Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration.

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Discovery of AZD4625, a Covalent Allosteric Inhibitor of the Mutant GTPase KRASG12C.

KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Diversity-orientated synthesis of macrocyclic heterocycles using a double SNAr approach.

An efficient macrocyclisation approach based on the double aromatic nucleophilic substitution (SNACK) was developed. This methodology allows a facile incorporation of heterocyclic motifs into macrocyclic rings and rapid synthesis of a significant number of structurally diverse macrocycles. SNACK macrocyclisation enables preparation of stable diastereoisomers of conformationally restricted macrocycles (atropisomers). Practical application of SNACK macrocyclisation in a drug discovery project was exemplified by the identification of high affinity macrocyclic binders of B-cell lymphoma 6 (BCL6).

Impact of oxetane incorporation on the structure and stability of alpha-helical peptides.

Peptide-based drugs combine advantages of larger biological therapeutics with those of small molecule drugs, but they generally display poor permeability and metabolic stability. Recently, we introduced a new type of peptide bond isostere, in which the backbone carbonyl is replaced with a 3-amino oxetane heterocycle, into short linear peptides with the aim of improving their therapeutic potential. In this study, we have explored the impact of oxetane modification on α-helical peptides to establish whether or not this modification is tolerated in this biologically important structural motif. The oxetane modification was introduced at two positions in a well-characterised helical peptide sequence, and circular dichroism and NMR spectroscopy were used to measure the resulting secondary structure content under different experimental conditions. Our data demonstrated that introduction of an oxetane into the peptide backbone results in a significant loss of helicity, regardless of where in the sequence the modification is placed. The molecular determinants of this destabilisation were then explored using steered molecular dynamics simulations, a computational method analogous to single molecule spectroscopy. Our simulations indicated that oxetane modification introduces a kink in the helical axis, alters the dihedral angles of residues up to three positions away from the modification, and disrupts the (i, i + 4) hydrogen bonding pattern characteristic of α-helices in favour of new, short-range hydrogen bonds. The detailed structural understanding provided in this work can direct future design of chemically modified peptides.

The discovery and evaluation of 3-amino-2(1H)-pyrazinones as a novel series of selective p38α MAP kinase inhibitors.

The discovery and optimisation of a novel series of potent and selective p38α inhibitors is described. Evaluating the structure-activity relationship of an aminoalkyl substituent at the 3 position of the 2(1H)-pyrazinone core, p38α potency was increased 20000-fold. The most advanced compound (25) demonstrated excellent in vivo properties suitable for an inhaled route of administration.

Development of oxetane modified building blocks for peptide synthesis.

The synthesis and use of oxetane modified dipeptide building blocks in solution and solid-phase peptide synthesis (SPPS) is reported. The preparation of building blocks containing non-glycine residues at the N-terminus in a stereochemically controlled manner is challenging. Here, a practical 4-step route to such building blocks is demonstrated, through the synthesis of dipeptides containing contiguous alanine residues. The incorporation of these new derivatives at specific sites along the backbone of an alanine-rich peptide sequence containing eighteen amino acids is demonstrated via solid-phase peptide synthesis. Additionally, new methods to enable the incorporation of all 20 of the proteinogenic amino acids into such dipeptide building blocks are reported through modifications of the synthetic route (for Cys and Met) and by changes to the protecting group strategy (for His, Ser and Thr).

Free Ligand 1D NMR Conformational Signatures To Enhance Structure Based Drug Design of a Mcl-1 Inhibitor (AZD5991) and Other Synthetic Macrocycles.

The three-dimensional conformations adopted by a free ligand in solution impact bioactivity and physicochemical properties. Solution 1D NMR spectra inherently contain information on ligand conformational flexibility and three-dimensional shape, as well as the propensity of the free ligand to fully preorganize into the bioactive conformation. Herein we discuss some key learnings, distilled from our experience developing potent and selective synthetic macrocyclic inhibitors, including Mcl-1 clinical candidate AZD5991. Case studies have been selected from recent oncology research projects, demonstrating how 1D NMR conformational signatures can complement X-ray protein-ligand structural information to guide medicinal chemistry optimization. Learning to extract free ligand conformational information from routinely available 1D NMR signatures has proven to be fast enough to guide medicinal chemistry decisions within design cycles for compound optimization.

Discovery of a Thiadiazole-Pyridazine-Based Allosteric Glutaminase 1 Inhibitor Series That Demonstrates Oral Bioavailability and Activity in Tumor Xenograft Models.

Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell's ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

Enzymatically-stable oxetane-based dipeptide hydrogels.

Low molecular weight gelators that are not easily degraded by enzymes have a range of potential applications. Here, we report new Fmoc-protected dipeptides in which the amide carbonyl group has been replaced by an oxetane ring. Remarkably one of these peptidomimetics, but not the corresponding dipeptide, is an effective gelator, forming hydrogels at a concentration of 3 mg mL-1. On assembly, there is a lack of beta-sheet structure, implying that there is no requirement for this motif in such a gel. Furthermore, the modified dipeptide is also stable to proteolysis compared to the parent dipeptide.

