Novel pyrrolopyrimidines as Mps1/TTK kinase inhibitors for breast cancer.

New targeted therapy approaches for certain subtypes of breast cancer, such as triple-negative breast cancers and other aggressive phenotypes, are desired. High levels of the mitotic checkpoint kinase Mps1/TTK have correlated with high histologic grade in breast cancer, suggesting a potential new therapeutic target for aggressive breast cancers (BC). Novel small molecules targeting Mps1 were designed by computer assisted docking analyses, and several candidate compounds were synthesized. These compounds were evaluated in anti-proliferative assays of a panel of 15 breast cancer cell lines and further examined for their ability to inhibit a variety of Mps1-dependent biological functions. The results indicate that the lead compounds have strong anti-proliferative potential through Mps1/TTK inhibition in both basal and luminal BC cell lines, exhibiting IC50 values ranging from 0.05 to 1.0µM. In addition, the lead compounds 1 and 13 inhibit Mps1 kinase enzymatic activity with IC50 values from 0.356µM to 0.809µM, and inhibited Mps1-associated cellular functions such as centrosome duplication and the spindle checkpoint in triple negative breast cancer cells. The most promising analog, compound 13, significantly decreased tumor growth in nude mice containing Cal-51 triple negative breast cancer cell xenografts. Using drug discovery technologies, computational modeling, medicinal chemistry, cell culture and in vivo assays, novel small molecule Mps1/TTK inhibitors have been identified as potential targeted therapies for breast cancers.

Synthesis of N3-substituted carboranyl thymidine bioconjugates and their evaluation as substrates of recombinant human thymidine kinase 1.

Four different libraries of overall twenty three N3-substituted thymidine (dThd) analogues, including eleven 3-carboranyl thymidine analogues (3CTAs), were synthesized. The latter are potential agents for Boron Neutron Capture Therapy (BNCT) of cancer. Linker between the dThd scaffold and the m-carborane cluster at the N3-position of the 3CTAs contained amidinyl-(3e and 3f), guanidyl-(7e-7g), tetrazolylmethyl-(9b1/2-9d1/2), or tetrazolyl groups (11b1/2-11d1/2) to improve human thymidine kinase 1 (hTK1) substrate characteristics and water solubilities compared with 1st generation 3CTAs, such as N5 and N5-2OH. The amidinyl- and guanidyl-type N3-substitued dThd analogues (3a-3f and 7a-7g) had hTK1 phosphorylation rates of <30% relative to that of dThd, the endogenous hTK1 substrate, whereas the tetrazolyl-type N3-substitued dThd analogues (9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2) had relative phosphorylation rates (rPRs) of >40%. Compounds 9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2 were subjected to in-depth enzyme kinetics studies and the obtained rk(cat)/K(m) (k(cat)/K(m) relative to that of dThd) ranged from 2.5 to 26%. The tetrazolyl-type N3-substitued dThd analogues 9b1/2 and 11d1/2 were the best substrates of hTK1 with rPRs of 52.4% and 42.5% and rk(cat)/K(m) values of 14.9% and 19.7% respectively. In comparison, the rPR and rk(cat)/K(m) values of N5-2OH in this specific study were 41.5% and 10.8%, respectively. Compounds 3e and 3f were >1900 and >1500 times, respectively, better soluble in PBS (pH 7.4) than N5-2OH whereas solubilities for 9b1/2-9d1/2 and 11b1/2-11d1/2 were only 1.3-13 times better.

Synthesis, biological evaluation, and radioiodination of halogenated closo-carboranylthymidine analogues.

The synthesis and initial biological evaluation of 3-carboranylthymidine analogues (3CTAs) that are (radio)halogenated at the closo-carborane cluster are described. Radiohalogenated 3CTAs have the potential to be used in the radiotherapy and imaging of cancer because they may be selectively entrapped in tumor cells through monophosphorylation by human thymidine kinase 1 (hTK1). Two strategies for the synthesis of a (127)I-labeled form of a specific 3CTA, previously designated as N5, are described: (1) direct iodination of N5 with iodine monochloride and aluminum chloride to obtain N5-(127)I and (2) initial monoiodination of o-carborane to 9-iodo-o-carborane followed by its functionalization to N5-(127)I. The former strategy produced N5-(127)I in low yields along with di-, tri-, and tetraiodinated N5 as well as decomposition products, whereas the latter method produced only N5-(127)I in high yields. N5-(127)I was subjected to nucleophilic halogen- and isotope-exchange reactions using Na(79/81)Br and Na(125)I, respectively, in the presence of Herrmann's catalyst to obtain N5-(79/81)Br and N5-(125)I, respectively. Two intermediate products formed using the second strategy, 1-(tert-butyldimethylsilyl)-9-iodo-o-carborane and 1-(tert-butyldimethylsilyl)-12-iodo-o-carborane, were subjected to X-ray diffraction studies to confirm that substitution at a single carbon atom of 9-iodo-o-carborane resulted in the formation of two structural isomers. To the best of our knowledge, this is the first report of halogen- and isotope-exchange reactions of B-halocarboranes that have been conjugated to a complex biomolecule. Human TK1 phosphorylation rates of N5, N5-(127)I, and N5-(79/81)Br ranged from 38.0% to 29.6% relative to that of thymidine, the endogenous hTK1 substrate. The in vitro uptake of N5, N5-(127)I, and N5-(79/81)Br in L929 TK1(+) cells was 2.0, 1.8, and 1.4 times greater than that in L929 TK1(-) cells.

