figure imgf000019_0001
5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbox- ylic acid (300 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and β-alanine methyl ester hydrochloride (168.6 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyljamino-propionic acid methyl ester (333 mg, 87% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CH3), 2.58 (d, J= 8.0 Hz, 2H, CH2CO), 3.50 (m, 2H, NCH2), 3.63 (s, 3H, COOCH3), 6.84 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.67 (t, J= 4.0Hz, 1H, CONH), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 10.88 (s, 1 H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 386 (M+1).
Example 7
Preparation of 3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-propionic acid
Figure imgf000019_0002
3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-propionic acid methyl ester (385 mg, 1 mmol) and 30OmI of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole- 3-carbonyl}amino-propionic acid (371 mg, 94% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CH3), 2.48 (d, J= 8.0 Hz, 2H, CH2CO), 3.41 (m, 2H, NCH2), 6.84 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.67 (t, J= 4.0Hz, 1 H, CONH), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 10.89 (s, 1H, indolinone-NH), 12.25 (s, 1H, COOH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 372 (M+1).
Example 8
Preparation of N-hydroxy-3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-propanamide
Figure imgf000020_0001
3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-propionic acid (371 mg, 1 mmol), triethylamine (151 mg, 1.5 mmol) and 20 ml of DMF were stirred at O0C while ethyl chloroformate (163 mg, 1.5 mmol) was added. The mixture was stirred at O0C for 2 hours, and then 50% aqueous solution of hydroxylamine (1.32 g, 20 mmol) was added. The mixture was stirred for 3 hours at room temperature, and then diluted with 1000 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-hydroxy-3-{5-[5-fluoro-2-oxo-1 ,2-di- hydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carbonyl}amino-propanamide (355 mg, 92%) as a brown solid.. 1H NMR (DMSO-d6)δ2.23 (d, J= 4.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CHs), 2.41 (s, 3H, pyrrole-CH3), 3.34 (m, 2H, NCH2), 6.83 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.90 (td, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.70 (s, 1 H, vinyl-H), 7.68 (t, J= 4.0Hz, 1H, CONH), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 8.70 (br s, 1H, N-OH), 10.69 (s, 2H, NH-O and indolinone-NH), 13.69 (s, 1H, pyrrole-NH). LC-MS (m/z) 387 (M+1).
Example 9 Preparation of
N-(2-aminophenyl)-3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-propanamide
Figure imgf000021_0001
3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyljamino-propionic acid (371 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimeth- yl-1 H-pyrrole-3-carbonyl}amino-propanamide (387 mg, 84% yield) as a brown solid. 1H NMR (DMSO-d6)δ2.40 (s, 3H, pyrrole-CH3), 2.42 (s, 3H, pyrrole-CH3), 2.60 (d, J= 8.0 Hz, 2H, CH2CO), 3.51 (m, 2H, NCH2), 4.83 (s, 2H, benzene-NH2), 6.52 (m, 1H1 Ar-H), 6.70 (d, J= 8.0 Hz, 1H, Ar-H), 6.82 (m, 3H, Ar-H), 7.16 (d, J= 8.0 Hz, 1H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.74 (m, 2H, CONH and Ar-H), 9.18 (s, 1H, benzene-NH), 10.89 (s, 1H, indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 462 (M+1).
Example 10 Preparation of
N-(2-amino-4-fluorophenyl)-3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-propanamide
Figure imgf000021_0002
S-tS-tδ-fluoro^-oxo-i ^-dihydro-indol^SZJ-ylidenemethylJ^^-dimethyl-IH-pyrrole-S-car- bonyl}amino-propionic acid (371 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-fluoro-o-phenylenediamine (151 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-fluorophenyl)-3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemeth-yl]-2, 4-dimethyl-1H-pyrrole-3-carbonyl} amino-propanamide (373 mg, 78% yield) as a brown solid.1H NMR (DMSO-d6)δ2.40 (s, 3H, pyrrole-CH3), 2.42 (s, 3H, pyrrole-CH3), 2.62 (d, J= 8.0 Hz, 2H, CH2CO), 3.53 (m, 2H, NCH2), 5.26 (s, 2H, benzene-NH2), 6.29 (m, 1H, Ar-H), 6.46 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.84 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.06 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.72 (s, 1H, vinyl-H), 7.76 (dd, J= 4.0 and 8.0 Hz1 1H, Ar-H), 7.84 (t, J= 4.0Hz, 1H, CONH), 9.18 (s, 1H, benzene-NH), 10.86 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 480 (M+1).
Example 11
Preparation of 6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester
Figure imgf000022_0001
5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyI-1H-pyrroIe-3-carbox- ylic acid (300 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyI)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester (346 mg, 81% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.51 (m, 2H, CH2), 1.55 (m, 2H, CH2), 2.31 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.40 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 3.57 (s, 3H, COOCH3), 6.82 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.70 (s, 1 H, vinyl-H), 7.63 (t, J= 4.0Hz, 1 H, CONH), 7.76 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 10.89 (s, 1 H, indolinone-NH), 13.66 (s, 1 H, pyrrole-NH). LC-MS (m/z) 428 (M+1).
Example 12
Preparation of 6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid
Figure imgf000022_0002
6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-hexanoic acid methyl ester (427 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole- 3-carbonyl}amino-hexanoic acid (377 mg, 91% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.50 (m, 4H, 2*CH2), 2.20 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CHs), 2.41 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 6.82 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.70 (s, 1 H, vinyl-H), 7.63 (t, J= 4.0Hz, 1 H, CONH), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 10.89 (s, 1 H, indolinone-NH), 12.00 (s, 1H, COOH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 414 (M+1).
Example 13
Preparation of N-hydroxy-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000023_0001
e-fδ-tS-fluoro^-oxo-i^-dihydro-indol^SZJ-ylidenemethylJ^^-dimethyl-IH-pyrrole-S-car- bonyl}amino-hexanoic acid (413 mg, 1 mmol), triethylamine (151 mg, 1.5 mmol) and 20 ml of DMF were stirred at O0C while ethyl chloroformate (163 mg, 1.5 mmol) was added. The mixture was stirred at O0C for 2 hours, and then 50% aqueous solution of hydroxylamine (1.32 g, 20 mmol) was added. The mixture was stirred for 3 hours at room temperature, and then diluted with 1000 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-hydroxy-6-{5-[5-fluoro-2-oxo-1 ,2-di- hydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide (385 mg, 90%) as a brown solid. 1H NMR (DMSO-d6)δ1.29 (m, 2H, CH2), 1.50 (m, 4H, 2χCH2), 1.94 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CHa), 3.19 (m, 2H, NCH2), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.63 (t, J= 4.0Hz, 1H, CONH), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 8.70 (br s, 1 H, N-OH), 10.70 (s, 2H, NH-O and indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 429 (M+1).
Example 14 Preparation of
N-(2-aminophenyl)-6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000023_0002
6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-hexanoic acid (413 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimeth- yl-1H-pyrrole-3-carbonyl}amino-hexanamide (437 mg, 87% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.41 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 4.82 (s, 2H, benzene-NH2), 6.53 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz, 1H, Ar-H), 6.85 (m, 3H, Ar-H), 7.14 (d, J= 8.0 Hz, 1H, Ar-H), 7.65 (t, J= 4.0 Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 9.11 (s, 1H, benzene-NH), 10.90 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrroie-NH). LC-MS (m/z) 504 (M+1).
Example 15 Preparation of
N-(2-amino-4-fluorophenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000024_0001
6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-hexanoic acid (413 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-fluoro-o-phenylenediamine (151 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-fluorophenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemeth-yl]-2, 4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide (354 mg, 68% yield) as a brown solid.1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.41 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 5.25 (s, 2H, benzene-NH2), 6.29 (m, 1H, Ar-H), 6.47 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.06 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.71 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.84 (t, J= 4.0Hz, 1H, CONH), 9.18 (s, 1H, benzene-NH), 10.86 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 522 (M+1).
Example 16 Preparation of 2-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-yIidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-acetic acid methyl ester
Figure imgf000025_0001
2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-acetic acid (357 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and glycine methyl ester hydrochloride (151.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 2-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-di- methyl-1H-pyrrole-3-carbonyl}amino}acetylamino-acetic acid methyl ester (342 mg, 80% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.47 (s, 3H, pyrrole-CH3), 2.49 (s, 3H1 pyrrole-CHs), 3.63 (s, 3H, COOCH3), 3.88 (d, J= 8.0 Hz, 2H, N-CH2), 3.90 (d, J= 8.0 Hz, 2H, CH2CO), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.72 (s, 1 H, vinyl-H), 7.74 (t, J= 8.0Hz, 1 H, CONH), 7.75 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 8.31 (t, J= 8.0Hz, 1H, CONH), 10.90 (s, 1H, indolinone-NH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 429 (M+1).
Example 17
Preparation of 2-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-yIidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-acetic acid
Figure imgf000025_0002
2-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-acetic acid methyl ester (428 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 2-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-di- methyl-IH-pyrrole-S-carbonylJaminolacetylamino-ace- tic acid (363 mg, 88% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.45 (s, 3H, pyrrole-CH3), 2.47 (s, 3H, pyrrole-CH3), 3.78 (d, J= 4.0 Hz, 2H, N-CH2), 3.89 (d, J= 4.0 Hz, 2H, CH2CO), 6.83 (del, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.71 (s, 1H, vinyl-H), 7.74 (t, J= 4.0Hz, 1H, CONH), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 8.14 (t, J= 4.0Hz, 1H, CONH), 10.90 (s, 1H, indolinone-NH), 12.57 (s, 1H, COOH), 13.70 (s, 1H, pyrrole-NH). LC-MS (m/z) 415 (M+1).
