Skeletal Editing Approach to Bridge-Functionalized Bicyclo[1.1.1]pentanes from Azabicyclo[2.1.1]hexanes.

Azabicyclo[2.1.1]hexanes (aza-BCHs) and bicyclo[1.1.1]pentanes (BCPs) have emerged as attractive classes of sp3-rich cores for replacing flat, aromatic groups with metabolically resistant, three-dimensional frameworks in drug scaffolds. Strategies to directly convert, or "scaffold hop", between these bioisosteric subclasses through single-atom skeletal editing would enable efficient interpolation within this valuable chemical space. Herein, we describe a strategy to "scaffold hop" between aza-BCH and BCP cores through a nitrogen-deleting skeletal edit. Photochemical [2+2] cycloadditions, used to prepare multifunctionalized aza-BCH frameworks, are coupled with a subsequent deamination step to afford bridge-functionalized BCPs, for which few synthetic solutions currently exist. The modular sequence provides access to various privileged bridged bicycles of pharmaceutical relevance.

Pharmacologic Characterization of JNJ-42226314, [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone, a Reversible, Selective, and Potent Monoacylglycerol Lipase Inhibitor.

The serine hydrolase monoacylglycerol lipase (MAGL) is the rate-limiting enzyme responsible for the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. Inhibition of 2-AG degradation leads to elevation of 2-AG, the most abundant endogenous agonist of the cannabinoid receptors (CBs) CB1 and CB2. Activation of these receptors has demonstrated beneficial effects on mood, appetite, pain, and inflammation. Therefore, MAGL inhibitors have the potential to produce therapeutic effects in a vast array of complex human diseases. The present report describes the pharmacologic characterization of [1-(4-fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone (JNJ-42226314), a reversible and highly selective MAGL inhibitor. JNJ-42226314 inhibits MAGL in a competitive mode with respect to the 2-AG substrate. In rodent brain, the compound time- and dose-dependently bound to MAGL, indirectly led to CB1 occupancy by raising 2-AG levels, and raised norepinephrine levels in cortex. In vivo, the compound exhibited antinociceptive efficacy in both the rat complete Freund's adjuvant-induced radiant heat hypersensitivity and chronic constriction injury-induced cold hypersensitivity models of inflammatory and neuropathic pain, respectively. Though 30 mg/kg induced hippocampal synaptic depression, altered sleep onset, and decreased electroencephalogram gamma power, 3 mg/kg still provided approximately 80% enzyme occupancy, significantly increased 2-AG and norepinephrine levels, and produced neuropathic antinociception without synaptic depression or decreased gamma power. Thus, it is anticipated that the profile exhibited by this compound will allow for precise modulation of 2-AG levels in vivo, supporting potential therapeutic application in several central nervous system disorders. SIGNIFICANCE STATEMENT: Potentiation of endocannabinoid signaling activity via inhibition of the serine hydrolase monoacylglycerol lipase (MAGL) is an appealing strategy in the development of treatments for several disorders, including ones related to mood, pain, and inflammation. [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone is presented in this report to be a novel, potent, selective, and reversible noncovalent MAGL inhibitor that demonstrates dose-dependent enhancement of the major endocannabinoid 2-arachidonoylglycerol as well as efficacy in models of neuropathic and inflammatory pain.

D3R grand challenge 4: blind prediction of protein-ligand poses, affinity rankings, and relative binding free energies.

The Drug Design Data Resource (D3R) aims to identify best practice methods for computer aided drug design through blinded ligand pose prediction and affinity challenges. Herein, we report on the results of Grand Challenge 4 (GC4). GC4 focused on proteins beta secretase 1 and Cathepsin S, and was run in an analogous manner to prior challenges. In Stage 1, participant ability to predict the pose and affinity of BACE1 ligands were assessed. Following the completion of Stage 1, all BACE1 co-crystal structures were released, and Stage 2 tested affinity rankings with co-crystal structures. We provide an analysis of the results and discuss insights into determined best practice methods.

D3R Grand Challenge 3: blind prediction of protein-ligand poses and affinity rankings.

The Drug Design Data Resource aims to test and advance the state of the art in protein-ligand modeling by holding community-wide blinded, prediction challenges. Here, we report on our third major round, Grand Challenge 3 (GC3). Held 2017-2018, GC3 centered on the protein Cathepsin S and the kinases VEGFR2, JAK2, p38-α, TIE2, and ABL1, and included both pose-prediction and affinity-ranking components. GC3 was structured much like the prior challenges GC2015 and GC2. First, Stage 1 tested pose prediction and affinity ranking methods; then all available crystal structures were released, and Stage 2 tested only affinity rankings, now in the context of the available structures. Unique to GC3 was the addition of a Stage 1b self-docking subchallenge, in which the protein coordinates from all of the cocrystal structures used in the cross-docking challenge were released, and participants were asked to predict the pose of CatS ligands using these newly released structures. We provide an overview of the outcomes and discuss insights into trends and best-practices.

Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-γ8.

