Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

Light-Driven Radiochemistry with Fluorine-18, Carbon-11 and Zirconium-89.

This review discusses recent advances in light-driven radiochemistry for three key isotopes: fluorine-18, carbon-11, and zirconium-89, and their applications in positron emission tomography (PET). In the case of fluorine-18, the predominant approach involves the use of cyclotron-produced [18F]fluoride or reagents derived thereof. Light serves to activate either the substrate or the fluorine-18 labeled reagent. Advancements in carbon-11 photo-mediated radiochemistry have been leveraged for the radiolabeling of small molecules, achieving various transformations, including 11C-methylation, 11C-carboxylation, 11C-carbonylation, and 11C-cyanation. Contrastingly, zirconium-89 photo-mediated radiochemistry differs from fluorine-18 and carbon-11 approaches. In these cases, light facilitates a postlabeling click reaction, which has proven valuable for the labeling of large biomolecules such as monoclonal antibodies (mAbs). New technological developments, such as the incorporation of photoreactors in commercial radiosynthesizers, illustrate the commitment the field is making in embracing photochemistry. Taken together, these advances in photo-mediated radiochemistry enable radiochemists to apply new retrosynthetic strategies in accessing novel PET radiotracers.

Ni-Catalyzed Reductive Cross-Coupling of Cyclopropylamines and Other Strained Ring NHP Esters with (Hetero)Aryl Halides.

A nickel-catalyzed reductive cross-coupling of cyclopropylamine NHP esters with (hetero)aryl halides is reported. This efficient protocol provides direct access to 1-arylcyclopropylamines, a bioisosteric motif commonly used in small molecule drug discovery. The reaction proceeds rapidly (<2 h) with excellent functional group tolerance and without requiring heat- or air-sensitive reagents. The method can also be extended to the arylation of four-membered strained rings. The NHP esters are easily obtained from the corresponding commercially available carboxylic acids in one step with high yields and no column chromatography.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Photoredox/Nickel Dual Catalytic Cross-Coupling of Potassium Thiomethyltrifluoroborates with Aryl and Heteroaryl Bromides.

The cross-coupling of S-aryl and S-alkyl potassium thiomethyltrifluoroborates with aryl and heteroaryl bromides is reported via photoredox/nickel dual catalysis. The transformation is achieved under mild conditions with commercially available or readily prepared, air stable reagents and affords benzylthioether products in moderate to good yields with good functional group tolerance. A practical and improved synthesis of potassium thiomethyltrifluoroborates is also reported that affords access to previously undescribed reagents.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Design and Evaluation of Highly Selective Human Immunoproteasome Inhibitors Reveal a Compensatory Process That Preserves Immune Cell Viability.

The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against nonimmune cells, which limits its use for autoimmune diseases. An attractive alternative is to selectively inhibit the immune cell-specific immunoproteasome to deplete pathogenic immune cells and spare nonhematopoietic cells. Here, we disclose the development of highly subunit-selective immunoproteasome inhibitors using insights obtained from the first bona fide human immunoproteasome cocrystal structures. Evaluation of these inhibitors revealed that immunoproteasome-specific inhibition does not lead to immune cell death as anticipated and that targeting viability requires inhibition of both immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knockout experiments confirmed upregulation of the constitutive proteasome upon disruption of the immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition alone is not a suitable approach to deplete immune cells.

Rhodium(III)-Catalyzed C-H Functionalization of 1-(2H)-Phthalazinones at C8.

The rhodium(III) catalyst tris(acetonitrile)pentamethylcyclopentadienylrhodium(III) hexafluoroantimonate ([Cp*Rh(MeCN)3](SbF6)2) reacts with 1-(2H)-phthalazinones to promote a C-H functionalization event at C8. Preparation of a set of compounds arising from oxidative alkenylation with olefins, hydroarylation with alkynes, and iodination with N-iodosuccinimide is reported here. Oxidative alkenylation proceeds in very good yield, and the scope and limitations of the hydroarylation and halogenation reactions are discussed. Notably, this strategy enables rapid preparation of C8-substituted phthalazinones without requiring phthalazinone ring synthesis starting from a prefunctionalized arene.

Discovery of dual leucine zipper kinase (DLK, MAP3K12) inhibitors with activity in neurodegeneration models.

Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.

Synthesis, characterization, and PK/PD studies of a series of spirocyclic pyranochromene BACE1 inhibitors.