Example 18
Preparation of N-hydroxy-2-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-acetamide
Figure imgf000026_0001
2-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-acetic acid (414 mg, 1 mmol), triethylamine (151 mg, 1.5 mmol) and 20 ml of DMF were stirred at O0C while ethyl chloroformate (163 mg, 1.5 mmol) was added. The mixture was stirred at O0C for 2 hours, and then 50% aqueous solution of hydroxylamine (1.32 g, 20 mmol) was added. The mixture was stirred for 3 hours at room temperature, and then diluted with 1000 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-hydroxy-2-{2-{5-[5-fluoro- 2-0X0-1 ,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino}- acetylamino-acetamide (365 mg, 85%) as a brown solid.. 1H NMR (DMSO-d6)δ2.45 (s, 3H, pyrrole-CHs), 2.47 (s, 3H1 pyrrole-CH3), 3.65 (d, J= 4.0 Hz, 2H, N-CH2), 3.88 (d, J= 4.0 Hz, 2H, CH2CO), 6.83 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.91 (td, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.71 (S, 1H, vinyl-H), 7.74 (t, J= 4.0Hz, 1H, CONH), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 8.14 (t, J= 4.0Hz, 1H, CONH), 8.83 (br s, 1H, N-OH), 10.86 (br s, 2H, NH-O and indolinone-NH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 430 (M+1).
Example 19 Preparation of
N-(2-aminophenyl)-2-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-acetamide
Figure imgf000026_0002
2-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-acetic acid (414 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-2-{2-{5-[5- fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carbonyl}amino} acetylamino-acetamide (423 mg, 84% yield) as a brown solid. 1H NMR (DMSO-d6)δ2.45 (s, 3H, pyrrole-CH3), 2.47 (s, 3H, pyrrole-CH3), 3.91 (d, J= 4.0 Hz, 2H, N-CH2), 3.93 (d, J= 4.0 Hz, 2H, CH2CO), 4.89 (s, 2H, benzene-NH2), 6.53 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz, 1H, Ar-H), 6.83 (m, 1H, Ar-H), 6.90 (m, 2H, Ar-H), 7.10 (d, J= 8.0 Hz, 1H, Ar-H), 7.72 (s, 1 H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.83 (t, J= 4.0 Hz, 1H, CONH), 8.25 (t, J= 4.0 Hz, 1H, CONH), 9.10 (s, 1H, benzene-NH), 10.91 (s, 1H, indolinone-NH), 13.72 (s, 1H, pyrrole-NH). LC-MS (m/z) 505 (M+1).
Example 20 Preparation of
N-(2-amino-4-fluorophenyl)-2-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-acetamide
Figure imgf000027_0001
2-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-acetic acid (414 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-fluoro-o-phenylenediamine (151 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-fluorophenyl)-2-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)- ylidenemethyll^^-dimethyl-IH-pyrrole-S-carbonytyaminoJacetylamino-acetamide (381 mg, 73% yield) as a brown solid.1H NMR (DMSO-d6)δ2.45 (s, 3H, pyrrole-CH3), 2.46 (s, 3H, pyrrole-CHs), 3.91 (d, J= 4.0 Hz, 4H, 2*CH2), 5.22 (s, 2H, benzene-NH2), 6.28 (m, 1H, Ar-H), 6.45 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.90 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.02 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.72 (s, 1H, vinyl-H), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.82 (t, J= 4.0Hz, 1H, CONH), 8.25 (t, J= 4.0 Hz, 1H, CONH), 9.03 (s, 1H, benzene-NH), 10.90 (s, 1H, indolinone-NH), 13.72 (s, 1H, pyrrole-NH). LC-MS (m/z) 523 (M+1).
Example 21 Preparation of 2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2, 4-di methyl- 1 H-pyrrole-3-carbonyl}amino}propionylamino-acetic acid methyl ester
Figure imgf000028_0001
3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-propionic acid (371 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and glycine methyl ester hydrochloride (151.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 2-{3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carbonyl}amino}propionyl- amino-acetic acid methyl ester (372 mg, 84% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.35(s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CHs), 2.49 (d, J= 8.0 Hz, 2H, N-CH2), 3.41 (m, 2H, N-CH2), 3.63 (s, 3H, COOCH3), 3.83 (d, J= 4.0 Hz, 2H, N-CH2), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.60 (t, J= 8.0Hz, 1H, CONH), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 8.41 (t, J= 8.0Hz, 1H, CONH), 10.89 (s, 1H, indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 443 (M+1).
Example 22
Preparation of 2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}propionylamino-acetic acid
Figure imgf000028_0002
2-{3-{5-t5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}propionylamino-acetic acid methyl ester (442 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-IH-pyrrole-S-carbonylJaminoJpropionylamino-acetic acid (404 mg, 94% yield) as a yellow solid. 1H NMR (DMSO-d6)δ2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CH3), 2.49 (d, J= 4.0 Hz, 2H, N-CH2), 3.43 (m, 2H, N-CH2), 3.74 (d, J= 4.0 Hz, 2H, CH2CO), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.59 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 8.28 (t, J= 4.0Hz, 1H, CONH), 10.89 (s, 1H1 indolinone-NH), 12.60 (s, 1H, COOH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 429 (M+1).
Example 23
Preparation of N-hydroxy-2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}propionylamino-acetamide
Figure imgf000029_0001
2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}propionylamino-acetic acid (428 mg, 1 mmol), triethylamine (151 mg, 1.5 mmol) and 20 ml of DMF were stirred at O0C while ethyl chloroformate (163 mg, 1.5 mmol) was added. The mixture was stirred at O0C for 2 hours, and then 50% aqueous solution of hydroxylamine (1.32 g, 20 mmol) was added. The mixture was stirred for 3 hours at room temperature, and then diluted with 1000 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-hydroxy-2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carbonyl}amino}propionylamino-acetamide (372 mg, 84%) as a brown solid. 1H NMR (DMSO-d6)δ2.28 (d, J= 4.0 Hz, 2H, N-CH2), 2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CHs), 3.60 (m, 2H, N-CH2), 3.92 (d, J= 4.0 Hz, 2H, CH2CO), 6.82 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.91 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.63 (t, J= 4.0Hz, 1 H, CONH), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 8.18 (t, J= 4.0Hz, 1H, CONH), 8.75 (br s, 1H, N-OH), 10.86 (br s, 2H, NH-O and indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 444 (M+1).
Example 24 Preparation of
N-(2-aminophenyl)-2-{3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}propionylamino-acetamide
Figure imgf000029_0002
2-{3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}propionylamino-acetic acid (428 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino}propionylamino-acetamide (419 mg, 81% yield) as a brown solid.1H NMR (DMSO-d6)δ2.40 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CH3), 2.496(d, J= 4.0 Hz, 2H, N-CH2), 3.45 (m, 2H, N-CH2), 3.89 (d, J= 4.0 Hz, 2H, CH2CO), 4.88 (s, 2H, benzene-NH2), 6.51 (m, 1H, Ar-H), 6.69 (d, J= 8.0 Hz, 1H, Ar-H), 6.86 (m, 3H, Ar-H), 7.09 (d, J= 8.0 Hz, 1H, Ar-H), 7.64 (t, J= 4.0 Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 8.30 (t, J= 4.0 Hz, 1H, CONH), 9.18 (s, 1H, benzene-NH), 10.89 (s, 1 H, indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 519 (M+1).
Example 25 Preparation of
N-(2-amino-4-fluorophenyl)-2-{3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}propionylamino-acetamide
Figure imgf000030_0001
2-{3-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}propionylamino-acetic acid (428 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-fluoro-o-phenylenediamine (151 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-fluorophenyl)-2-{3-{5-[5-fluoro-2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}propionylamino-acet amide (407 mg, 76% yield) as a brown solid. 1H NMR (DMSO-d6)δ2.39 (s, 3H, pyrrole-CHs), 2.41 (s, 3H, pyrrole-CH3), 2.45 (d, J= 4.0 Hz, 2H, N-CH2), 3.45 (m, 2H, N-CH2), 3.87 (d, J= 4.0 Hz, 2H, CH2CO), 5.21 (s, 2H, benzene-NH2), 6.26 (m, 1H, Ar-H), 6.45 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.82 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.91 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.01 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.65 (t, J= 4.0Hz, 1H, CONH), 7.69 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 8.31 (t, J= 4.0 Hz, 1H, CONH), 9.10 (s, 1H, benzene-NH), 10.89 (s, 1H, indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 537 (M+1).
Example 26
Preparation of 6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino}acetylamino-hexanoic acid methyl ester
Figure imgf000031_0001
2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyljamino-acetic acid (357 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-hexanoic acid methyl ester (415 mg, 86% yield) as a yellow solid.1H NMR (DMSO-d6)δ1.27 (m, 2H, CH2), 1.39 (m, 2H, CH2), 1.52 (m, 2H, CH2), 2.28 (t, J= 8.0 Hz, 2H, CH2CO), 2.45(s, 3H, pyrrole-CH3), 2.47 (s, 3H, pyrrole-CHs), 3.06 (m, 2H, N-CH2), 3.57 (s, 3H, COOCH3), 3.81 (d, J= 4.0 Hz, 2H, N-CH2), 6.83 (dd, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 6.91 (td, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 7.72 (s, 1H, vinyl-H), 7.67 (t, J= 8.0Hz, 1H, CONH), 7.75 (dd, J= 4.0 and 8.0 Hz1 1H, Ar-H), 7.87 (t, J= 4.0Hz, 1H, CONH), 10.92 (s, 1H, indolinone-NH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 485 (M+1).