Selective regulation of aromatase expression for drug discovery.

Aromatase is a particularly attractive drug target in the treatment of hormone-responsive breast cancer, and aromatase activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Additionally, COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase can act locally to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer. Two pharmacological approaches are being developed, one approach utilizing small molecule drug design and the second approach involving mRNA silencing technology. The small molecule drug design approach focuses on the synthesis and biological evaluation of a novel series of sulfonanilide analogs derived from COX-2 selective inhibitors. Combinatorial chemistry approaches were used to generate diversely substituted novel sulfonanilides. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose and time dependent manner, and structure activity analysis does not find a correlation between aromatase suppression and COX inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogs decrease aromatase gene transcription in breast cells. Furthermore, the sulfonanilide compounds selectively decrease aromatase gene expression in several breast cancer cells, without exhibiting cytotoxic or apoptotic effects at low micromole concentrations. A ligand-based pharmacophore model for selective aromatase modulation (SAM) by the novel sulfonanilides identified an aromatic ring, two hydrogen bond acceptors, and a hydrophobic function as four key chemical features. In the second approach, short interfering RNAs (siRNA) were designed targeting human aromatase mRNA. Treatment of breast cancer cells with siRNAs targeting aromatase (siAROMs) completely masked the aromatase enzyme activity and resulted in suppression of CYP19 mRNA. Thus, these results suggest that the novel sulfonanilides and the siRNAs targeting aromatase expression may be valuable tools for selective regulation of aromatase in breast cancer.

Pharmacokinetics, biodistribution and metabolism of a novel selective androgen receptor modulator designed for prostate cancer imaging.

Knowledge of the presence and extent of disease plays a major role in clinical management of prostate cancer, as it provides meaningful information as to which therapy to choose and who might benefit from this therapy. The wide expression of androgen receptor (AR) in primary and metastatic prostate tumors offers a cellular target for receptor-mediated imaging of prostate cancer. In our previous study, a non-steroidal AR ligand, S-26 [S-3-(4-fluorophenoxy)-2-hydroxy-2-methyl-N-(4-cyano-3-iodophenyl)-propionamide] showed promising in vitro pharmacological properties as an AR-mediated imaging agent, with high AR binding affinity and AR specificity. The overall goal of this study was to characterize the in vivo metabolic and biodistribution profile of S-26 in rats. Non-compartmental pharmacokinetic analysis of S-26 in rat plasma showed that clearance (CL), volume of distribution (Vd(ss)), and half-life (T(1/2)) of S-26 were 0.30 + or - 0.07 l/h/kg, 1.44 + or - 0.33 l/kg, and 4 h, respectively, after intravenous (i.v.) administration. Dose proportionality (1, 10 and 30 mg/kg) studies suggested that the pharmacokinetics of S-26 are dose-independent. The plasma concentrations of all 3 doses were further simultaneously fitted with a two-compartmental model and the results were similar to those obtained from non-compartmental analysis. Biodistribution studies using (125)I-labeled S-26 indicated that it did not specifically target AR-rich tissue (e.g. prostate). A substantial amount of radioactivity recovered from thyroid gland indicated the release of free iodine. In metabolism studies, unchanged S-26 and its metabolites were detected in rat urine and fecal samples. Oxidation, de-iodination, hydrolysis, and sulfate conjugation were the major metabolic pathways of S-26 in rats, with de-iodination representing a unique metabolic pathway of S-26 among other selective androgen receptor modulators. In conclusion, the extensive plasma clearance and de-iodination of S-26 likely contribute to its lack of AR tissue selectivity in vivo. Future studies using metabolically stable ligands with less lipophilicity and higher AR binding affinity may represent a promising and rational approach for AR-mediated imaging.

Synthesis and biological evaluation of novel sulfonanilide compounds as antiproliferative agents for breast cancer.

