Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue.

Irreversible enzyme inhibitors and covalent chemical biology probes often utilize the reaction of a protein cysteine residue with an appropriately positioned electrophile (e.g., acrylamide) on the ligand template. However, cysteine residues are not always available for site-specific protein labeling, and therefore new approaches are needed to expand the toolkit of appropriate electrophiles ("warheads") that target alternative amino acids. We previously described the rational targeting of tyrosine residues in the active site of a protein (the mRNA decapping scavenger enzyme, DcpS) using inhibitors armed with a sulfonyl fluoride electrophile. These inhibitors subsequently enabled the development of clickable probe technology to measure drug-target occupancy in live cells. Here we describe a fluorosulfate-containing inhibitor (aryl fluorosulfate probe (FS-p1)) with excellent chemical and metabolic stability that reacts selectively with a noncatalytic serine residue in the same active site of DcpS as confirmed by peptide mapping experiments. Our results suggest that noncatalytic serine targeting using fluorosulfate electrophilic warheads could be a suitable strategy for the development of covalent inhibitor drugs and chemical probes.

Synthetic Approaches to the New Drugs Approved During 2015.

New drugs introduced to the market every year represent privileged structures for particular biological targets. These new chemical entities (NCEs) provide insight into molecular recognition while serving as leads for designing future new drugs. This annual review describes the most likely process-scale synthetic approaches to 29 new chemical entities (NCEs) that were approved for the first time in 2015.

Design of Potent mRNA Decapping Scavenger Enzyme (DcpS) Inhibitors with Improved Physicochemical Properties To Investigate the Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA).

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.

Chemoselective Preparation of Clickable Aryl Sulfonyl Fluoride Monomers: A Toolbox of Highly Functionalized Intermediates for Chemical Biology Probe Synthesis.

Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.

Selectivity Determination of a Small Molecule Chemical Probe Using Protein Microarray and Affinity Capture Techniques.

Small molecule selectivity is an essential component of candidate drug selection and target validation. New technologies are required to better understand off-target effects, with particular emphasis needed on broad protein profiling. Here, we describe the use of a tritiated chemical probe and a 9000 human protein microarray to discern the binding selectivity of an inhibitor of the mRNA decapping scavenger enzyme DcpS. An immobilized m7GTP resin was also used to assess the selectivity of a DcpS inhibitor against mRNA cap-associated proteins in whole cell extracts. These studies confirm the exquisite selectivity of diaminoquinazoline DcpS inhibitors, and highlight the utility of relatively simple protein microarray and affinity enrichment technologies in drug discovery and chemical biology.

Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells.

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies, the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case, the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity.

Synthetic approaches to the 2014 new drugs.

New drugs introduced to the market every year represent privileged structures for particular biological targets. These new chemical entities (NCEs) provide insight into molecular recognition and also serve as leads for designing future new drugs. This annual review covers the synthesis of thirty-seven NCEs that were approved for the first time in 2014 and one drug which was approved in 2013 and was not covered in a previous edition of this review.

A library approach to rapidly discover photoaffinity probes of the mRNA decapping scavenger enzyme DcpS.

Despite its diverse applications, such as identification of the protein binding partners of small molecules and investigation of intracellular drug-target engagement, photoaffinity labelling (PAL) is intrinsically challenging, primarily due to the difficulty in discovering functionally active photoaffinity probes. Here we describe the creation of a chemoproteomic library to discover a novel photoaffinity probe for DcpS, an mRNA decapping enzyme that is a putative target for Spinal Muscular Atrophy. This library approach expedites the discovery of photoaffinity probes and expands the chemical biology toolbox to include RNA cap-binding proteins.

Synthetic approaches to the 2013 new drugs.

New drugs introduced to the market every year represent privileged structures for particular biological targets. These new chemical entities (NCEs) provide insight into molecular recognition and also serve as leads for designing future new drugs. This annual review covers the synthesis of twenty-four NCEs that were approved for the first time in 2013 and two 2012 drugs which were not covered during the previous edition of this review.

Synthesis of pyrazoles from 1,3-diols via hydrogen transfer catalysis.

1,3-Diols engage in ruthenium-catalyzed hydrogen transfer in the presence of alkyl hydrazines to provide 1,4-disubstituted pyrazoles. Regioselective synthesis of unsymmetrical pyrazoles from ß-hydroxy ketones is also described.

Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes.

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.

Synthetic approaches to the 2012 new drugs.

New drugs introduced to the market every year represent a privileged structure for a particular biological target. These new chemical entities (NCEs) provide insights into molecular recognition and also serve as leads for designing future new drugs. This review covers the synthesis of twenty-six NCEs that were launched or approved worldwide in 2012 and two additional drugs which were launched at the end of 2011.

Catalytic enantioselective allyl-allyl cross-coupling with a borylated allylboronate.

Catalytic enantioselective allyl-allyl cross-coupling of a borylated allylboronate reagent gives versatile borylated chiral 1,5-hexadienes. These compounds may be manipulated in a number of useful ways to give functionalized chiral building blocks for asymmetric synthesis.

Enantioselective construction of all-carbon quaternary centers by branch-selective Pd-catalyzed allyl-allyl cross-coupling.

The Pd-catalyzed cross-coupling of racemic tertiary allylic carbonates and allylboronates is described. This reaction generates all-carbon quaternary centers in a highly regioselective and enantioselective fashion. The outcome of these reactions is consistent with a process that proceeds by way of 3,3'-reductive elimination of bis(η(1)-allyl)palladium intermediates. Strategies for distinguishing the product alkenes and application to the synthesis of (+)-α-cuparenone are also described.

Allylation of nitrosobenzene with pinacol allylboronates. A regioselective complement to peroxide oxidation.

Addition of nitrosobenzene to pinacol allylboronates leads to oxidation of the organoboron with concomitant rearrangement of the substrate alkene. This reaction appears to proceed by allylboration of the nitroso group in analogy to carbonyl and imine allylation reactions. Remarkably, the N-O bond is cleaved during the reaction such that simple alcohols are the final reaction product.

Intramolecular thermal allenyne [2 + 2] cycloadditions; facile construction of the 5-6-4 ring core of sterpurene.

A variety of 1-allenyl-2-propargyl-substituted cyclopentanol derivatives were found to undergo facile intramolecular microwave-assisted 2+2 alleneyne cycloaddition reactions to generate tricyclic 5-6-4 ring systems present in the sterpurenes.

Synthesis of seven-membered carbocyclic rings via a microwave-assisted tandem oxyanionic 5-exo dig cyclization-claisen rearrangement process.

Appropriately substituted 1-alkenyl-4-pentyn-1-ol systems, readily prepared from simple starting materials, serve as useful precursors to a number of substituted cyclohept-4-enone derivatives via a microwave-assisted tandem oxyanionic 5-exo cyclization/Claisen rearrangement sequence. The reactions involving terminally substituted 4-pentyn-1-ols were found to be highly stereoselective, with the alpha and beta groups in the final product showing a strong preference for the trans orientation.

Progress toward the total synthesis of frondosin C.

A straightforward approach toward the total synthesis of frondosin C is described. This strategy involves a key one-pot, microwave-assisted 5-exo cyclization-Claisen rearrangement sequence that was used for the expedient assembly of the frondosic C scaffold. Subsequent manipulation of the tetracyclic core allowed the synthesis of an advanced intermediate bearing the characteristic diene moiety in the B ring. [reaction: see text]