Example 27
Preparation of 6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-hexanoic acid
Figure imgf000031_0002
6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-hexanoic acid methyl ester (484 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-hexanoic acid (431 mg, 92% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.27 (m, 2H, CH2), 1.40 (m, 2H, CH2), 1.49 (m, 2H, CH2), 2.18 (t, J= 8.0 Hz, 2H1 CH2CO)1 2.44 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.06 (m, 2H, N-CH2), 3.81 (d, J= 4.0 Hz, 2H, N-CH2), 6.83 (dd, J= 4.0 and 8.0 Hz1 1H1 Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.65 (t, J= 4.0Hz, 1H, CONH), 7.72 (s, 1H, vinyl-H), 7.77 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.85 (t, J= 4.0Hz, 1H, CONH), 10.90 (s, 1H, indolinone-NH), 12.01 (s, 1H, COOH), 13.66 (s, 1 H, pyrrole-NH). LC-MS (m/z) 471 (M+1).
Example 28
Preparation of N-hydroxy-6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-hexanamide
Figure imgf000032_0001
6-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-hexanoic acid (470 mg, 1 mmol), triethylamine (151 mg, 1.5 mmol) and 20 ml of DMF were stirred at O0C while ethyl chloroformate (163 mg, 1.5 mmol) was added. The mixture was stirred at O0C for 2 hours, and then 50% aqueous solution of hydroxyiamine (1.32 g, 20 mmol) was added. The mixture was stirred for 3 hours at room temperature, and then diluted with 1000 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-hydroxy-6-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carbonyl}amino}acetylamino-hexanamide (378 mg, 78%) as a brown solid. 1H NMR (DMSO-d6)δ1.23 (m, 2H, CH2), 1.38 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.92 (t, J= 8.0 Hz, 2H, CH2CO), 2.44 (s, 3H, pyrrole-CH3), 2.47 (s, 3H, pyrrole-CH3), 3.06 (m, 2H, N-CH2), 3.81 (d, J= 4.0 Hz, 2H, N-CH2), 6.84 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.72 (s, 1H, vinyl-H), 7.67 (t, J= 4.0Hz, 1H, CONH), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.85 (t, J= 4.0Hz, 1H, CONH), 8.70 (br s, 1H, N-OH), 10.64 (br s, 2H, NH-O and indolinone-NH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 486 (M+1).
Example 29 Preparation of
N-(2-aminophenyl)-6-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-hexanamide
Figure imgf000032_0002
6-{2-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-hexanoic acid (470 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-6-{2-{5-[5- fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino} acetylamino-hexanamide (456 mg, 80% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.32 (m, 2H, CH2), 1.44 (m, 2H, CH2), 1.59 (m, 2H, CH2), 2.30 (t, J= 8.0 Hz, 2H, CH2CO), 2.47 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.09 (m, 2H, N-CH2), 3.81 (d, J= 4.0 Hz, 2H, N-CH2), 4.81 (s, 2H, benzene-NH2), 6.53 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz, 1H, Ar-H), 6.83 (m, 3H, Ar-H), 7.14 (d, J= 8.0 Hz, 1H, Ar-H), 7.67 (t, J= 4.0 Hz, 1H, CONH), 7.72 (s, 1H, vinyl-H), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.88 (t, J= 4.0 Hz, 1H, CONH), 9.10 (s, 1H, benzene-NH), 10.91 (s, 1H, indolinone-NH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 561 (M+1).
Example 30 Preparation of
N-(2-amino-4-fluorophenyl)-6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino}acetylamino-hexanamide
Figure imgf000033_0001
6-{2-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino}acetylamino-hexanoic acid (470 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-fluoro-o-phenylenediamine (151 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-fluorophenyl)-6-{2-{5-[5-fluoro-2-oxo-1 ,2- dihydro-indol^SZJ-ylidenemethyll^^-dimethyl-IH-pyrrole-S-carbony^aminoJacetylamino- hexanamide (416 mg, 72% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.32 (m, 2H, CH2), 1.44 (m, 2H, CH2), 1.58 (m, 2H, CH2), 2.30 (t, J= 8.0 Hz, 2H, CH2CO), 2.47 (s, 3H, pyrrole-CHs), 2.49 (s, 3H, pyrrole-CH3), 3.08 (m, 2H, N-CH2), 3.82 (d, J= 4.0 Hz, 2H, N-CH2), 5.21 (s, 2H, benzene-NH2), 6.26 (m, 1H, Ar-H), 6.45 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.82 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 6.91 (td, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.02 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.65 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.76 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 8.31 (t, J= 4.0 Hz, 1H, CONH), 9.10 (s, 1H, benzene-NH), 10.89 (s, 1H, indolinone-NH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 579 (M+1). Example 31 Preparation of
N-(2-amino-4-chlorophenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000034_0001
δ^S-IS-fluoro^-oxo-i ^-dihydro-indol-CSZJ-ylidenemethyl^.Φdimethyl-IH-pyrrole-S-car- bonyljamino-hexanoic acid (413 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-chloro-o-phenylenediamine (171 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-chlorophenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidene-methyl]-2, 4-dimethyl-1 H-pyrrole-3-carbonyl}-amino-hexanamide (414 mg, 78% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.47 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 4.86 (s, 2H, benzene-NH2), 6.65 (d, J= 8.0 Hz, 1 H, Ar-H), 6.85 (m, 3H, Ar-H), 7.28 (d, J= 8.0 Hz, 1H, Ar-H), 7.65 (t, J= 4.0 Hz, 1 H, CONH), 7.70 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 9.12 (s, 1H, benzene-NH), 10.90 (s, 1H, indoiinone-NH), 13.67 (S1 1H1 pyrrole-NH). LC-MS (m/z) 539 (M+1).
Example 32 Preparation of
N-(2-amino-4-methylphenyl)-6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000034_0002
6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyljamino-hexanoic acid (413 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-methyl-o-phenylenediamine (146 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-methylphenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}- amino-hexanamide (418 mg, 81% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H1 CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.34 (s, 3H, benzene-CH3), 2.46 (s, 3H, pyrrole-CHs), 2.48 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 4.75 (s, 2H, benzene-NH2), 6.55 (m, 2H, Ar-H), 6.82 (dd, 1H, J= 4.0 and 8.0 Hz, Ar-H), 6.92 (td, J= 4.0 and 8.0 Hz, Ar-H), 7.05 (d, J= 8.0 Hz, 1H, Ar-H),7.65 (t, J= 4.0 Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 9.11 (s, 1H, benzene-NH), 10.90 (s, 1H, indo!inone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 518 (M+1).
Example 33 Preparation of
N-(2-amino-4-methoxyphenyl)-6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-yIidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000035_0001
6-{5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-hexanoic acid (413 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-methoxy-o-phenylenediamine (166 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-methoxyphenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino-hexanamide (442 mg, 83% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.49 (s, 3H, pyrrole-CH3), 2.51 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 3.83 (s, 3H, O-CH3), 4.82 (s, 2H, benzene-NH2), 6.16 (d, J= 4.0 Hz, 1H, Ar-H), 6.33 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.83 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.92 (m, 1H, Ar-H), 7.45 (d, J= 8.0 Hz, 1H, Ar-H), 7.65 (t, J= 4.0 Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 9.11 (s, 1H, benzene-NH), 10.90 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 534 (M+1).
Example 34 Preparation of
N-(2-amino-4-trifluoromethylphenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indoI-(3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000036_0001
6-{5-[5-fluoro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-hexanoic acid (413 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-trifluoromethyl-o-phenylenediamine (211 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-amino-4-trifluoromethyl-phenyl)-6-{5-[5-fluoro-2-oxo-1 ,2-di-hydro- indol-(3Z)-yiidenemethyl]-2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanamide (451 mg, 79% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.49 (s, 3H, pyrrole-CH3), 2.52 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 4.75 (s, 2H, benzene-NH2), 6.83 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 6.94 (m, 3H, Ar-H), 7.10 (d, J= 8.0 Hz, 1 H, Ar-H), 7.65 (t, J= 4.0 Hz, 1 H, CONH), 7.70 (s, 1H, vinyl-H), 7.75 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 9.11 (s, 1H, benzene-NH), 10.90 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 572 (M+1).
Example 35
Preparation of 6-{5-[2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester
Figure imgf000036_0002
5-[2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (282 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl- 3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[2-oxo-1 ,2-dihydro- indol-(3Z)-ylidenemethyl]-2,4-dimethyl- 1H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester (303 mg, 74% yield) as a yellow solid.1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.51 (m, 2H, CH2), 1.55 (m, 2H, CH2), 2.31 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.40 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 3.57 (s, 3H, COOCH3), 6.88 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.01 (m, 1H, Ar-H), 7.01 (m, 1H1 Ar-H), 7.63 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.82 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 10.89 (s, 1 H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 410 (M+1).
Example 36
Preparation of 6-{5-[2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrroIe-3-carbonyl}amino-hexanoic acid
Figure imgf000037_0001
6-{5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}- amino-hexanoic acid methyl ester (409 mg, 1 mmol) and 300ml Of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[2-oxo-1 ,2- dihydro-indol-CSZJ-ylidenemethyll^^-dimethyl-IH-pyrrole-S-carbonylJamino-hexanoic acid (354 mg, 90% yield) as a yellow solid.1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.50 (m, 4H, 2χCH2), 2.20 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 6.88 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.01 (m, 1H, Ar-H), 7.01 (m, 1 H, Ar-H), 7.63 (t, J= 4.0Hz, 1 H, CONH), 7.71 (s, 1 H, vinyl-H), 7.82 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 10.89 (s, 1 H, indolinone-NH), 12.06 (s, 1H, COOH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 396 (M+1).