The acute glucose lowering effect of specific GPR120 activation in mice is mainly driven by glucagon-like peptide 1.

The mechanism behind the glucose lowering effect occurring after specific activation of GPR120 is not completely understood. In this study, a potent and selective GPR120 agonist was developed and its pharmacological properties were compared with the previously described GPR120 agonist Metabolex-36. Effects of both compounds on signaling pathways and GLP-1 secretion were investigated in vitro. The acute glucose lowering effect was studied in lean wild-type and GPR120 null mice following oral or intravenous glucose tolerance tests. In vitro, in GPR120 overexpressing cells, both agonists signaled through Gαq, Gαs and the ß-arrestin pathway. However, in mouse islets the signaling pathway was different since the agonists reduced cAMP production. The GPR120 agonists stimulated GLP-1 secretion both in vitro in STC-1 cells and in vivo following oral administration. In vivo GPR120 activation induced significant glucose lowering and increased insulin secretion after intravenous glucose administration in lean mice, while the agonists had no effect in GPR120 null mice. Exendin 9-39, a GLP-1 receptor antagonist, abolished the GPR120 induced effects on glucose and insulin following an intravenous glucose challenge. In conclusion, GLP-1 secretion is an important mechanism behind the acute glucose lowering effect following specific GPR120 activation.

Solid-Phase Synthesis of Oxetane Modified Peptides.

Solid-phase peptide synthesis (SPPS) is used to create peptidomimetics in which one of the backbone amide C═O bonds is replaced by a four-membered oxetane ring. The oxetane containing dipeptide building blocks are made in three steps in solution, then integrated into peptide chains by conventional Fmoc SPPS. This methodology is used to make a range of peptides in high purity including backbone modified derivatives of the nonapeptide bradykinin and Met- and Leu-enkephalin.

Discovery of Pyrazolo[1,5-a]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-a]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.

Indazole-6-phenylcyclopropylcarboxylic Acids as Selective GPR120 Agonists with in Vivo Efficacy.

GPR120 agonists have therapeutic potential for the treatment of diabetes, but few selective agonists have been reported. We identified an indazole-6-phenylcyclopropylcarboxylic acid series of GPR120 agonists and conducted SAR studies to optimize GPR120 potency. Furthermore, we identified a (S,S)-cyclopropylcarboxylic acid structural motif which gave selectivity against GPR40. Good oral exposure was obtained with some compounds displaying unexpected high CNS penetration. Increased MDCK efflux was utilized to identify compounds such as 33 with lower CNS penetration, and activity in oral glucose tolerance studies was demonstrated. Differential activity was observed in GPR120 null and wild-type mice indicating that this effect operates through a mechanism involving GPR120 agonism.

An iron-catalysed C-C bond-forming spirocyclization cascade providing sustainable access to new 3D heterocyclic frameworks.

Heterocyclic architectures offer powerful creative possibilities to a range of chemistry end-users. This is particularly true of heterocycles containing a high proportion of sp3-carbon atoms, which confer precise spatial definition upon chemical probes, drug substances, chiral monomers and the like. Nonetheless, simple catalytic routes to new heterocyclic cores are infrequently reported, and methods making use of biomass-accessible starting materials are also rare. Here, we demonstrate a new method allowing rapid entry to spirocyclic bis-heterocycles, in which inexpensive iron(III) catalysts mediate a highly stereoselective C-C bond-forming cyclization cascade reaction using (2-halo)aryl ethers and amines constructed using feedstock chemicals readily available from plant sources. Fe(acac)3 mediates the deiodinative cyclization of (2-halo)aryloxy furfuranyl ethers, followed by capture of the intermediate metal species by Grignard reagents, to deliver spirocycles containing two asymmetric centres. The reactions offer potential entry to key structural motifs present in bioactive natural products.

Discovery of potent, selective small molecule inhibitors of α-subtype of type III phosphatidylinositol-4-kinase (PI4KIIIα).

The discovery and optimisation of novel, potent and selective small molecule inhibitors of the α-isoform of type III phosphatidylinositol-4-kinase (PI4Kα) are described. Lead compounds show cellular activity consistent with their PI4Kα potency inhibiting the accumulation of IP1 after PDGF stimulation and reducing cellular PIP, PIP2 and PIP3 levels. Hence, these compounds are useful in vitro tools to delineate the complex biological pathways involved in signalling through PI4Kα.

Synthesis and indole coupling reactions of azetidine and oxetane sulfinate salts.

Azetidine and oxetane sulfinate salts are easily prepared from commercially available 3-iodoheterocycle precursors in a three-step sequence. They undergo smooth coupling reactions thereby providing an expedient route for the introduction of these four-membered heterocycles into indoles.