Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Diminishing GSH-Adduct Formation of Tricyclic Diazepine-based Mutant IDH1 Inhibitors.

Mutant isocitrate dehydrogenase 1 (IDH1) has been identified as an attractive oncology target for which >70% of grade II and III gliomas and ∼10% of acute myeloid leukemia (AML) harbor somatic IDH1 mutations. These mutations confer a neomorphic gain of function, leading to the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). We identified and developed a potent, selective, and orally bioavailable brain-penetrant tricyclic diazepine scaffold that inhibits mutant IDH1. During the course of in vitro metabolism studies, GSH-adduct metabolites were observed. The hypothesis for GSH-adduct formation was driven by the electron-rich nature of the tricyclic core. Herein, we describe our efforts to reduce the electron-rich nature of the core. Ultimately, a strategy focused on core modifications to block metabolic hot spots coupled with substitution pattern changes (C8 N → C linked) led to the identification of new tricyclic analogues with minimal GSH-adduct formation across species while maintaining an overall balanced profile.

Oxetane Promise Delivered: Discovery of Long-Acting IDO1 Inhibitors Suitable for Q3W Oral or Parenteral Dosing.

3,3-Disubstituted oxetanes have been utilized as bioisosteres for gem-dimethyl and cyclobutane functionalities. We report the discovery of a novel class of oxetane indole-amine 2,3-dioxygenase (IDO1) inhibitors suitable for Q3W (once every 3 weeks) oral and parenteral dosing. A diamide class of IDO inhibitors was discovered through an automated ligand identification system (ALIS). Installation of an oxetane and fluorophenyl dramatically improved the potency. Identification of a biaryl moiety as an unconventional amide isostere addressed the metabolic liability of amide hydrolysis. Metabolism identification (Met-ID)-guided target design and the introduction of polarity resulted in the discovery of potent IDO inhibitors with excellent pharmacokinetic (PK) profiles in multiple species. To enable rapid synthesis of the key oxetane intermediate, a novel oxetane ring cyclization was also developed, as well as optimization of a literature route on kg scale. These IDO inhibitors may enable unambiguous proof-of-concept testing for the IDO1 inhibition mechanism for oncology.

Comprehensive Strategies to Bicyclic Prolines: Applications in the Synthesis of Potent Arginase Inhibitors.

Comprehensive synthetic strategies afforded a diverse set of structurally unique bicyclic proline-containing arginase inhibitors with a high degree of three-dimensionality. The analogs that favored the Cγ-exo conformation of the proline improved the arginase potency over the initial lead. The novel synthetic strategies reported here not only enable access to previously unknown stereochemically complex proline derivatives but also provide a foundation for the future synthesis of bicyclic proline analogs, which incorporate inherent three-dimensional character into building blocks, medicine, and catalysts and could have a profound impact on the conformation of proline-containing peptides and macrocycles.

Structure-Based Discovery of Proline-Derived Arginase Inhibitors with Improved Oral Bioavailability for Immuno-Oncology.

Recent data suggest that the inhibition of arginase (ARG) has therapeutic potential for the treatment of a number of indications ranging from pulmonary and vascular disease to cancer. Thus, high demand exists for selective small molecule ARG inhibitors with favorable druglike properties and good oral bioavailability. In light of the significant challenges associated with the unique physicochemical properties of previously disclosed ARG inhibitors, we use structure-based drug design combined with a focused optimization strategy to discover a class of boronic acids featuring a privileged proline scaffold with superior potency and oral bioavailability. These compounds, exemplified by inhibitors 4a, 18, and 27, demonstrated a favorable overall profile, and 4a was well tolerated following multiple days of dosing at concentrations that exceed those required for serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model.

Discovery of IDO1 inhibitors containing a decahydroquinoline, decahydro-1,6-naphthyridine, or octahydro-1H-pyrrolo[3,2-c]pyridine scaffold.

A series of IDO1 inhibitors containing a decahydroquinoline, decahydro-1,6-naphthyridine, or octahydro-1H-pyrrolo[3,2-c]pyridine scaffold were identified with good cellular and human whole blood activity against IDO1. These inhibitors contain multiple chiral centers and all diastereomers were separated. The absolute stereochemistry of each isomers were not determined. Compounds 15 and 27 stood out as leads due to their good cellular as well as human whole blood IDO1 inhibition activity, low unbound clearance, and reasonable mean residence time in rat cassette PK studies.

SAR towards indoline and 3-azaindoline classes of IDO1 inhibitors.

A novel series of IDO1 inhibitors have been identified with good IDO1 Hela cell and human whole blood activity. These inhibitors contain an indoline or a 3-azaindoline scaffold. Their structure-activity-relationship studies have been explored. Compounds 37 and 41 stood out as leads due to their good potency in IDO1 Hela assay, good IDO1 unbound hWB IC50s, reasonable unbound clearance, and good MRT in rat and dog PK studies.

Combination of EP4 antagonist MF-766 and anti-PD-1 promotes anti-tumor efficacy by modulating both lymphocytes and myeloid cells.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2-mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

The Discovery of Two Novel Classes of 5,5-Bicyclic Nucleoside-Derived PRMT5 Inhibitors for the Treatment of Cancer.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the post-translational symmetric dimethylation of protein substrates. PRMT5 plays a critical role in regulating biological processes including transcription, cell cycle progression, RNA splicing, and DNA repair. As such, dysregulation of PRMT5 activity is implicated in the development and progression of multiple cancers and is a target of growing clinical interest. Described herein are the structure-based drug designs, robust synthetic efforts, and lead optimization strategies toward the identification of two novel 5,5-fused bicyclic nucleoside-derived classes of potent and efficacious PRMT5 inhibitors. Utilization of compound docking and strain energy calculations inspired novel designs, and the development of flexible synthetic approaches enabled access to complex chemotypes with five contiguous stereocenters. Additional efforts in balancing bioavailability, solubility, potency, and CYP3A4 inhibition led to the identification of diverse lead compounds with favorable profiles, promising in vivo activity, and low human dose projections.

Discovery of Potent and Orally Available Bicyclo[1.1.1]pentane-Derived Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitors.

Indoleamine-2,3-dioxygenase 1 (IDO1) inhibition and its combination with immune checkpoint inhibitors like pembrolizumab have drawn considerable attention from both academia and the pharmaceutical industry. Here, we describe the discovery of a novel class of highly potent IDO1 heme-displacing inhibitors featuring a unique bicyclo[1.1.1]pentane motif. Compound 1, evolving from an ALIS (automated ligand identification system) hit, exhibited excellent potency but lacked the desired pharmacokinetic profile due to extensive amide hydrolysis of the benzamide moiety. Replacing the central phenyl ring in 1 with a bicyclo[1.1.1]pentane bioisostere effectively circumvented the amide hydrolysis issue, resulting in the discovery of compound 2 with a favorable overall profile such as excellent potency, selectivity, pharmacokinetics, and a low predicted human dose.

Discovery and Optimization of Rationally Designed Bicyclic Inhibitors of Human Arginase to Enhance Cancer Immunotherapy.

The action of arginase, a metalloenzyme responsible for the hydrolysis of arginine to urea and ornithine, is hypothesized to suppress immune-cell activity within the tumor microenvironment, and thus its inhibition may constitute a means by which to potentiate the efficacy of immunotherapeutics such as anti-PD-1 checkpoint inhibitors. Taking inspiration from reported enzyme-inhibitor cocrystal structures, we designed and synthesized novel inhibitors of human arginase possessing a fused 5,5-bicyclic ring system. The prototypical member of this class, 3, when dosed orally, successfully demonstrated serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model, despite modest oral bioavailability. Structure-based design strategies to improve the bioavailability of this class, including scaffold modification, fluorination, and installation of active-transport recognition motifs were explored.

A Selective Synthesis of 2,2-Difluorobicyclo[1.1.1]pentane Analogues: "BCP-F2".

The bicyclo[1.1.1]pentane (BCP) motif has been utilized as bioisosteres in drug candidates to replace phenyl, tert-butyl, and alkynyl fragments in order to improve physicochemical properties. However, bceause of the difficulty of synthesis, most BCP analogues prepared only bear 1,3-"para"-substituents. We report the first selective synthesis of 2,2-difluorobicyclo[1.1.1]pentanes via difluorocarbene insertion into bicyclo[1.1.0]butanes. Moreover, this methodology should inspire future studies on synthesis of other "ortho/meta-substituted" BCPs via similar mechanisms.

Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer's disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

Polycyclic xanthone natural products: structure, biological activity and chemical synthesis.

Polycyclic xanthone natural products are a family of polyketides which are characterized by highly oxygenated, angular hexacyclic frameworks. In the last decade, this novel class of molecules has attracted noticeable attention from the synthetic and biological communities due to emerging reports of their potential use as antitumour agents. The aim of this article is to highlight the most recent developments of this subset of the xanthone family by detailing the innate challenges of the construction of this class of natural products, new synthetic approaches, and pharmacological data.

Total synthesis and absolute stereochemical assignment of kibdelone C.

Kibdelones are hexacyclic tetrahydroxanthones and potent anticancer agents isolated from an Australian microbe. Herein, we describe the synthesis of a chiral, nonracemic iodocyclohexene carboxylate EF ring fragment of the kibdelones employing an intramolecular iodo halo-Michael aldol reaction and its merger with an ABCD ring fragment to afford the congener kibdelone C.

Fluorinated piperidine acetic acids as gamma-secretase modulators.

We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.

Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2).

We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.

The discovery of 6-amino nicotinamides as potent and selective histone deacetylase inhibitors.

This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.