Example 37
Preparation of N-(2-aminophenyl)-6-{5-[2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyi}amino-hexanarnide
Figure imgf000037_0002
6-{5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}- amino-hexanoic acid (395 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-6-{5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H- pyrrole-3-carbonyl}amino-hexanamide (433 mg, 89% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H1 CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.41 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 4.82 (s, 2H, benzene-NH2), 6.52 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz, 1H, Ar-H), 6.89 (m, 2H, Ar-H), 7.01 (m, 2H, Ar-H), 7.16 (m, 2H, Ar-H), 7.65 (t, J= 4.0 Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.82 (d, J= 8.0 Hz, 1H, Ar-H), 9.11 (s, 1H, benzene-NH), 10.90 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 486 (M+1).
Example 38
Preparation of 6-{5-[5-chloro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethylJ- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester
Figure imgf000038_0001
5-[5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- boxylic acid (316 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[5-chloro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester (356 mg, 80% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.51 (m, 2H, CH2), 1.55 (m, 2H, CH2), 2.31 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.40 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 3.57 (s, 3H, COOCH3), 6.87 (d, J= 8.0 Hz, 1H1 Ar-H), 7.18 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.63 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.98 (d, J= 4.0 Hz, 1H, Ar-H), 10.88 (s, 1H, indolinone-NH), 13.66 (s, 1 H, pyrrole-NH). LC-MS (m/z) 444 (M+1).
Example 39
Preparation of 6-{5-[5-chloro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid
Figure imgf000038_0002
6-{5-[5-choro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl}amino-hexanoic acid methyl ester (443 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[5-chloro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole- 3-carbonyl}amino-hexanoic acid (382 mg, 89% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.50 (m, 4H, 2χCH2), 2.20 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CHs), 2.41 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 6.87 (d, J= 8.0 Hz, 1H, Ar-H), 7.18 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.63 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.98 (d, J= 4.0 Hz, 1H, Ar-H), 10.89 (s, 1H, indolinone-NH), 12.00 (s, 1H, COOH), 13.67 (s, 1 H, pyrrole-NH). LC-MS (m/z) 430 (M+1).
Example 40 Preparation of
N-(2-aminophenyl)-6-{5-[5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000039_0001
6-{5-[5-choro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyI-1H-pyrrole-3-car- bonyljamino-hexanoic acid (430 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2- aminophenyl)-6-{5-[5-chloro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H -pyrrole-3-carbonyl}amino-hexanamide (436 mg, 84% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.41 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 4.82 (s, 2H, benzene-NH2), 6.52 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz, 1H, Ar-H), 6.88 (m, 2H, Ar-H), 7.16 (m, 2H, Ar-H), 7.65 (t, J= 4.0 Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.95 (d, J= 4.0 Hz, 1H, Ar-H), 9.11 (s, 1H, benzene-NH), 10.90 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 520 (M+1).
Example 41
Preparation of 6-{5-[4-methyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester
Figure imgf000040_0001
S-^-methyl^-oxo-i^-dihydro-indol^SZJ-ylidenemethyll^^-dimethyl-IH-pyrrole-S-car- boxylic acid (296 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[4-methyl-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester (312 mg, 74% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.51 (m, 2H1 CH2), 1.55 (m, 2H, CH2), 2.31 (d, J= 8.0 Hz, 2H, CH2CO)1 2.39 (s, 3H, pyrrole-CH3), 2.40 (s, 3H, pyrrole-CH3), 2.48 (s, 3H, indolinone-CH3), 3.18 (m, 2H, NCH2), 3.57 (s, 3H, COOCH3), 6.75 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 7.19 (t, J= 8.0 Hz, 1 H1 Ar-H), 7.63 (t, J= 4.0Hz, 1 H1 CONH), 7.70 (s, 1H, vinyl-H), 7.95 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 10.89 (s, 1H1 indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 424 (M+1).
Example 42
Preparation of 6-{5-[4-methyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid
Figure imgf000040_0002
6-{5-[4-methyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino-hexanoic acid methyl ester (423 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[4-methy 1-2-0X0-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole- 3-carbonyl}amino-hexanoic acid (355 mg, 87% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.50 (m, 4H, 2*CH2), 2.20 (d, J= 8.0 Hz, 2H, CH2CO)1 2.39 (s, 3H, pyrrole-CHa), 2.41 (s, 3H, pyrrole-CH3), 2.48 (s, 3H, indolinone-CH3), 3.18 (m, 2H1 NCH2), 6.75 (dd, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 7.19 (t, J= 8.0 Hz, 1H1 Ar-H), 7.63 (t, J= 4.0Hz1 1H, CONH)1 7.70 (s, 1H, vinyl-H), 7.95 (dd, J= 4.0 and 8.0 Hz1 1H1 Ar-H)1 10.89 (s, 1H, indolinone-NH), 12.00 (s, 1 H, COOH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 410 (M+1).
Example 43
Preparation of N-hydroxy-6-{5-[4-methyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000041_0001
6-{5-[4-methyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl} amino-hexanoic acid (409 mg, 1 mmol), triethylamine (151 mg, 1.5 mmol) and 20 ml of DMF were stirred at O0C while ethyl chloroformate (163 mg, 1.5 mmol) was added. The mixture was stirred at O0C for 2 hours, and then 50% aqueous solution of hydroxylamine (1.32 g, 20 mmol) was added. The mixture was stirred for 3 hours at room temperature, and then diluted with 1000 ml_ of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-hydroxy-6-{5-[4-methyl- 2-OXO-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino- hexanamide (364 mg, 86%) as a brown solid. 1H NMR (DMSO-d6)δ1.29 (m, 2H, CH2), 1.50 (m, 4H, 2*CH2), 1.94 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CHs), 2.48 (s, 3H, indolinone-CH3), 3.19 (m, 2H, NCH2), 6.75 (dd, J= 4.0 and 8.0 Hz, 1H1 Ar-H), 7.19 (t, J= 8.0 Hz, 1H, Ar-H), 7.64 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1 H, vinyl-H), 7.95 (dd, J= 4.0 and 8.0 Hz, 1 H, Ar-H), 8.70 (br s, 1 H, N-OH), 10.70 (s, 2H, NH-O and indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 429 (M+1).
Example 44
Preparation of 6-{5-[5-nitro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester
Figure imgf000041_0002
5-[5-nitro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxy- Nc acid (327 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl- 3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[5-nitro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyMH-pyrrole-S-carbonylJamino-hexanoic acid methyl ester (321 mg, 71% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.51 (m, 2H, CH2), 1.55 (m, 2H, CH2), 2.31 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.40 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 3.57 (s, 3H, COOCH3), 7.63 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H1 vinyl-H), 7.91 (d, J= 4.0 Hz, 1H, Ar-H), 8.02 (d, J= 8.0 Hz, 1H, Ar-H), 8.12 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 10.88 (s, 1H, indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 455 (M+1).
Example 45
Preparation of 6-{5-[5-nitro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid
Figure imgf000042_0001
6-{5-[5-nitro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl} amino-hexanoic acid methyl ester (454 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[5-nitro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole-3- carbonyljamino-hexanoic acid (409 mg, 93% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.50 (m, 4H, 2*CH2), 2.20 (d, J= 8.0 Hz1 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 7.63 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.91 (d, J= 4.0 Hz, 1H, Ar-H), 8.02 (d, J= 8.0 Hz, 1H, Ar-H), 8.12 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 10.89 (s, 1H, indolinone-NH), 12.03 (s, 1H, COOH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 441 (M+1).
Example 46 Preparation of
N-(2-aminophenyl)-6-{5-[5-nitro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000042_0002
6-{5-[5-nitro-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- bonyl} amino-hexanoic acid (440 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyI)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-6-{5-[5-nitro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl- 1H-pyrrole-3-carbonyl}amino-hexanamide (434 mg, 82% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.41 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 4.82 (s, 2H, benzene-NH2), 6.52 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz, 1H, Ar-H), 6.89 (m, 3H, Ar-H), 7.16 (d, J= 8.0 Hz, 1 H, Ar-H), 7.65 (t, J= 4.0 Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 7.91 (d, J= 4.0 Hz, 1H, Ar-H), 8.02 (d, J= 8.0 Hz, 1H, Ar-H), 8.12 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 9.11 (s, 1H, benzene-NH), 10.90 (s, 1H, indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 531 (M+1).
Example 47
Preparation of 6-{5-[6-methoxy-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester
Figure imgf000043_0001
5-[6-methoxy-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car- boxylic acid (312 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[6-methoxy-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester (328 mg, 75% yield) as a yellow solid.1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.51 (m, 2H, CH2), 1.55 (m, 2H, CH2), 2.31 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.40 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 3.57 (s, 3H, COOCH3), 3.83 (s, 3H, O-CH3), 6.68 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.25 (d, J= 8.0 Hz, 1H, Ar-H), 7.39 (d, J= 4.0 Hz, 1H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.63 (t, J= 4.0Hz, 1H, CONH), 10.89 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 440 (M+1).
Example 48
Preparation of 6-{5-[6-methoxy-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid
Figure imgf000044_0001
6-{5-[6-methoxy-2-oxo-1,2-dihydro-indoI-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl}amino-hexanoic acid methyl ester (439 mg, 1 mmol) and 300m! of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[6-methoxy-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrro- le-3-carbonyl}amino-hexanoic acid (361 mg, 85% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.50 (m, 4H, 2χCH2), 2.20 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CHs), 2.41 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 3.83 (s, 3H, 0-CH3), 6.68 (dd, J= 4.0 and 8.0 Hz, 1H, Ar-H), 7.25 (d, J= 8.0 Hz, 1H, Ar-H), 7.39 (d, J= 4.0 Hz, 1H, Ar-H), 7.70 (s, 1H, vinyl-H), 7.63 (t, J= 4.0Hz, 1H, CONH), 10.89 (s, 1H, indolinone-NH), 12.01 (s, 1H, COOH), 13.71 (s, 1H, pyrrole-NH). LC-MS (m/z) 426 (M+1).
Example 49 Preparation of
N-(2-aminophenyl)-6-{5-[6-methoxy-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000044_0002
6-{5-[6-methoxy-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carbonyl} amino-hexanoic acid (425 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2- aminophenyl)-6-{5-[6-methoxy-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl- 1H-pyrrole-3-carbonyl}amino-hexanamide (443 mg, 86% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.41 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H, NCH2), 3.83 (s, 3H, 0-CH3), 4.82 (s, 2H, benzene-NH2), 6.52 (m, 1H, Ar-H), 6.69 (m, 2H, Ar-H), 6.89 (m, 3H1 Ar-H), 7.16 (d, J= 8.0 Hz, 1H1 Ar-H), 7.25 (d, J= 8.0 Hz, 1H1 Ar-H), 7.32 (d, J= 4.0 Hz, 1H, Ar-H), 7.65 (t, J= 4.0 Hz1 1H1 CONH), 7.70 (s, 1H, vinyl-H), 9.10 (s, 1H, benzene-NH), 10.89 (s, 1H, indolinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 516 (M+1).
Example 50
Preparation of 6-{5-[6-trifluoromethyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-S-carbonytyamino-hexanoic acid methyl ester
Figure imgf000045_0001
5-[6-trifluoromethyl-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3 -carboxylic acid (350 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 6-aminocaproic acid methyl ester hydrochloride (217.8 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[6-trifluoromethyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanoic acid methyl ester (344 mg, 72% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.51 (m, 2H, CH2), 1.55 (m, 2H, CH2), 2.31 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.40 (s, 3H, pyrrole-CHs), 3.18 (m, 2H, NCH2), 3.57 (s, 3H, COOCH3), 7.30 (m, 2H, Ar-H), 7.63 (t, J= 4.0Hz, 1H, CONH), 7.70 (s, 1H, vinyl-H), 8.07 (d, J= 4.0 Hz1 1H, Ar-H), 10.86 (s, 1H, indoiinone-NH), 13.66 (s, 1H, pyrrole-NH). LC-MS (m/z) 478 (M+1).
Example 51
Preparation of 6-{5-[6-trifluoromethyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-1 H-pyrrole-3-carbonyl}amino-hexanoic acid
Figure imgf000045_0002
6-{5-[6-trifluoromethyl-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrro- le-3-carbonyl}amino-hexanoic acid methyl ester (477 mg, 1 mmol) and 300ml of CH3OH were stirred at room temperature while 25 ml of 4 N solution of LiOH in H2O was added. The mixture was stirred for 24 hours at room temperature. The mixture is neutralized with concentrated hydrochloric acid to pH 7 and evaporated under vacuum to remove methanol. The residue was adjusted to pH 3 with concentrated hydrochloric acid. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give 6-{5-[6-trifluoromethyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H- pyrrole-S-carbonyljamino-hexanoic acid (380 mg, 82% yield) as a yellow solid. 1H NMR (DMSO-d6)δ1.30 (m, 2H, CH2), 1.50 (m, 4H, 2*CH2), 2.20 (d, J= 8.0 Hz, 2H, CH2CO), 2.39 (s, 3H, pyrrole-CH3), 2.41 (s, 3H, pyrrole-CH3), 3.18 (m, 2H, NCH2), 7.30 (m, 2H, Ar-H), 7.63 (t, J= 4.0Hz, 1 H, CONH), 7.70 (s, 1H, vinyl-H), 8.07 (d, J= 4.0 Hz, 1H, Ar-H), 10.89 (s, 1H, indolinone-NH), 12.01 (s, 1H, COOH), 13.69 (s, 1H, pyrrole-NH). LC-MS (m/z) 464 (M+1).
Example 52 Preparation of
N-(2-aminophenyl)-6-{5-[6-trifluoromethyl-2-oxo-1 ,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide
Figure imgf000046_0001
6-{5-[6-trifluoromethyl-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrro- le-3-carbonyl}amino-hexanoic acid (463 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (432 mg, 4 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 mL of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give N-(2-aminophenyl)-6-{5-[6-trifluoromethyl-2-oxo-1,2-dihydro-indol-(3Z)-ylidene- methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}amino-hexanamide (448 mg, 81% yield) as a brown solid. 1H NMR (DMSO-d6)δ1.38 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.32 (d, J= 8.0 Hz, 2H, CH2CO), 2.41 (s, 3H, pyrrole-CH3), 2.49 (s, 3H, pyrrole-CH3), 3.21 (m, 2H1 NCH2), 4.82 (s, 2H, benzene-NH2), 6.52 (m, 1H, Ar-H), 6.70 (d, J= 8.0 Hz, 1 H, Ar-H), 6.89 (m, 3H, Ar-H), 7.16 (d, J= 8.0 Hz, 1H, Ar-H), 7.30 (m, 2H, Ar-H), 7.65 (t, J= 4.0Hz, 1H, CONH), 7.71 (s, 1H, vinyl-H), 8.06 (d, J= 4.0 Hz, 1H, Ar-H), 10.89 (s, 1H, indolinone-NH), 13.67 (s, 1H, pyrrole-NH). LC-MS (m/z) 554 (M+1).
Example 53
In vitro inhibition on enzyme activities of receptor tyrosine kinase c-Kit, PDGFR, and VEGFR and in vivo inhibition on receptor ligand-dependent cell proliferation by the
Compound 3-52
Figure imgf000046_0002
Example ICsonM ICsonM ICsonM (PDGF (VEGF
(compound) (c-kit) (PDGFR) (VEGFR) ligand-dependent cell ligand-dependent cell proliferation) proliferation)
87 100 139 34
168 58
21 14 933 81
154 63
161 72
17 55 178 692 5 2 5 151 71 8 3 5 178 92 91 513 6 MOOOO >10000 17 3 5 912 457 20 5 6 1051 471 5 4 4 447 1000 15 12 14 550 126 18 12 15 651 171 18 2 13 603 688 72 63 18 1202 93 75 82 25 1324 123 31 16 12 15 234 143
32 170 101
33 198 95
34 11 15 10 203 89
37 13 22 15 313 217
40 9 6 13 336 152
43 7 19 54 171 712
46 30 27 45 431 755
49 15 17 25 352 183
52 16 11 22 323 124
Measurement of in vitro inhibition on enzyme activity of receptor tyrosine kinase: PDGFRQ Bioassay:
This assay is used to measure in vitro kinase activity of PDGFR in an ELISA assay. Materials and Reagent:
1. Streptavidin coated-96-well-white plate
2. Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) (Cell Signaling)
3. HRP-labeled anti-mouse IgG (Upstate)
4. HTScan™ Tyrosine Kinase Buffer (4 X)
5. DTT (1000 X. 1.25 M)
6. ATP (IO mM)
7. FLT3 (Tyr589) Biotinylated Peptide Substrate (Cell Signaling)
8. PDGF Receptor Kinase (Cell Signaling)
9. Wash Buffer: 1 X PBS, 0.05% Tween-20 (PBS/T)
10. Bovine Serum Albumin (BSA)
11. Stop Buffer: 50 mM EDTA, pH 8
12. Enhanced chemiluminescence (ECL) (Amersham)
Procedure for performing the assay in 96-well plate:
1. Add 10 μl 10 mM ATP to 1.25 ml 6 μM substrate peptide. Dilute the mixture with dH20 to 2.5 ml to make 2X ATP/substrate cocktail ([ATP]=40 μM, [substrate]=3 μm).
2. Immediately transfer enzyme from -800C to ice. Allow enzyme to thaw on ice.
3. Microcentrifuge briefly at 4°C to bring liquid to the bottom of the vial. Return immediately to ice.
4. Add 10 μl of DTT (1.25 M) to 2.5 ml of 4X HTScan™ Tyrosine Kinase Buffer (240 mM HEPES pH 7.5, 20 mM MgCI2, 20 mM MnCI2, 12 μM Na3VO4) to make DTT/Kinase buffer.
5. Transfer 1.25 ml of DTT/Kinase buffer to enzyme tube to make 4X reaction cocktail ([enzyme]=4 ng/μL in 4X reaction cocktail).
6. Incubate 12.5 μl of the 4X reaction cocktail with 12.5 μl/well of prediluted compound of interest (usually around 10 μM) for 5 minutes at room temperature.
7. Add 25 μl of 2X ATP/substrate cocktail to 25 μl/well preincubated reaction cocktail/compound. Final Assay Conditions for a 50 μl Reaction:
60 mM HEPES pH 7.5
5 mM MgCI2
5 mM MnCI2
3 μM Na3VO4
1.25 mM DTT
20 μMATP
1.5 μM peptide
50 ng PDGF Receptor Kinase
1. Incubate reaction plate at room temperature for 30 minutes.
2. Add 50 μl/well Stop Buffer (50 mM EDTA, pH 8) to stop the reaction.
3. Transfer 25 μl of each reaction and 75 μl dH2O/well to a 96-well streptavidin-coated plate and incubate at room temperature for 60 minutes.
