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1.
Bioorg Med Chem Lett ; 22(12): 4033-7, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22607669

ABSTRACT

This Letter reports the optimization of a pyrrolopyrimidine series as dual inhibitors of Aurora A/B kinases. This series derived from a pyrazolopyrimidine series previously reported as inhibitors of aurora kinases and CDKs. In an effort to improve the selectivity of this chemotype, we switched to the pyrrolopyrimidine core which allowed functionalization on C-2. In addition, the modeling rationale was based on superimposing the structures of Aurora-A kinase and CDK2 which revealed enough differences leading to a path for selectivity improvement. The synthesis of the new series of pyrrolopyrimidine analogs relied on the development of a different route for the two key intermediates 7 and 19 which led to analogs with both tunable activity against CDK1 and maintained cell potency.


Subject(s)
Antineoplastic Agents/chemical synthesis , CDC2 Protein Kinase/chemistry , Cyclin-Dependent Kinase 2/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrroles/chemical synthesis , Antineoplastic Agents/pharmacology , Aurora Kinases , Binding Sites , Cell Cycle Checkpoints/drug effects , Cell Line , Drug Design , Humans , Models, Molecular , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/chemistry , Pyrimidines/pharmacology , Pyrroles/pharmacology , Structural Homology, Protein , Structure-Activity Relationship
2.
Bioorg Med Chem Lett ; 22(5): 2070-4, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22326168

ABSTRACT

Since the early 2000s, the Aurora kinases have become major targets of oncology drug discovery particularly Aurora-A and Aurora-B kinases (AKA/AKB) for which the selective inhibition in cells lead to different phenotypes. In addition to targeting these Aurora kinases involved in mitosis, CDK1 has been added as a primary inhibition target in hopes of enhancing the cytotoxicity of our chemotypes harboring the pyrazolopyrimidine core. SAR optimization of this series using the AKA, AKB and CDK1 biochemical assays led to the discovery of the compound 7h which combines strong potency against the 3 kinases with an acceptable microsomal stability. Finally, switching from a primary amide to a two-substituted pyrrolidine amide gave rise to compound 15a which exhibited the desired AKA/CDK1 inhibition phenotype in cells but showed moderate activity in animal models using HCT116 tumor cell lines.


Subject(s)
CDC2 Protein Kinase/antagonists & inhibitors , Colonic Neoplasms/drug therapy , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Aurora Kinase A , Aurora Kinase B , Aurora Kinases , CDC2 Protein Kinase/metabolism , Cell Line , Colon/drug effects , Colon/pathology , Colonic Neoplasms/pathology , HCT116 Cells , Humans , Mice , Models, Molecular , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Rats , Structure-Activity Relationship
3.
Cancer Lett ; 274(2): 243-9, 2009 Feb 18.
Article in English | MEDLINE | ID: mdl-19008038

ABSTRACT

Oncogene addiction due to Myc deregulation has been identified in a variety of tumor types. In order to identify pharmacological agents that cause selective apoptosis in tumors with deregulated Myc expression, we designed a cell-based screening assay based on our Anti-cancer Screening Apoptosis Program (ASAP) technology targeting increased activity in a "Myc-addicted" cancer cell panel. We have identified a novel set of substituted 4-aryl-3-(3-aryl-1-oxo-2-propenyl)-2(1H)-quinolinones that activates apoptosis in cancer cell lines with deregulated Myc, but show low activity in cell lines where Myc is not deregulated. Apoptosis induced by these compounds is rapid, and is associated with a significant downregulation of Myc protein. Selective knockdown of Myc levels in these cells by RNA interference increased sensitivity to apoptosis with compound treatment. By targeting the Myc pathway in Myc-addicted cancer cells, we have identified a novel class of apoptotic inducers that selectively and efficiently target cancer cells with deregulated Myc.


Subject(s)
Apoptosis/drug effects , Genes, myc , Quinolones/pharmacology , Caspases/metabolism , Cell Cycle , Cell Line, Tumor , Down-Regulation , Enzyme Activation , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Polymerase Chain Reaction , RNA Interference , RNA, Small Interfering , Ubiquitination
4.
Bioorg Med Chem ; 16(1): 222-31, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-17950608

ABSTRACT

We report the discovery and SAR study of a series of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as potent inducers of apoptosis. N-(3-Acetylphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine (2) was discovered through our cell- and caspase-based HTS assays as an inducer of apoptosis. Compound 2 is active against cancer cells derived from several human solid tumors, with EC(50) values ranging from 400 to 700 nM. SAR study of hit 2 led to the discovery of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a novel series of potent apoptosis inducers, with 1,3-dimethyl-N-(4-propionylphenyl)-1H-pyrazolo[3,4-b]quinolin-amine (6b) having EC(50) values ranging from 30 to 70 nM in cancer cells. These compounds also demonstrated potent activity in the cell growth inhibition assay, with GI(50) values of 16-42 nM for compound 6b.