Combinatorial chemistry approaches facilitate drug discovery processes and result in structural modifications of lead compounds that enhance pharmacological activity, improve pharmacokinetic properties, or reduce unwanted side effects. Epidemiological and animal model studies have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) can act as chemopreventive agents. The cyclooxygenase-2 (COX-2) inhibitor nimesulide shows anticancer effects in several cancer cell lines via COX-2-dependent and -independent mechanisms. The molecular structure of nimesulide was used as a starting scaffold to design novel sulfonanilide analogs and examine the structural features that contribute to this anticancer effect. A systematic combinatorial chemical approach was used to generate diversely substituted sulfonanilide derivatives that were tested for their effects on the proliferation of human breast cancer cells. Structure-function analysis indicated that the inhibition of cell growth by compounds derived from the novel sulfonanilides required a bulky terminal phenyl ring, a methanesulfonamide, and a hydrophobic carboxamide moiety.

Novel sulfonanilide analogs decrease aromatase activity in breast cancer cells: synthesis, biological evaluation, and ligand-based pharmacophore identification.

Aromatase converts androgens to estrogens and is a particularly attractive target in the treatment of estrogen receptor positive breast cancer. Previously, the COX-2 selective inhibitor nimesulide and analogs decreased aromatase expression and enzyme activity independent of COX-2 inhibition. In this manuscript, a combinatorial approach was used to generate diversely substituted novel sulfonanilides by parallel synthesis. Their pharmacological evaluation as agents for suppression of aromatase activity in SK-BR-3 breast cancer cells was extensively explored. A ligand-based pharmacophore model was elaborated for selective aromatase modulation (SAM) using the Catalyst HipHop algorithms. The best qualitative model consisted of four features: one aromatic ring, two hydrogen bond acceptors, and one hydrophobic function. Several lead compounds have also been tested in aromatase transfected MCF-7 cells, and they significantly suppressed cellular aromatase activity. The results suggest that both genomic and nongenomic mechanisms of these compounds are involved within the aromatase suppression effect.

Potent inhibition of tau fibrillization with a multivalent ligand.

Small-molecule inhibitors of tau fibrillization are under investigation as tools for interrogating the tau aggregation pathway and as potential therapeutic agents for Alzheimer's disease. Established inhibitors include thiacarbocyanine dyes, which can inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers. In an effort to increase inhibitory potency, a cyclic bis-thiacarbocyanine molecule containing two thiacarbocyanine moieties was synthesized and characterized with respect to tau fibrillization inhibitory activity by electron microscopy and ligand aggregation state by absorbance spectroscopy. Results showed that the inhibitory activity of the bis-thiacarbocyanine was qualitatively similar to a monomeric cyanine dye, but was more potent with 50% inhibition achieved at approximately 80nM concentration. At all concentrations tested in aqueous solution, the bis-thiacarbocyanine collapsed to form a closed clamshell structure. However, the presence of tau protein selectively stabilized the open conformation. These results suggest that the inhibitory activity of bis-thiacarbocyanine results from multivalency, and reveal a route to more potent tau aggregation inhibitors.

Intestinal epithelial cell accumulation of the cancer preventive polyphenol ellagic acid--extensive binding to protein and DNA.

Ellagic acid (EA), a polyphenol present in many berries, has been demonstrated to be preventive of esophageal cancer in animals both at the initiation and promotion stages. To be able to extrapolate these findings to humans we have studied the transcellular absorption and epithelial cell accumulation of [14C]EA in the human intestinal Caco-2 cells. The apical (mucosal) to basolateral (serosal) transcellular transport of 10 microM [14C]EA was minimal with a P(app) of only 0.13 x 10(-6)cm/s, which is less than for the paracellular transport marker mannitol. In spite of observations of basolateral to apical efflux, Caco-2 cell uptake studies showed high accumulation of EA in the cells (1054+/-136 pmol/mg protein), indicating facile absorptive transport across the apical membrane. Surprisingly, as much as 93% of the cellular EA was irreversibly bound to macromolecules (982+/-151 pmol/mg protein). To confirm the irreversible nature of the binding to protein, Caco-2 cells treated with 10 microM [14C]EA were subjected to SDS-PAGE analysis. This resulted in radiolabeled protein bands trapped in the stacking gel, consistent with [14C]EA-crosslinked proteins. Treatment of Caco-2 cells with 10 microM [14C]EA also revealed irreversible binding of EA to cellular DNA as much as five times higher than for protein (5020+/-773 pmol/mg DNA). Whereas the irreversible binding to protein required oxidation of EA by reactive oxygen species, this did not seem to be the case with the DNA binding. The avid irreversible binding to cellular DNA and protein may be the reason for its highly limited transcellular absorption. Thus, EA appears to accumulate selectively in the epithelial cells of the aerodigestive tract, where its cancer preventive actions may be displayed.