Discovery of an In Vivo Chemical Probe for BCL6 Inhibition by Optimization of Tricyclic Quinolinones.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays.

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

Optimizing Shape Complementarity Enables the Discovery of Potent Tricyclic BCL6 Inhibitors.

To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.

Improved Binding Affinity and Pharmacokinetics Enable Sustained Degradation of BCL6 In Vivo.

The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.

Into Deep Water: Optimizing BCL6 Inhibitors by Growing into a Solvated Pocket.

We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Synthesis of Substituted 1,4-Dioxenes through O-H Insertion and Cyclization Using Keto-Diazo Compounds.

1,4-Dioxenes present interesting potential as synthetic intermediates and as unusual motifs for incorporation into biologically active compounds. Here, an efficient synthesis of functionalized 1,4-dioxenes is achieved in two steps. Using keto-diazo compounds, a ruthenium catalyzed O-H insertion with ß-halohydrins followed by treatment with base results in cyclization with excellent selectivity, through O-alkylation of the keto-enolate. A variety of halohydrins and anion-stabilizing groups in the diazo-component are tolerated, affording novel functionalized dioxenes. Enantioenriched ß-bromohydrins provide enantioenriched 1,4-dioxenes.

Oxetanes: Recent Advances in Synthesis, Reactivity, and Medicinal Chemistry.

The four-membered oxetane ring has been increasingly exploited for its contrasting behaviors: its influence on physicochemical properties as a stable motif in medicinal chemistry and its propensity to undergo ring-opening reactions as a synthetic intermediate. These applications have driven numerous studies into the synthesis of new oxetane derivatives. This review takes an overview of the literature for the synthesis of oxetane derivatives, concentrating on advances in the last five years up to the end of 2015. These methods are clustered by strategies for preparation of the ring and further derivatization of preformed oxetane-containing building blocks. Examples of the use of oxetanes in medicinal chemistry are reported, including a collation of oxetane derivatives appearing in recent patents for medicinal chemistry applications. Finally, examples of oxetane derivatives in ring-opening and ring-expansion reactions are described.

Synthesis of diversely functionalised 2,2-disubstituted oxetanes: fragment motifs in new chemical space.

Di-, tri- and tetra-substituted oxetane derivatives with combinations of ester, amide, nitrile, aryl, sulfone and phosphonate substituents are prepared as fragments or building blocks for drug discovery. The synthesis of these novel oxetane functional groups, in new chemical space, is achieved via rhodium-catalysed O-H insertion and C-C bond forming cyclisation.

Synthesis of di-, tri-, and tetrasubstituted oxetanes by rhodium-catalyzed O-H insertion and C-C bond-forming cyclization.

Oxetanes offer exciting potential as structural motifs and intermediates in drug discovery and materials science. Here an efficient strategy for the synthesis of oxetane rings incorporating pendant functional groups is described. A wide variety of oxetane 2,2-dicarboxylates were accessed in high yields, including functionalized 3-/4-aryl- and alkyl-substituted oxetanes and fused oxetane bicycles. Enantioenriched alcohols provided enantioenriched oxetanes with complete retention of configuration. The oxetane products were further derivatized, while the ring was maintained intact, thus highlighting their potential as building blocks for medicinal chemistry.

Regio- and stereospecific synthesis of C-3 functionalized proline derivatives by palladium catalyzed directed C(sp3)-H arylation.

Functionalization of C(sp(3))-H bonds at the unactivated 3-position of proline derivatives has been achieved using aryl iodides and palladium catalysis. This directly affords cis-2,3-disubstituted pyrrolidines as single stereoisomers. 3-Arylation occurs in high yield under solvent-free conditions with aminoquinoline and methoxyaminoquinoline directing groups. The latter was readily removed to give primary amide derivatives with physicochemical properties appropriate for use as fragments in drug discovery.

Copper-catalyzed N-arylation of 2-imidazolines with aryl iodides.

The first copper-catalyzed N-arylation of 2-imidazolines is described. The reaction affords compounds with desirable lead-like characteristics in high yield with practical simplicity under inexpensive, "ligand-free" conditions. The cross coupling was successful with electron-rich and electron-poor aromatic iodides. Substrates bearing halides, esters, nitriles, and free hydroxyls are well tolerated, providing reactive handles for further functionalization, as are pyridines. In addition, the regioselective N-arylation of a 4-substituted imidazoline is reported.