Subject(s)
Antineoplastic Agents/chemistry , Apoptosis/drug effects , Quinolines/chemistry , Quinolines/pharmacology , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Structure-Activity Relationship , Tumor Cells, Cultured
5.
Bioorg Med Chem ; 14(23): 7761-73, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-16919962

ABSTRACT

A series of 4-anilino-2-(2-pyridyl)pyrimidines has been discovered as a new class of potent inducers of apoptosis using a cell-based HTS assay. Compound 5a was found to arrest T47D cells in G2/M and induced apoptosis. SAR studies showed that a small and electron-donating group at the meta-position of the anilino ring is important for activity. A 20-fold increase in potency, from hit compound 4-(3-methoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5a) to lead compound 4-(2,5-dimethoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5l), was obtained through the SAR studies. Compound 5l is highly active with an EC50 value of 18 nM in the caspase activation assay in T47D breast cells. Interestingly, 5a and other meta-mono-substituted compounds were active against T47D cells but were not active against H1299 and HT29 cells, while 5l and other 2,5-disubstituted compounds were active against all the three cells. In a tubulin polymerization assay, compound 5l inhibited tubulin polymerization with an IC50 value of < 0.5 microM, while 5a was not active up to 50 microM.


Subject(s)
Apoptosis/drug effects , Breast Neoplasms/drug therapy , Drug Screening Assays, Antitumor/methods , Pyrimidines/pharmacology , Aniline Compounds/chemistry , Aniline Compounds/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Caspases/drug effects , Caspases/metabolism , Cell Line, Tumor , Female , Humans , Inhibitory Concentration 50 , Pyrimidines/chemistry , Structure-Activity Relationship , Tubulin/drug effects , Tubulin/metabolism
6.
Cancer Res ; 64(22): 8208-12, 2004 Nov 15.
Article in English | MEDLINE | ID: mdl-15548686

ABSTRACT

HeLaHF cells are transformation revertants of cervical cancer HeLa cells and have lost anchorage-independent growth potential and tumorigenicity. Activation of tumor suppressor(s) was implicated previously in this transformation reversion. In this study, expression profiling analysis was carried out to identify potential oncogenes that are down-regulated in HeLaHF cells. We found that all three members of the NR4A1/Nur77/NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) were down-regulated in the HeLaHF revertant. Small interfering RNA-mediated down-regulation of NR4A2 in HeLa cells, either transiently or stably, resulted in reduced anchorage-independent growth that was largely attributable to increased anoikis. Furthermore, down-regulation of NR4A2 as well as NR4A1 promoted intrinsic apoptosis. These phenotypes were also observed in several other experimental cancer cells, suggesting the observed apoptosis suppression is a more general property of NR4A2 and NR4A1. These phenotypes also suggest that the Nur77/NGFIB subfamily of orphan receptors exhibit certain oncogenic functionalities with regards to cell proliferation and apoptosis and could therefore be evaluated as potential cancer therapeutic targets.


Subject(s)
Apoptosis/physiology , Cell Transformation, Neoplastic , DNA-Binding Proteins/physiology , Transcription Factors/physiology , Down-Regulation , HeLa Cells , Humans , Nuclear Receptor Subfamily 4, Group A, Member 2 , RNA, Small Interfering
7.
Biotechniques ; 36(5): 826-8, 830, 832-3, 2004 May.
Article in English | MEDLINE | ID: mdl-15152603

ABSTRACT

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.


Subject(s)
Cell Count/methods , Cell Culture Techniques/methods , Cell Division , Drug Delivery Systems/methods , Neoplasms/pathology , Robotics/methods , Agar , Cell Culture Techniques/instrumentation , Cell Differentiation , Drug Delivery Systems/instrumentation , HeLa Cells , Humans , Robotics/instrumentation
8.
Oncogene ; 22(12): 1783-94, 2003 Mar 27.
Article in English | MEDLINE | ID: mdl-12660814

ABSTRACT

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M(+)HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M(+)HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M(+)HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.


Subject(s)
Cell Adhesion Molecules/metabolism , Neoplasm Proteins/metabolism , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antigens, CD , Antigens, Neoplasm , Base Sequence , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/immunology , DNA Primers , Humans , Immunohistochemistry , Molecular Sequence Data , Neoplasm Metastasis , Neoplasm Proteins/chemistry , Neoplasm Proteins/immunology , Phosphorylation , Subtraction Technique , Tumor Cells, Cultured
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