11. Wash three times with 200 μl/well PBS/T
12. Dilute primary antibody, Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100), 1 :1000 in PBS/T with 1% BSA. Add 100 μl/well of primary antibody.
13. Incubate at room temperature for 60 minutes.
14. Wash three times with 200 μl/well PBS/T
15. Dilute HRP labeled anti-mouse IgG 1:500 in PBS/T with 1% BSA. Add 100 μl/well diluted antibody.
16. Incubate at room temperature for 30 minutes.
17. Wash five times with 200 μl/well PBS/T.
18. Add 100 μl/well ECL Solution.
19. Detect luminescence with appropriate Plate Reader.
VEGFR2 Bioassay
This assay is used to measure in vitro kinase activity of VEGFR2 in an ELISA assay.
Materials and Reagent:
1. Streptavidin coated, 96-well, white plate
2. Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) (Cell Signaling)
3. HRP-labeled anti-mouse IgG (Upstate)
4. HTScan™ Tyrosine Kinase Buffer (4 X) 5. DTT (IOOO X. 1.25 M)
6. ATP (IO mM)
7. Gastrin Precursor (Tyr87) Biotinylated Peptide Substrate (Cell Signaling)
8. VEGF Receptor 2 Kinase (recombinant, human) (Cell Signaling)
9. Wash Buffer: 1 X PBS, 0.05% Tween-20 (PBS/T)
10. Bovine Serum Albumin (BSA)
11. Stop Buffer: 50 mM EDTA pH 8
12. Enhanced chemiluminescence (ECL) (Amersham)
Procedure for performing the assay in 96-well plate:
1. Add 10 μl 10 mM ATP to 1.25 ml 6 μM substrate peptide. Dilute the mixture with dH20 to 2.5 ml to make 2X ATP/substrate cocktail ([ATP]=40 μM, [substrate]=3 μm).
2. Immediately transfer enzyme from -800C to ice. Allow enzyme to thaw on ice.
3. Microcentrifuge briefly at 4°C to bring liquid to the bottom of the vial. Return immediately to ice.
4. Add 10 μl of DTT (1.25 M) to 2.5 ml of 4X HTScan™ Tyrosine Kinase Buffer (240 mM HEPES pH 7.5, 20 mM MgCI2, 20 mM MnCI2, 12 μM Na3VO4) to make DTT/Kinase buffer.
5. Transfer 1.25 ml of DTT/Kinase buffer to enzyme tube to make 4X reaction cocktail ([enzyme]=4 ng/μL in 4X reaction cocktail).
6. Incubate 12.5 μl of the 4X reaction cocktail with 12.5 μl/well of prediluted compound of interest (usually around 10 μM) for 5 minutes at room temperature.
7. Add 25 μl of 2X ATP/substrate cocktail to 25 μl/well preincubated reaction cocktail/compound. Final Assay Conditions for a 50 μl Reaction:
60 mM HEPES pH 7.5
5 mM MgCI2
5 mM MnCI2
3 μM Na3VO4
1.25 mM DTT
20 μM ATP
1.5 μM peptide
100 ng VEGFR2 Kinase
8. Incubate reaction plate at room temperature for 30 minutes.
9. Add 50 μl/well Stop Buffer (50 mM EDTA, pH 8) to stop the reaction.
10. Transfer 25 μl of each reaction and 75 μl dH2O/well to a 96-well streptavidincoated plate and incubate at room temperature for 60 minutes.
11. Wash three times with 200 μl/well PBS/T
12. Dilute primary antibody, Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100), 1:1000 in PBS/T with 1% BSA. Add 100 μl/well of primary antibody.
13. Incubate at room temperature for 60 minutes.
14. Wash three times with 200 μl/well PBS/T
15. Dilute HRP labeled anti-mouse IgG 1:500 in PBS/T with 1% BSA. Add 100 μl/well diluted antibody.
16. Incubate at room temperature for 30 minutes. 17. Wash five times with 200 μl/well PBS/T.
18. Add 100 μl/well ECL Solution.
19. Detect luminescence with appropriate Plate Reader.
c-KIT Bioassay
This assay is used to measure in vitro kinase activity of c-KIT in an ELISA assay.
Materials and Reagent:
1. Streptavidin coated, 96-well, white plate
2. Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) (Cell Signaling)
3. HRP-labeled anti-mouse IgG (Upstate)
4. HTScan™ Tyrosine Kinase Buffer (4 X)
5. DTT (1000 X. 1.25 M)
6. ATP (IO mM)
7. KDR (Tyr996) Biotinylated Peptide Substrate (Cell Signaling)
8. c-KIT Kinase (recombinant, human) (Cell Signaling)
9. Wash Buffer: 1 X PBS, 0.05% Tween-20 (PBSAT)
10. Bovine Serum Albumin (BSA)
11. Stop Buffer: 50 mM EDTA pH 8
12. Enhanced chemiluminescence (ECL) (Amersham)
Procedure for performing the assay in 96-well plate:
1. Add 10 μl 10 mM ATP to 1.25 ml 6 μM substrate peptide. Dilute the mixture with dH20 to 2.5 ml to make 2X ATP/substrate cocktail ([ATP]=40 μM, [substrate]=3 μm).
2. Immediately transfer enzyme from -800C to ice. Allow enzyme to thaw on ice.
3. Microcentrifuge briefly at 4°C to bring liquid to the bottom of the vial. Return immediately to ice.
4. Add 10 μl of DTT (1.25 M) to 2.5 ml of 4X HTScan™ Tyrosine Kinase Buffer (240 mM HEPES pH 7.5, 20 mM MgCI2, 20 mM MnCI2, 12 μM Na3VO4) to make DTT/Kinase buffer.
5. Transfer 1.25 ml of DTT/Kinase buffer to enzyme tube to make 4X reaction cocktail ([enzyme]=4 ng/μL in 4X reaction cocktail).
6. Incubate 12.5 μl of the 4X reaction cocktail with 12.5 μl/well of prediluted compound of interest (usually around 10 μM) for 5 minutes at room temperature.
7. Add 25 μl of 2X ATP/substrate cocktail to 25 μl/well preincubated reaction cocktail/compound. Final Assay Conditions for a 50 μl Reaction:
60 mM HEPES pH 7.5 5 mM MgCI2 5 mM MnCI2 3 μM Na3VO4 1.25 mM DTT 20 μM ATP 1.5 μM peptide 100 ng c-KIT Kinase
8. Incubate reaction plate at room temperature for 30 minutes. 9. Add 50 μl/well Stop Buffer (50 mM EDTA, pH 8) to stop the reaction.
10. Transfer 25 μl of each reaction and 75 μl dH2O/well to a 96-well streptavidincoated plate and incubate at room temperature for 60 minutes.
11. Wash three times with 200 μl/well PBS/T
12. Dilute primary antibody, Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100), 1:1000 in PBS/T with 1% BSA. Add 100 μl/well of primary antibody.
13. Incubate at room temperature for 60 minutes.
14. Wash three times with 200 μl/well PBS/T
15. Dilute HRP labeled anti-mouse IgG 1:500 in PBS/T with 1% BSA. Add 100 μl/well diluted antibody.
16. Incubate at room temperature for 30 minutes.
17. Wash five times with 200 μl/well PBS/T.
18. Add 100 μl/well ECL Solution.
19. Detect luminescence with appropriate Plate Reader.
Measurement of in vivo inhibition on receptor ligand-dependent cell proliferation: PDGF dependent cell proliferation:
NIH-3T3 mouse fibroblasts cell line engineered to stably express human PDGFRC was constructed and used to evaluate PDGF dependent cell proliferation. PDGFRDNIH-3T3 cells were plated into 96-well plates at 5,000 per well and incubated with serum-free medium for 24 hours. Compounds and PDGF BB (50ng/ml) were added and incubated for 72 hours in serum-free medium. The effects on proliferation were determined by addition of MTS reagent (Promega) according to the instruction, incubation for 2 hours at 37°C in CO2 incubator, and record the absorbance at 490nm using an ELISA plate reader.
VEGF dependent cell proliferation:
HUVEC cells were plated into 96-well plates at 6,000 per well and incubated with serum-free medium for 2 hours. Compounds and VEGF 165 (50ng/ml) were added and incubated for 72 hours in serum-free medium. The effects on proliferation were determined by addition of MTS reagent (Promega) according to the instruction, incubation for 2 hours at 37°C in CO2 incubator, and record the absorbance at 490nm using an ELISA plate reader.
