Discovery and Optimization of a Series of Sulfonamide Inverse Agonists for the Retinoic Acid Receptor-Related Orphan Receptor-α.

BACKGROUND: Despite a massive industry endeavor to develop RORγ-modulators for autoimmune disorders, there has been no indication of efforts to target the close family member RORα for similar indications. This may be due to the misconception that RORα is redundant to RORγ, or the inherent difficulty in cultivating tractable starting points for RORα. RORα-selective modulators would be useful tools to interrogate the biology of this understudied orphan nuclear receptor. OBJECTIVE: The goal of this research effort was to identify and optimize synthetic ligands for RORα starting from the known LXR agonist T0901317. METHODS: Fourty-five analogs of the sulfonamide lead (1) were synthesized and evaluated for their ability to suppress the transcriptional activity of RORα, RORγ, and LXRα in cell-based assays. Analogs were characterized by 1H-NMR, 13C-NMR, and LC-MS analysis. The pharmacokinetic profile of the most selective RORα inverse agonist was evaluated in rats with intraperitoneal (i.p.) and per oral (p.o.)dosing. RESULTS: Structure-activity relationship studies led to potent dual RORα/RORγ inverse agonists as well as RORα-selective inverse agonists (20, 28). LXR activity could be reduced by removing the sulfonamide nitrogen substituent. Attempts to improve the potency of these selective leads by varying substitution patterns throughout the molecule proved challenging. CONCLUSION: The synthetic RORα-selective inverse agonists identified (20, 28) can be utilized as chemical tools to probe the function of RORα in vitro and in vivo.

Design, Synthesis, and Biological Evaluation of Indole Biphenylcarboxylic Acids as PPARγ Antagonists.

The thiazolidinediones (TZD) typified by rosiglitazone are the only approved therapeutics targeting PPARγ for the treatment of type-2 diabetes (T2DM). Unfortunately, despite robust insulin sensitizing properties, they are accompanied by a number of severe side effects including congestive heart failure, edema, weight gain, and osteoporosis. We recently identified PPARγ antagonists that bind reversibly with high affinity but do not induce transactivation of the receptor, yet they act as insulin sensitizers in mouse models of diabetes (SR1664).1 This Letter details our synthetic exploration around this novel series of PPARγ antagonists based on an N-biphenylmethylindole scaffold. Structure-activity relationship studies led to the identification of compound 46 as a high affinity PPARγ antagonist that exhibits antidiabetic properties following oral administration in diet-induced obese mice.

The discovery of indole full agonists of the neurotensin receptor 1 (NTSR1).

Neurotensin (NT) is an endogenous tridecapeptide found in the central nervous system (CNS) and in peripheral tissues. Neurotensin exerts a wide range of physiological effects and it has been found to play a critical role in a number of human diseases, such as schizophrenia, Parkinson's disease and drug addiction. The discovery of small-molecule non-peptide neurotensin receptor (NTSR) modulators would represent an important breakthrough as such compounds could be used as pharmacological tools, to further decipher the cellular functions of neurotensin, and potentially as therapeutic agents to treat human disease. Herein, we report the identification of non-peptide low-micromolar neurotensin receptor 1 (NTSR1) full agonists, discovered through structural optimization of the known NTSR1 partial agonist 1. In vitro cellular screenings, based on an intracellular Ca(2+) mobilization assay, revealed our best hit molecule 8 (SR-12062) to have an EC50 of 2 µM at NTSR1 with full agonist behaviour (Emax=100%), showing a higher efficacy and ∼90-fold potency improvement compared to parent compound 1 (EC50=178 µM; Emax=17%).

Synthesis and SAR of novel isoxazoles as potent c-jun N-terminal kinase (JNK) inhibitors.

The design and synthesis of isoxazole 3 is described, a potent JNK inhibitor with two fold selectivity over p38. Optimization of this scaffold led to compounds 27 and 28 which showed greatly improved selectivity over p38 by maintaining the JNK3 potency of compound 3. Extensive SAR studies will be described as well as preliminary in vivo data of the two lead compounds.

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker.

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.

A small molecule bidentate-binding dual inhibitor probe of the LRRK2 and JNK kinases.

