Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists.

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.

BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe.

The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.

Rational Design and Synthesis of Selective PRMT4 Inhibitors: A New Chemotype for Development of Cancer Therapeutics*.

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates the arginine residues of histone H3 and nonhistone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and, potentially, therapeutics. Although several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Herein, we report the structure-based design of a new class of alanine-containing 3-arylindoles as potent and selective PRMT4 inhibitors, and describe key structure-activity relationships for this class of compounds.

Concentration Dependence of the Unbound Partition Coefficient Kpuu and Its Application to Correct for Exposure-Related Discrepancies between Biochemical and Cellular Potency of KAT6A Inhibitors.

The unbound partition coefficient (Kpuu) allows the estimation of intracellular target exposure from free extracellular drug concentrations. Although the active mechanisms controlling Kpuu are saturable, Kpuu is commonly determined at a single concentration, which may not be appropriate in cases in which drug concentrations can largely vary, e.g., in plasma in vivo or in vitro IC50 assays. We examined the concentration dependence of Kpuu in vitro using KAT6A inhibitors with varying potency drop-off in ZR75-1 breast cancer cells to account for exposure-related discrepancies between cellular and biochemical IC50 Considering saturability resulted in a better quantitative bridge between both IC50 values and gave way to a simplified method to determine Kpuu that is suitable for the prediction of unbound cytosolic drug concentrations without the need to generate fu,cell estimates from binding studies in cell homogenates. As opposed to the binding method, which destroys cellular integrity, this approach provides an alternative fu,cell estimate and directly reflects the fraction of unbound drug in the cell cytosol based on Kp saturation (fu,cyto) of intact cells. In contrast to the binding method, prediction of intracellular KAT6A exposure with this more physiologic approach was able to bridge the average exposure gap between biochemical and cellular IC50 values from 73-fold down to only 5.4-fold. The concept of concentration-dependent Kpuu provides a solid rationale for early drug discovery to discriminate between pharmacology and target exposure-related IC50 discrepancies. The attractiveness of the approach also lies in the use of the same assay format for cellular IC50, fu,cyto, and the unbound partition coefficient based on fu,cyto (Kpuu,cyto) determination. SIGNIFICANCE STATEMENT: Examination of the yet-unexplored concentration dependence of the unbound partition coefficient led to a new experimental approach that resulted in more reliable predictions of intracellular target exposure and is well suited for routine drug discovery projects.

Benzoisoquinolinediones as Potent and Selective Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains.

Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure-activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.

Synthesis of new enantiopure poly(hydroxy)aminooxepanes as building blocks for multivalent carbohydrate mimetics.

New compounds with carbohydrate-similar structure (carbohydrate mimetics) are presented in this article. Starting from enantiopure nitrones and lithiated TMSE-allene we prepared three 1,2-oxazine derivatives which underwent a highly stereoselective Lewis acid-induced rearrangement to give bicyclic products in good yield. Subsequent reductive transformations delivered a library of new poly(hydroxy)aminooxepane derivatives. The crucial final palladium-catalyzed hydrogenolysis of the 1,2-oxazine moiety was optimized resulting in a reasonably efficient approach to a series of new seven-membered carbohydrate mimetics.