Synthesis of 3‑(Hetero)aryl Tetrahydropyrazolo[3,4‑<i>c</i>]pyridines by Suzuki–Miyaura Cross-Coupling Methodology

A new synthetic route to 3-(heteroaryl) tetrahydropyrazolo­[3,4-<i>c</i>]­pyridines has been developed that uses the Suzuki–Miyaura cross-coupling of a triflate <b>6</b> with (hetero)­aryl boronic acids or esters. Using Pd­(OAc)₂ and XPhos or an XPhos precatalyst, a diverse range of substituents at the C3 position of the tetrahydropyrazolo­[3,4-<i>c</i>]­pyridine skeleton were prepared. The use of pivaloyloxymethyl and benzyl protection also offers the potential to differentially functionalize the pyrazole and tetrahydropyridine nitrogens

Discovery of 4‑{4-[(3<i>R</i>)‑3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}‑1<i>H</i>‑indole (AZ20): A Potent and Selective Inhibitor of ATR Protein Kinase with Monotherapy in Vivo Antitumor Activity

ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound <b>6</b> (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC₅₀ of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC₅₀ of 50 nM. Compound <b>6</b> potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses <b>6</b> leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound <b>6</b> is a useful compound to explore ATR pharmacology in vivo

Small Molecule Binding Sites on the Ras:SOS Complex Can Be Exploited for Inhibition of Ras Activation

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras

Discovery of Pyrazolo[1,5‑<i>a</i>]pyrimidine B‑Cell Lymphoma 6 (BCL6) Binders and Optimization to High Affinity Macrocyclic Inhibitors

Inhibition of the protein–protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo­[1,5-<i>a</i>]­pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand–protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven