Discovery and optimization of heteroaryl piperazines as potent and selective PI3Kδ inhibitors.

A high-throughput screening (HTS) campaign identified a class of heteroaryl piperazines with excellent baseline affinity and selectivity for phosphoinositide 3-kinase δ (PI3Kδ) over closely related isoforms. Rapid evaluation and optimization of structure-activity relationships (SAR) for this class, leveraging the modular nature of this scaffold, facilitated development of this hit class into a series of potent and selective inhibitors of PI3Kδ. This effort culminated in the identification of 29, which displayed excellent potency in enzyme and cell-based assays, as well as favorable pharmacokinetic and off-target profiles.

Conformational-Design-Driven Discovery of EZM0414: A Selective, Potent SETD2 Inhibitor for Clinical Studies.

SETD2, a lysine N-methyltransferase, is a histone methyltransferase that plays an important role in various cellular processes and was identified as a target of interest in multiple myeloma that features a t(4,14) translocation. We recently reported the discovery of a novel small-molecule SETD2 inhibitor tool compound that is suitable for preclinical studies. Herein we describe the conformational-design-driven evolution of the advanced chemistry lead, which resulted in compounds appropriate for clinical evaluation. Further optimization of this chemical series led to the discovery of EZM0414, which is a potent, selective, and orally bioavailable inhibitor of SETD2 with good pharmacokinetic properties and robust pharmacodynamic activity in a mouse xenograft model.

Discovery of a First-in-Class Inhibitor of the Histone Methyltransferase SETD2 Suitable for Preclinical Studies.

SET domain-containing protein 2 (SETD2), a histone methyltransferase, has been identified as a target of interest in certain hematological malignancies, including multiple myeloma. This account details the discovery of EPZ-719, a novel and potent SETD2 inhibitor with a high selectivity over other histone methyltransferases. A screening campaign of the Epizyme proprietary histone methyltransferase-biased library identified potential leads based on a 2-amidoindole core. Structure-based drug design (SBDD) and drug metabolism/pharmacokinetics (DMPK) optimization resulted in EPZ-719, an attractive tool compound for the interrogation of SETD2 biology that enables in vivo target validation studies.

Indazoles: regioselective protection and subsequent amine coupling reactions.

Indazoles are unselectively protected under strongly basic conditions to give a mixture at N-1 and N-2. Under mildly acidic conditions, regioselective protection at N-2 takes place. Thermodynamic conditions lead to regioselective protection at N-1. This trend applies to various substituted indazoles. Protected 5-bromoindazoles participate in Buchwald reactions with a range of amines to generate novel derivatives.

Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

Synthesis and activity of new aryl- and heteroaryl-substituted pyrazole inhibitors of the transforming growth factor-beta type I receptor kinase domain.

Pyrazole-based inhibitors of the transforming growth factor-beta type I receptor kinase domain (TbetaR-I) are described. Examination of the SAR in both enzyme- and cell-based in vitro assays resulted in the emergence of two subseries featuring differing selectivity versus p38 MAP kinase. A common binding mode at the active site has been established by successful cocrystallization and X-ray analysis of potent inhibitors with the TbetaR-I receptor kinase domain.

Synthesis and protein kinase inhibitory activity of balanol analogues with modified benzophenone subunits.

A series of analogues of the protein kinase C (PKC) inhibitory natural product balanol which bear modified benzophenone subunits are described. The analogues were designed with the goal of uncovering structure-activity features that could be used in the development of PKC inhibitors with a reduced polar character compared to balanol itself. The results of these studies suggest that most of the benzophenone features found in the natural product are important for obtaining potent PKC inhibitory compounds. However, several modifications were found to lead to selective inhibitors of the related enzyme protein kinase A (PKA), and several specific modifications to the polar structural elements of the benzophenone were found to provide potent PKC inhibitors. In particular, it was found that replacement of the benzophenone carboxylate with bioisosteric equivalents could lead to potent analogues. Further, a tolerance for lipophilic substituents on the terminal benzophenone ring was uncovered. These results are discussed in light of recently available structural information for PKA.

Total Synthesis of (-)- and (+)-Balanol(1).

Two total syntheses of the potent protein kinase C inhibitory fungal metabolite balanol are described. In the first approach, the core aminohydroxyazepane subunit was prepared in racemic form by stereospecific functionalization of N-benzyl-epsilon-caprolactam. Resolution prior to coupling to the benzophenone subunit provided access to both enantiomers of balanol. In the second approach, an efficient silicon-mediated cyclization of (2S,3R)-3-hydroxylysine followed by reduction provided the azepane subunit in enantiomerically pure form. The sterically congested benzophenone subunit was assembled from two highly substituted aromatic precursors by way of an anionic homo-Fries rearrangement.

Kinetic characterization of novel pyrazole TGF-beta receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition.

Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate, we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease.