Methyl 3-((6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (GN39482) as a tubulin polymerization inhibitor identified by MorphoBase and ChemProteoBase profiling methods.

A series of indenopyrazoles was synthesized from the corresponding indanones and phenyl isothiocyanates in two steps. Among the compounds synthesized, methyl 3-((6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate 6m (GN39482) was found to possess a promising antiproliferative activity toward human cancer cells without affecting any antimicrobial and antimalarial activities at 100 nM. Both a methoxy group at R(1) position and a methoxycarbonyl group at R(2) position of the anilinoquinazoline framework are essential for the high cell growth inhibition. Both MorphoBase and ChemProteoBase profiling analyses suggested that compound 6m was classified as a tubulin inhibitor. Indeed, compound 6m inhibited the acetylated tubulin accumulation and the microtubule formation and induced G2/M cell cycle arrest in HeLa cells, revealing that a promising antiproliferative activity of compound 6m toward human cancer cells is probably caused by the tubulin polymerization inhibition.

ortho-Carboranylphenoxyacetanilides as inhibitors of hypoxia-inducible factor (HIF)-1 transcriptional activity and heat shock protein (HSP) 60 chaperon activity.

ortho-Carboranylphenoxy derivatives were synthesized and evaluated for their ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter gene assay. Among the compounds synthesized, compound 1d showed the most significant inhibition of hypoxia-induced HIF-1 transcriptional activity with the IC50 of 0.53µM. Furthermore, compound 1h was found to possess the most significant inhibition of heat shock protein (HSP) 60 chaperon activity among the reported inhibitors: the IC50 toward the porcine heart malate dehydrogenase (MDH) refolding assay was 0.35µM.

Synthesis and biological evaluation of diaryl-substituted carboranes as inhibitors of hypoxia inducible factor (HIF)-1 transcriptional activity.

Diaromatic-substituted ortho- and meta-carboranes were synthesized as mimics of manassantin A. Among the carboranes synthesized, compounds 1 and 2 showed significant inhibition of hypoxia-induced HIF-1 transcriptional activity, with IC(50) values of 3.2 and 2.2 µM, respectively. Compounds 1 and 2 similarly suppressed hypoxia-induced HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of HIF-1α mRNA. The hypoxia-induced accumulation and translocation of HIF-1α into nuclei were not observed in HeLa cells treated with compounds 1 and 2 by immunofluorescence analysis, revealing that the inhibition of hypoxia-induced HIF-1 transcriptional activity is induced by compounds 1 and 2 through a degradation pathway of the HIF-1α protein under hypoxic conditions.

Development of hypoxia-inducible factor (HIF)-1α inhibitors: effect of ortho-carborane substituents on HIF transcriptional activity under hypoxia.

A series of substituted ortho-carboranylphenoxyacetanilides were synthesized and evaluated for their ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter assay in HeLa cells expressing the HRE-dependent firefly luciferase reporter construct (HRE-Luc) and constitutively expressing CMV-driven Renilla luciferase reporter, and their ability to inhibit cell growth (GI(50)) using the MTT assay. Among the compounds synthesized, 1g and 1l showed significant inhibition of hypoxia-induced HIF-1 transcriptional activity (IC(50): 1.9 ± 0.4 and 1.4 ± 0.2 µM, respectively). Both compounds suppressed HIF-1α accumulation in a concentration-dependent manner. The porcine heart malate dehydrogenase (MDH) refolding assay revealed that compound 1l inhibited human Hsp60 chaperone activity (IC(50): 6.80 ± 0.25 µM) and this inhibition activity was higher than that of ETB (IC(50): 10.9 ± 0.63 µM).

HSP60 as a drug target.

Heat shock proteins (HSPs) are ubiquitous and evolutionary conserved proteins induced by cell stress. HSP60, in particular, is a typical mitochondrial molecular chaperone that is known to assist nascent polypeptides to reach a native conformation. HSP60 is also known to interact with HSP10. In the last decade, HSP60 has been detected in the cytosol, the cell surface, the extracellular space, and biological fluids. HSP60 elicits potent proinflammatory response in cells of the innate immune system and serves as a danger signal of stressed or damaged cells. As cytosolic HSP60 levels gradually increase or decrease during carcinogenesis in various organs, HSP60 can be used as a biomarker for the diagnosis and prognosis of preneoplastic and neoplastic lesions. In this review, we summarize recent discoveries on the important roles of HSP60 in various diseases ranging from autoimmune diseases to tumors. Furthermore, small molecules targeting HSP60, which were the target of intensive investigations in the last few years, are also summarized. The possibility of utilizing HSP60 as a new drug target for the treatment of certain diseases is examined.

Discovery of Indenopyrazoles as a New Class of Hypoxia Inducible Factor (HIF)-1 Inhibitors.

The indenopyrazole framework was investigated as a new class of HIF-1α inhibitors. Indenopyrazole 2l was found to most strongly inhibit the hypoxia-induced HIF-1α transcriptional activity (IC50 = 0.014 µM) among all of the known compounds having relatively simple structures, unlike manassantins. Indenopyrazole 2l suppressed HIF-1α transcriptional activity without affecting both HIF-1α protein accumulation and HIF-1α/HIF-1ß heterodimerization in nuclei under the hypoxic conditions, suggesting that 2l probably affected the transcriptional pathway induced by the HIF-1α/HIF-1ß heterodimer.

Identification of HSP60 as a primary target of o-carboranylphenoxyacetanilide, an HIF-1alpha inhibitor.

We succeeded in the design and synthesis of multifunctional chemical probes of the HIF-1alpha inhibitor carboranylphenoxyacetanilide (1) that combine photoaffinity labeling and click reaction to identify the target protein. HSP60 was identified as a primary target protein of 1 using the chemical probes 2 and 3. Furthermore, HSP60 inhibitor 4 suppressed hypoxia-induced HIF activation, indicating that HSP60 affects HIF-1alpha accumulation directly or indirectly.

Boron-containing phenoxyacetanilide derivatives as hypoxia-inducible factor (HIF)-1alpha inhibitors.

A series of boron-containing phenoxyacetanilide derivatives 8a-f, 9a-f, 15, and 16 were synthesized as hypoxia-inducible factor (HIF)-1alpha inhibitors. Among the compounds synthesized, carboranylphenoxyacetanilide 16 (GN26361) was found to be a potent inhibitor against HIF-1alpha accumulation under hypoxic conditions and inhibited the hypoxia-induced HIF-1 transcriptional activity in HeLa cells (IC50=0.74 microM). Compound 16 suppressed hypoxia-induced HIF-1alpha accumulation and vascular endothelial growth factor mRNA expression in a concentration-dependent manner without affecting the expression of HIF-1alpha mRNA.