The development of 1,3,4,4a,5,10a-hexahydropyrano[3,4-b]chromene analogs as BACE1 inhibitors is described. Introduction of the spirocyclic pyranochromene scaffold yielded several advantages over previous generation cores, including increased potency, reduced efflux, and reduced CYP2D6 inhibition. Compound 13 (BACE1 IC50=110 nM) demonstrated a reduction in CSF Aß in wild type rats after a single dose.

Cyanide anion as a leaving group in nucleophilic aromatic substitution: synthesis of quaternary centers at azine heterocycles.

Nucleophilic aromatic substitution of 2- or 4-cyanoazines with the anions derived from aliphatic α,α-disubstituted esters and nitriles leads to displacement of the cyanide function. Enabling cyanides to be used as highly active leaving groups in S(N)Ar reactions provides additional flexibility in starting materials for synthesis. We show that, in many cases, the cyanide leaving group is displaced preferentially in the presence of halogens. The resulting heteroaryl iodides, bromides, and chlorides subsequently can be used as handles for further chemical diversification.

Site-selective azaindole arylation at the azine and azole rings via N-oxide activation.

Subjection of N-methyl 6- and 7-azaindole N-oxides to a Pd(OAc)2/DavePhos catalyst system enables regioselective direct arylation of the azine ring. Following deoxygenation, 7-azaindole substrates undergo an additional regioselective azole direct arylation event in good yield.

Enzyme-catalyzed synthesis of isosteric phosphono-analogues of sugar nucleotides.

Efficient enzymatic syntheses of isosteric phosphono analogues of sugar nucleotides have been accomplished using a thymidylyltransferase.

Engineering ribonucleoside triphosphate specificity in a thymidylyltransferase.

Nature's glycosylation catalysts, glycosyltransferases, indirectly manipulate and control many important biological processes by transferring sugar nucleotide donors onto acceptors. Challenging chemical synthesis impedes synthetic access to sugar nucleotides and limits the study of many glycosyltransferases. Enzymatic access to sugar nucleotides is a rapidly expanding avenue of research, limited only by the substrate specificity of the enzyme. We have explored the promiscuous thymidylyltransferase from Streptococcus pneumoniae, Cps2L, and enhanced its uridylyltransferase and guanidyltransferase activities by active site engineering. Mutagenesis at position Q24 resulted in a variant with 10-, 3-, and 2-fold enhancement of UDP-glucosamine, UDP-mannose, and UDP- N-acetylglucosamine production, respectively. New catalytic activities were observed for the Cps2L variant over the wild-type enzyme, including the formation of GDP-mannose. The variant was evaluated as a catalyst for the formation of a series of dTDP- and UDP-furanoses and notably produced dTDP-Gal f in 90% yield and UDP-Ara f in 30% yield after 12 h. A series of 3- O-alkylglucose 1-phosphates were also evaluated as substrates, and notable conversions to UDP-3- O-methylglucose and UDP-3- O-dodecylglucose were achieved with the variant but not the wild-type enzyme. The Q24S variant also enhanced essentially all thymidylyltransferase activities relative to the wild-type enzyme. Comparison of active sites of uridylyltransferases and thymidylyltransferases with products bound indicate the Q24S variant to be a new approach in broadening nucleotidylyltransferase activity.

Intramolecular Pd(II)-catalyzed oxidative biaryl synthesis under air: reaction development and scope.

New reaction conditions for intramolecular palladium(II)-catalyzed oxidative carbon-carbon bond formation under air are described. The use of pivalic acid as the reaction solvent, instead of acetic acid, results in greater reproducibility, higher yields, and broader scope. This includes the use of electron-rich diarylamines as illustrated in the synthesis of three naturally occurring carbazole products: Murrayafoline A, Mukonine, and Clausenine. A variety of side products have also been isolated, casting light on competing reaction pathways and revealing new reactivity with palladium(II) catalysis.

Lipophilic sugar nucleotide synthesis by structure-based design of nucleotidylyltransferase substrates.

Structure-based design of alkyl sugar-1-phosphates provides an efficient nucleotidylyltransferase-catalyzed synthesis of a series of new lipophilic sugar nucleotides possessing long or branched alkyl chains, thereby demonstrating the utility of nucleotidylyltransferases to catalyze the synthesis of sugar nucleotides with potential applications in lipopolysaccharide and lipoglycopeptide biosynthesis.