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.

Discovery of TRPA1 Antagonist GDC-6599: Derisking Preclinical Toxicity and Aldehyde Oxidase Metabolism with a Potential First-in-Class Therapy for Respiratory Disease.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metabolite─generated by aldehyde oxidase (AO)─possessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.

Discovery of Selective Tertiary Amide Inhibitors of Cyclin-Dependent Kinase 2 (CDK2).

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and are frequently altered in cancer cells, thereby leading to uncontrolled proliferation. In this context, CDK2 has emerged as an appealing target for anticancer drug development. Herein, we describe the discovery of a series of selective small molecule inhibitors of CDK2 beginning with historical compounds from our ERK2 program (e.g., compound 6). Structure-based drug design led to the potent and selective tool compound 32, where excellent selectivity against ERK2 and CDK4 was achieved by filling the lipophilic DFG-1 pocket and targeting interactions with CDK2-specific lower hinge binding residues, respectively. Compound 32 demonstrated 112% tumor growth inhibition in mice bearing OVCAR3 tumors with 50 mg/kg bis in die (BID) oral dosing.

A Retrospective Look at the Impact of Binding Site Environment on the Optimization of TRPA1 Antagonists.

Transient receptor potential ankyrin 1 (TRPA1) antagonists have generated broad interest in the pharmaceutical industry for the treatment of both pain and asthma. Over the past decade, multiple antagonist classes have been reported in the literature with a wide range of structural diversity. Our own work has focused on the development of proline sulfonamide and hypoxanthine-based antagonists, two antagonist classes with distinct physicochemical properties and pharmacokinetic (PK) trends. Late in our discovery program, cryogenic electron microscopy (cryoEM) studies revealed two different antagonist binding sites: a membrane-exposed proline sulfonamide transmembrane site and an intracellular hypoxanthine site near the membrane interface. A retrospective look at the discovery program reveals how the different binding sites, and their location relative to the cell membrane, influenced the optimization trajectories and overall drug profiles of each antagonist class.

Tetrahydrofuran-Based Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists: Ligand-Based Discovery, Activity in a Rodent Asthma Model, and Mechanism-of-Action via Cryogenic Electron Microscopy.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.

Asymmetric total synthesis of (+)-virosine A via sequential nucleophilic cyclizations onto an activated formamide.

The first synthesis of tetracyclic alkaloid virosine A is reported. The natural alkaloid was prepared in only 13 steps, in an enantioenriched form. The azabicyclo[2.2.2]octane core was efficiently assembled using a key Vilsmeier-Haack and Mannich cyclizations sequence performed in one pot.

Use of alkyl 2,4,6-triisopropylbenzoates in the asymmetric homologation of challenging boronic esters.

(-)-Sparteine induced lithiation of primary 2,4,6-triisopropylbenzoates and subsequent homologation of boronic esters is reported. A comparative study with lithiated N,N-diisopropylcarbamates has demonstrated the superiority of the hindered benzoate.

Ate complexes of secondary boronic esters as chiral organometallic-type nucleophiles for asymmetric synthesis.

The addition of an aryllithium reagent to a secondary boronic ester leads to an intermediate boron-ate complex that behaves as a chiral nucleophile, reacting with a broad range of electrophiles with inversion of stereochemistry. Depending on the electrophile, the C-B bond can be converted into C-I, C-Br, C-Cl, C-N, C-O, and C-C, all with very high levels of stereocontrol. This discovery now adds a new, readily available, configurationally stable, chiral organometallic-type reagent to the arsenal of methods for use in asymmetric organic synthesis.

Rapid assembly of quinolizidines via consecutive nucleophilic cyclizations onto activated amides.

A new approach to the synthesis of quinolizidines involving a cascade of nucleophilic cyclizations triggered by chemoselective amide activation is reported. Particular attention was given to the effect of the nature of the tethered nucleophiles on the cascade of cyclizations. As a result, simple acyclic amides gave rapid access to functionalized quinolizidines bearing either a tertiary or quaternary center at the ring junction. Such a fused bicyclic motif is found in several alkaloids.

Synthesis of the tricyclic core of alkaloid securinol B using a cascade of Vilsmeier-Haack and Mannich cyclizations.

Iminium ions generated upon amide activation were trapped sequentially with tethered nucleophiles. This cascade of cyclizations constitutes a new synthetic strategy that was applied to the construction of the tricyclic core of alkaloid securinol B.

Intramolecular additions of various pi-nucleophiles to chemoselectively activated amides and application to the synthesis of (+/-)-tashiromine.

[reaction: see text] Vilsmeier-Haack type cyclizations proved to be particularly efficient for generating parts of the polycyclic cores of many alkaloids, although only monocyclizations have so far been reported. With the goal of rapidly and efficiently constructing polycyclic alkaloids, we decided to exploit the Vilsmeier-Haack reaction by utilizing iminium ions successively generated and trapped with tethered nucleophiles. To develop such a strategy, we had to set the first cyclization. This constitutes a great challenge in itself because amide activation conditions are usually not compatible with tethered nucleophiles, except for indoles and aromatic rings which have already been reported. This paper describes the comprehensive study of intramolecular addition of silyl enol ethers, allylsilanes, and enamines to chemoselectively activated formamides, aliphatic amides, and lactams. Good to excellent yields were obtained for the 5-exo, 6-exo, and 6-endo modes of cyclization. Moreover, we demonstrated that the species in solution after the cyclization are iminium ions. This is highly encouraging for the development of bis-cyclization strategies. An expeditious total synthesis of (+/-)-tashiromine is also reported.

Addition of tethered nonaromatic carbon nucleophiles to chemoselectively activated amides.

[reaction: see text] In an effort to develop new ways of synthesizing polycyclic alkaloids, we successfully added silyl enol ethers, allylsilanes, and enamines to iminium ions generated from amides. Because of their higher oxidation state, such iminiums show a yet unexploited advantage of potential double cyclizations over standard Mannich monocyclizations. We report herein the first example of tethered nonaromatic carbon nucleophiles adding to activated amides for the generation of enaminals of various ring sizes, with endo- or exo-cyclic nitrogen.