Both JNK and LRRK2 are associated with Parkinson's disease (PD). Here we report a reasonably selective and potent kinase inhibitor (compound 6) that bound to both JNK and LRRK2 (a dual inhibitor). A bidentate-binding strategy that simultaneously utilized the ATP hinge binding and a unique protein surface site outside of the ATP pocket was applied to the design and identification of this kind of inhibitor. Compound 6 was a potent JNK3 and modest LRRK2 dual inhibitor with an enzyme IC50 value of 12 nM and 99 nM (LRRK2-G2019S), respectively. Compound 6 also exhibited good cell potency, inhibited LRRK2:G2019S-induced mitochondrial dysfunction in SHSY5Y cells, and was demonstrated to be reasonably selective against a panel of 116 kinases from representative kinase families. Design of such a probe molecule may help enable testing if dual JNK and LRRK2 inhibitions have added or synergistic efficacy in protecting against neurodegeneration in PD.

Synthesis and SAR of novel quinazolines as potent and brain-penetrant c-jun N-terminal kinase (JNK) inhibitors.

Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC(50)=40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.

Synthesis and SAR of 4-(pyrazol-3-yl)-pyridines as novel c-jun N-terminal kinase inhibitors.

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK) inhibitors is described. The development of the 4-(pyrazol-3-yl)-pyridine series was discovered from an earlier pyrimidine series of JNK inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.

PND-1186 FAK inhibitor selectively promotes tumor cell apoptosis in three-dimensional environments.

Tumor cells can grow in an anchorage-independent manner. This is mediated in part through survival signals that bypass normal growth restraints controlled by integrin cell surface receptors. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that associates with integrins and modulates various cellular processes including growth, survival, and migration. As increased FAK expression and tyrosine phosphorylation are associated with tumor progression, inhibitors of FAK are being tested for anti-tumor effects. Here, we analyze PND-1186, a substituted pyridine reversible inhibitor of FAK activity with a 50% inhibitory concentration (IC50) of 1.5 nM in vitro. PND-1186 has an IC50 of ~100 nM in breast carcinoma cells as determined by anti-phospho-specific immunoblotting to FAK Tyr-397. PND-1186 did not alter c­Src or p130Cas tyrosine phosphorylation in adherent cells, yet functioned to restrain cell movement. Notably, 1.0 µM PND-1186 (>5-fold above IC50) had limited effects on cell proliferation. However, under non-adherent conditions as spheroids and as colonies in soft agar, 0.1 µM PND-1186 blocked FAK and p130Cas tyrosine phosphorylation, promoted caspase-3 activation, and triggered cell apoptosis. PND-1186 inhibited 4T1 breast carcinoma subcutaneous tumor growth correlated with elevated tumor cell apoptosis and caspase 3 activation. Addition of PND-1186 to the drinking water of mice was well tolerated and inhibited ascites- and peritoneal membrane-associated ovarian carcinoma tumor growth associated with the inhibition of FAK Tyr-397 phosphorylation. Our results with low-level PND-1186 treatment support the conclusion that FAK activity selectively promotes tumor cell survival in three-dimensional environments.

Synthesis and SAR of piperazine amides as novel c-jun N-terminal kinase (JNK) inhibitors.

A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.

Characterization of dasatinib and its structural analogs as CYP3A4 mechanism-based inactivators and the proposed bioactivation pathways.

Dasatinib was approved in 2006 for the treatment of imatinib-resistant chronic myelogenous leukemia and functions primarily through the inhibition of BCR-ABL and Src kinase. Dasatinib is extensively metabolized in humans by CYP3A4. In this study, we report that the bioactivation of dasatinib by CYP3A4 proceeds through a reactive intermediate that leads to CYP3A4 inactivation with K(I) = 6.3 microM and k(inact) = 0.034 min(-1). The major mechanism of inactivation proceeds through hydroxylation at the para-position of the 2-chloro-6-methylphenyl ring followed by further oxidation, forming a reactive quinone-imine, similar to the reactive intermediates formed by acetaminophen and diclofenac. Formation of a reactive imine-methide was also detected but appears to be a minor pathway. When glutathione was added to human liver microsomal incubations, dasatinib-glutathione adducts were detected. Numerous dasatinib analogs were synthesized in an effort to understand what modifications would block the formation of reactive intermediates during dasatinib metabolism. It is interesting to note that blocking the site of hydroxylation with a methyl group was not effective because a reactive imine-methide was formed, nor was blocking the site with fluorine because the fluorine was removed through an oxidative defluorination mechanism and the reactive quinone-imine was still formed. Numerous analogs are presented that did effectively block the formation of glutathione adducts and prevent the inactivation of CYP3A4.