Example 54
In vitro inhibition of total HDAC enzyme activity, in cell inhibition of HDAC subtype activity, and in cell acetylation of substrates by the Compound 3-52
ACsoμM ACsoμM
ICδoμM ACsoμM ACsoμM ACsoμM
For HDAC For
(total For HDAC2 For HDAC3 For HDAC6
Example Class I HDAC4/5
HHDDAACC ((SSRREE ((GGDDFF1111 (α-tubulin
(compound) ((PP2211 ((MMEEFF22 enzyme reporter reporter acetylation reporter reporter activity) assay) assay) assay) assay) assay) >60 19.95 ia 19.95 ia nd
>60 15.49 15.14 14.79 15.14 ia
>60 13.21 17.53 15.23 13.76 ia >60 12.8 ia ia ia ia
>60 14.74 13.69 13.54 16.75 ia >60 14.92 16.61 14.23 15.73 ia 0.83 8.32 6.46 8.91 5.37 17.78 >60 15.49 12.88 14.45 12.59 nd >60 16.04 12.92 15.9 13.11 nd >60 13.1 ia ia ia nd >60 13.6 nd ia nd nd >60 nd nd nd nd nd >60 ia ia 12.30 ia nd >60 15.14 17.78 15.85 14.13 ia >60 nd nd nd nd nd 0.7 14.79 10.96 15.14 14.45 ia >60 12.30 15.49 12.02 16.22 ia 30 >60 nd nd nd nd ia
31 >60 nd nd nd nd ia
32 >60 nd nd nd nd ia
33 >60 10.23 15.01 9.42 13.78 ia
34 >60 11.74 14.99 15.23 14.70 ia
37 >60 16.34 14.34 15.85 14.51 ia
40 >60 14.41 11.08 15.66 12.12 ia
43 1.14 8.48 7.52 8.16 5.87 16.21
46 >60 16.97 13.04 13.56 11.04 ia
49 >60 17.23 12.91 15.74 13.51 ia
52 >60 16.64 12.97 15.65 16.14 ia nd : not determined ia* : inactive
Measurement of in vitro inhibition of total HDAC enzyme activity:
The in vitro inhibition of total HDAC enzyme was determined by HDAC Fluorimetric Assay/Drug Discovery Kit (BIOMOL) according to manufacture's instruction. 1. Add Assay buffer, diluted trichostatin A or test inhibitor to appropriate wells of the microtiter plate. Following table lists examples of various assay types and the additions required for each test.
Figure imgf000054_0001
2. Add diluted HeLa extract or other HDAC sample to all wells except those that are to be "No Enzyme Controls" (Blank).
3. Allow diluted Fluor de Lys™ Substrate and the samples in the microtiter plate to equilibrate to assay temperature (25°C).
4. Initiate HDAC reactions by adding diluted substrate (25 μl) to each well and mixing thoroughly.
5. Allow HDAC reactions to proceed for desired length of time and then stop them by addition of Fluor de Lys™ Developer (50 μl). Incubate plate at room temperature (25°C) for 10-15 min.
6. Read samples in a microtiter-plate reading fluorimeter capable of excitation at a wavelength in the range 350- 380 nm and detection of emitted light in the range 440- 460 nm.
Measurement of in vivo inhibition of HDAC subtype activity:
HDAC subtype selectivity inhibition assay of tested compounds was carried out by several reporter gene assays experiments. Briefly, HeLa cells were seeded in 96-well plates the day before transfection to give a confluence of 50-80%. Cells were transfected with one of reporter gene plasmids containing a promoter sequences or response elements upstream of a luciferase gene construct using FuGeneθ transfection reagent according to the manufacturer's instructions (Roche). The promoter or response elements including p21-promoter, gdfH -promoter, serum response element (SRE), MEF-binding element were fused upstream to the luciferase gene reporter construct. For normalizing the transfection efficiency, a GFP expression plasmid was cotransfected. Cells were allowed to express protein for 24 hours followed by addition of individual compounds or the vehicle (DMSO). 24 hours later the cells were harvested, and the luciferase assays were performed using the luciferase assay kit according to the manufacturer's instructions (Promega). To normalize the data from the luciferase assays, β-galactosidase activity from transfected cells was measured using a kit (Promega) as instructed by the manufacturer.
Measurement of in vivo acetylation activity on substrates:
Cytoblot assay of acetylation D-tubulin
This assay is used to measure in vivo inhibition of HDAC6 in a cytoblot assay.
Materials and Reagent:
1. 96-well tissue culture white plate
2. A549 cell line
3. Anti-acetyl-tubulin (Upstate)
4. HRP-labeled anti-mouse IgG (Upstate)
5. Fixation solution: 95% ethanol, 5% acetic acid
6. TBS: 0.15M NaCI, 0.02M Tris-CI pH7.4
7. ADB: TBS + 2%BSA + 0.1% Triton X-100
8. Enhanced chemiluminescence (ECL) (Amersham)
Procedure for performing the assay in 96-well plate: 1. A549 cells were seeded at a density of 20000 cells/200μl/well in 96-well white plate and incubated at 37°C for 24 hours.
2. Compounds were added and incubated for 24 hours at 37°C.
3. After incubation with test compounds, culture medium was removed, then add 100μl/well of fixation solution for 5 min.
4. Wells were aspirated and washed twice with ADB.
5. After aspirating, 100μl/well of ADB containing anti-Ac-tubulin (1 :200) were added and incubated for 2 hour at room temperature.
6. Wells were aspirated and washed twice with 150μl ADB.
7. After aspirating, 10Oμl/well of IgG-HRP conjugated antibody (1 :1000) were added and incubated for 2 hour at room temperature.
8. Plates were washed three times with 150μl TBS.
9. Add 50μl/well of ECL mixture, then plates were read on the plate reader.
Cytoblot assay of acetylation lysine
This assay is used to measure the in vivo inhibition of total HDAC activity in a cytoblot assay.
Materials and Reagent:
9. 96-well tissue culture white plate
10. A549 cell line
11. Anti-acetyl- lysine (Upstate)
12. HRP-labeled anti-mouse IgG (Upstate)
13. Fixation solution: 95% ethanol, 5% acetic acid
14. TBS: 0.15M NaCI, 0.02M Tris-CI pH7.4
15. ADB: TBS + 2%BSA + 0.1% Triton X-100
16. Enhanced chemiluminescence (ECL) (Amersham)
Procedure for performing the assay in 96-well plate:
10. A549 cells were seeded at a density of 20000 cells/200μl/well in 96-well white plate and incubated at 37°C for 24 hours.
11. Compounds were added and incubated for 24 hours at 37°C.
12. After incubation with test compounds, culture medium was removed, then add 100μl/well of fixation solution for 5 min.
13. Wells were aspirated and washed twice with ADB.
14. After aspirating, 100μl/well of ADB containing anti-Ac-lysine (1 :200) were added and incubated for 2 hour at room temperature.
15. Wells were aspirated and washed twice with 150μl ADB.
16. After aspirating, 100μl/well of IgG-HRP conjugated antibody (1 :1000) were added and incubated for 2 hour at room temperature.
17. Plates were washed three times with 150μl TBS.
18. Add 50μl/well of ECL mixture, then plates were read on the analyst plate reader. Example 55 In vivo anti-cell proliferation by the Compound 3-52
Example GIsoμM GI5oμM GIsoμM GIsoμM GIsoμM GI5oμM
(compound) (MBA-MD- (Bel-7402) (A549) (HeLa) (HL60) (MCF7)
3 >100 >100 31.62 18.20 10.00 69.18
4 >100 10.45 >100 >100 >100 >100
5 >100 12.34 >100 >100 >100 >100
8 >100 >100 >100 >100 85.11 >100
9 >100 >100 >100 >100 >100 >100
10 >100 >100 >100 >100 >100 >100
13 6.03 14.79 >100 12.30 2.88 >100
14 >100 >100 >100 >100 <1 >100
15 >100 >100 >100 >100 <1 >100
18 33.88 19.95 >100 >100 >100 >100
19 >100 >100 >100 >100 >100 >100
20 >100 >100 >100 >100 >100 >100
23 >100 >100 >100 >100 2.19 >100
24 >100 >100 >100 >100 >100 >100
25 >100 >100 >100 >100 >100 >100 28 19.95 27.54 48.98 51.29 22.91 38.02
29 >100 >100 >100 >100 5.75 >100
30 >100 >100 >100 >100 4.32 >100
31 >100 >100 >100 >100 <1 >100
32 >100 >100 >100 >100 <1 >100
33 >100 >100 >100 >100 <1 >100
34 >100 >100 >100 >100 <1 >100
37 >100 >100 >100 >100 <1 >100
40 >100 >100 >100 >100 <1 >100
43 8.45 17.32 >100 13.33 1.19 >100
46 >100 >100 >100 >100 <1 >100
49 >100 >100 >100 >100 <1 >100
52 >100 >100 >100 >100 <1 >100
Measurement of in vivo cell proliferation:
Tumor cells were trypsinized and plated into 96-well plates at 3,000 per well and incubated in complete medium with 10% FBS for 24 hours. Compounds were added over a final concentration range of 100 μmol/L to 100 nmol/L in 0.1% DMSO and incubated for 72 hours in complete medium. The effects on proliferation were determined by addition of MTS reagent (Promega) according to the instruction, incubation for 2 hours at 37°C in CO2 incubator, and record the absorbance at 490nm using an ELISA plate reader. Example 56
In vitro inhibition of HDAC subtypes activity, in cell acetylation of histone H3 and α-tubulin by the Compound 13,14 and 29.
Figure imgf000059_0001
Detection of acetylated histone H 3 by Western blot:
About 2>«106 HeLa cells were cultured in each 10-cm-diameter tissue culture plate in DMEM medium supplemented with 10% calf serum. After cultured for 24 h, the cells were treated with CS055 and SAHA as a positive control and the same volume of DMSO as a negative control respectively for 24 h. The treated cells were harvested and histones of cultured cells were extracted according to standard procedure. Protein was quantitated using a protein assay kit (plusOne 2-D quant kit, Amersham Biosciences). About 10 μg of extracted cellular histones were separated by SDS-PAGE on a 12% separating gel and electrophoretically transferred to PVDF membrane. After the membrane was blocked to prevent non-specific binding, the total histone H3 proteins were identified by using anti-histone H3 antibody (Upstate,1:1000) as primary antibody and the goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Upstate, 1:2000) as secondary antibody. The bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak). After the membrane was stripped of bounded antibodies and blocked to prevent non-specific binding, the acetylated histone H3 proteins were detected by using anti-acetyl-histone H3 antibody (Upstate, 1 :50000) as primary antibody and the goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (Upstate, 1 :2000) as secondary antibody. The bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak).
Detection of acetylated α-Tubulin by Western blot:
About 3*105 HeLa cells were cultured in each well of 6-well plate in DMEM medium supplemented with 10% calf serum. After cultured for 24 h, the cells were treated with CS055 and SAHA as a positive control and the same volume of DMSO as a negative control respectively for 24 h. The treated cells were washed once with PBS and treated with 100 μl of M-PER mammalian protein extraction reagent (PIERCE). The lysate was collected and the protein was quantitated using a protein assay kit (plusOne 2-D quant kit, Amersham Biosciences). About 10 μg of the lysate was separated by SDS-PAGE on a 10% separating gel and electrophoretically transferred to PVDF membrane. After the membrane was blocked to prevent non-specific binding, the total α-Tubulin proteins were identified by using anti-α-Tubulin antibody (Santa Cruz, 1:500) as primary antibody and the goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Upstate, 1 :2000) as secondary antibody. The bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak). After the membrane was stripped of bounded antibodies and blocked to prevent non-specific binding, the acetylated α-Tubulin were detected by using anti-acetyl-α-Tubulin antibody (Santa Cruz, 1:1000) as primary antibody and the goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Upstate, 1:2000) as secondary antibody. The bands were detected with ECL Western blotting detection reagents (Amersham Bioscience) and exposed to a film (Kodak).
Measurement of in vitro inhibition of HDAC subtypes enzyme activity:
The in vitro inhibition of HDAC subtypes were determined by HDAC Fluorimetric Assay/Drug Discovery Kit (BIOMOL) according to manufacture's instruction.
1. Add Assay buffer, diluted trichostatin A or test inhibitor to appropriate wells of the microtiter plate. Following table lists examples of various assay types and the additions required for each test.
Figure imgf000060_0001
2. Add diluted HDAC subtype samples to all wells except those that are to be "No Enzyme Controls" (Blank).
3. Allow diluted Fluor de Lys™ Substrate and the samples in the microtiter plate to equilibrate to assay temperature (25°C).
4. Initiate HDAC reactions by adding diluted substrate (25 μl) to each well and mixing thoroughly.
5. Allow HDAC reactions to proceed for desired length of time and then stop them by addition of Fluor de Lys™ Developer (50 μl). Incubate plate at room temperature (25°C) for 10-15 min.
6. Read samples in a microtiter-plate reading fluorimeter capable of excitation at a wavelength in the range 350- 380 nm and detection of emitted light in the range 440- 460 nm.
Example 57
HCT-8 Human Colon Tumor Xenograft Experiment on Female NCr-nu/nu Mice Treated with Compound 13, 14 and 4. Effect of HDACi on HCT-8 tumor growth(Vt-V0)/V0*100
-Blank ~*-4 -±- 13 -*- 14
Figure imgf000061_0001
8 10 12 14 16 18 20 22 24 26 28
Days after beginning of treatment
Tumor Xenograft Experiments:
Female NCr-nu/nu mice were used for all studies. Tumors of HCT-8 model were established and maintained through in vivo passage of s.c. fragments (3x3 mm) implanted in the flank using a 12-gauge trocar. A new generation of the passage was initiated every three weeks, and studies were conducted between generations 3 and 12 of this line. Tumor bearing animal was randomized for grouping and treatment was initiated when tumor size exceeds 6 mm. The mice were divided into 4 groups and each contains 8. Treatment for all but the compound 13 was administered orally once daily at 80mg/kg body weight for the duration indicated in each experiment. The compound 13 was given by IP once daily at 40mg/kg body weight for the duration indicated in each experiment.
Example 58
MCF-7 Human Breast Cancer Xenograft Experiments on Female NCr-nu/nu Mice
Treated by Compound 13 and 14.
Effect ofHDACi on MCF-7 tumor growth
Figure imgf000062_0001
Contorl 14-120 14-240 13-30 13-60 Groups
*:P<0.05
Tumor Xenograft Experiments:
Female NCr-nu/nu mice were used for all studies Tumors of MCF-7 model were established and maintained through in vivo passage of s.c. fragments (3x3 mm) implanted in the flank using a 12-gauge trocar. A new generation of the passage was initiated every three weeks, and studies were conducted between generations 3 and 12 of this line. Tumor bearing animal was randomized for grouping and treatment was initiated when tumor size exceeds 6 mm. The mice were divided into 5 groups and each contains 8. The compound 14 was administered orally once daily at 120mg/kg (14-120) or 240mg/kg (14-240) body weight for the duration indicated in each experiment. The compound 13 was given by IP once daily at 30mg/kg (13-30) or 60mg/kg (13-60) body weight for the duration indicated in each experiment. Tumor weight was weighted at the day after the last treatment.

Claims

What is claimed is:
1. An isolated compound of formula I:
Figure imgf000063_0001
(I) or its stereoisomer, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof, wherein:
X is a valence bond or -C(O)-NH-(CH2)m-;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
R5 is -NHOH or
Figure imgf000063_0002
R6, R7, R8 and R9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; n is an integer ranging from 1 to 5; m is an integer ranging from 1 to 5.
2. A compound of claim 1 , wherein X is a valence bond;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl; R5 Js -NHOH; n is 4 or 5.
3. A compound of claim 1 , wherein X is a valence bond;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl; R5 is
Figure imgf000064_0001
R6, R7, R8 and R9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; n is 4 or 5.
4. A compound of claim 1 , wherein X is -C(O)-NH-(CH2)(T,-;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl; R5 is -NHOH; n is 3, 4 or 5; m is 1.
5. A compound of claim 1 , wherein X is -C(O)-NH-(CH2)m-;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
R5 is
Figure imgf000064_0002
R6, R7, R8 and R9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; n is 3, 4 or 5; m is 1.
6. A process for the preparation of a compound of formula I
Figure imgf000064_0003
(I) or its stereoisomer, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof, wherein
X is a valence bond or -C(O)-N H-(CH2)m-;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
R5 is -NHOH or
Figure imgf000065_0001
R6, R7, R8 and R9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; n is an integer ranging from 1 to 5; m is an integer ranging from 1 to 5; a stereoisomer, enantiomer, diastereomer, or pharmaceutically acceptable salts thereof comprising the steps of:
(a) condensing compound 1 with compound 2 to give compound 3;
Figure imgf000065_0002
(b) hydrolyzing compound 3 with lithium hydroxide to give compound 4;
Figure imgf000065_0003
(c) condensing compound 4 with hydroxylamine to give compound 5a;
NHOH
Figure imgf000065_0004
(d) condensing compound 4 with compound 6 to give compound 5b;
Figure imgf000066_0001
7. The process of claim 6, wherein the condensation reactions of steps (a) and (d) are conducted by using a peptide condensing agent.
8. The process of claim 7, wherein said peptide condensing agent is 1-Ethyl-3-(3-dimethyl- aminopropyl)carbodiimide, dicyclohexylcarbodiimide, or N,N'-carbonyldiimidazole.
9. The process of claim 6, wherein the condensation reaction of step (c) is conducted by using CICOOEt as a condensing agent.
10. The process of claim 6, wherein the hydrolysis reaction of step (b) is conducted by using a hydrolysis agent.
11. The process of claim 10, wherein said hydrolysis agent is lithium hydroxide, sodium hydroxide, or potassium hydroxide.
12. A pharmaceutical composition useful as a therapeutic and/or improving agent for diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities comprising an effective amount of a compound according to claim 1 and at least one pharmaceutically acceptable excipient, carrier or diluent.
13. The pharmaceutical composition of claim 12, wherein said disease associated with abnormal protein kinase activities or abnormal histone deacetylase activities is selected from the group consisting essentially of inflammatory diseases, autoimmune diseases, cancer, neurological and neurodegenerative diseases, cardiovascular diseases, allergies and asthma or hormone-related disease.
14. A dosage form unit of the pharmaceutical composition of claim 12 comprising an amount within the range of about 0.0001 to about 200 mg of said compound.
15. A pharmaceutical composition of claim 12 for administration by the oral, nasal, transdermal, pulmonary, or parenteral route.
16. An isolated compound of formula II:
A-Y-B (H) or its stereoisomer, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof, wherein: A is
Figure imgf000067_0001
B is
Figure imgf000067_0002
Y is a moiety having -CH2-, -CO-, -CS-, -SO- or -SO2-, which is linear with a length of 4.0 to 12.0 A and links A and B;
R1, R2, R3 and R4 are independently hydrogen, halo, alkyl, alkoxy, nitro or trifluoromethyl;
R6, R7, R8 and R9 are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl;
R10, R11, R12, R13, R14 and R15 are independently hydrogen or alkyl.
17. A pharmaceutical composition useful as a therapeutic and/or improving agent for diseases associated with abnormal protein kinase activities or abnormal histone deacetylase activities comprising an effective amount of a compound according to claim 16 and at least one pharmaceutically acceptable excipient, carrier or diluent.
18. The pharmaceutical composition of claim 17, wherein said disease associated with abnormal protein kinase activities or abnormal histone deacetylase activities is selected from the group consisting essentially of inflammatory diseases, autoimmune diseases, cancer, neurological and neurodegenerative diseases, cardiovascular diseases, allergies and asthma or hormone-related disease.
19. A dosage form unit of the pharmaceutical composition of claim 17 comprising an amount within the range of about 0.0001 to about 200 mg of said compound.
20. A pharmaceutical composition of claim 17 for administration by the oral, nasal, transdermal, pulmonary, or parenteral route.
PCT/US2008/070075 2007-07-24 2008-07-15 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinone derivatives as multi-target protein kinase inhibitors and histone deacetylase inhibitors WO2009014941A1 (en)

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