Members of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediate the majority of fast synaptic transmission within the mammalian brain and spinal cord, representing attractive targets for therapeutic intervention. Here, we describe novel AMPA receptor modulators that require the presence of the accessory protein CACNG8, also known as transmembrane AMPA receptor regulatory protein γ8 (TARP-γ8). Using calcium flux, radioligand binding, and electrophysiological assays of wild-type and mutant forms of TARP-γ8, we demonstrate that these compounds possess a novel mechanism of action consistent with a partial disruption of the interaction between the TARP and the pore-forming subunit of the channel. One of the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one (JNJ-55511118), had excellent pharmacokinetic properties and achieved high receptor occupancy following oral administration. This molecule showed strong, dose-dependent inhibition of neurotransmission within the hippocampus, and a strong anticonvulsant effect. At high levels of receptor occupancy in rodent in vivo models, JNJ-55511118 showed a strong reduction in certain bands on electroencephalogram, transient hyperlocomotion, no motor impairment on rotarod, and a mild impairment in learning and memory. JNJ-55511118 is a novel tool for reversible AMPA receptor inhibition, particularly within the hippocampus, with potential therapeutic utility as an anticonvulsant or neuroprotectant. The existence of a molecule with this mechanism of action demonstrates the possibility of pharmacological targeting of accessory proteins, increasing the potential number of druggable targets.

Preclinical characterization of substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists.

The synthesis, SAR, and preclinical characterization of a series of substituted 6,7-dihydro[1,2,4]triazolo[4,3]pyrazin-8(5H)-one P2X7 receptor antagonists are described. Optimized leads from this series comprise some of the most potent human P2X7R antagonists reported to date (IC50s<1nM). They also exhibit sufficient potency and oral bioavailability in rat to enable extensive in vivo profiling. Although many of the disclosed compounds are peripherally restricted, compound 11d is brain penetrant and upon oral administration demonstrated dose-dependent target engagement in rat hippocampus as determined by ex vivo receptor occupancy with radiotracer 5 (ED50=0.8mg/kg).

Novel methyl substituted 1-(5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanones are P2X7 antagonists.

The optimization efforts that led to a novel series of methyl substituted 1-(5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanones that are potent rat and human P2X7 antagonists are described. These efforts resulted in the discovery of compounds with good drug-like properties that are capable of high P2X7 receptor occupancy in rat following oral administration, including compounds 7n (P2X7 IC50 = 7.7 nM) and 7u (P2X7 IC50 =7 .7 nM). These compounds are expected to be useful tools for characterizing the effects of P2X7 antagonism in models of depression and epilepsy, and several of the compounds prepared are candidates for effective P2X7 PET tracers.

Convergent synthesis of thiodiazole dioxides from simple ketones and amines through an unusual nitrogen-migration mechanism.

We report the modular preparation of dihydro-1,2,5-thiodiazole dioxide heterocycles starting from methyl ketones and primary amines. This one-pot, three-component coupling employs 2,3-dimethylimidazole-1-sulfonyl azide triflate as a coupling reagent and oxidant. The transformation is scalable and various ketones and amines can be used, yielding thiodiazole dioxide products in up to 89% yield. In addition, 15N- and 13C-labeling studies suggest a mechanism involving a 1,2-nitrogen migration. Together with the mechanistic studies, DFT calculations provide insight into the reaction pathway and set the stage for further exploration of the mechanistic nuances of reactions that use sulfamoyl azides. In combination with the demonstrated modularity of the approach reported herein, the derivatization of the thiodiazole dioxide products highlights the potential of this methodology to rapidly access diverse chemical structures.

Development of a flexible strategy towards FR900482 and the mitomycins.

FR900482 and the mitomycins are two intriguing classes of alkaloid natural products that have analogous biological mechanisms and obvious structural similarity. Both classes possess potent anticancer activity, a feature that has led to their investigation and implementation for the clinical treatment of human cancer. Given the structural similarity between these natural products, we envisioned a common synthetic strategy by which both classes could be targeted through assembling the mitomycin skeleton prior to further oxidative functionalization. Realization of this strategy with respect to FR900482 was accomplished through the synthesis of 7-epi-FR900482, which displayed equal potency relative to the natural product against two human cancer cell lines. With the challenging goal of a synthesis of either mitomycin or FR900482 in mind, several methodologies were explored. While not all of these methods ultimately proved useful for our synthetic goal, a number of them led to intriguing findings that provide a more complete understanding of several methodologies. In particular, amination via π-allyl palladium complexes for the synthesis of tetrahydroquinolines, eight-membered heterocycle formation via carbonylative lactamization, and amination through late-stage C-H insertion via rhodium catalysis all featured prominently in our synthetic studies.

Diazinones as P2 replacements for pyrazole-based cathepsin S inhibitors.

A pyridazin-4-one fragment 4 (hCatS IC(50)=170 microM) discovered through Tethering was modeled into cathepsin S and predicted to overlap in S2 with the tetrahydropyridinepyrazole core of a previously disclosed series of CatS inhibitors. This fragment served as a template to design pyridazin-3-one 12 (hCatS IC(50)=430 nM), which also incorporates P3 and P5 binding elements. A crystal structure of 12 bound to Cys25Ser CatS led to the synthesis of the potent diazinone isomers 22 (hCatS IC(50)=60 nM) and 27 (hCatS IC(50)=40 nM).

Discovery and SAR of novel pyrazole-based thioethers as cathepsin S inhibitors: part 1.

A series of pyrazole-based thioethers were prepared and found to be potent cathepsin S inhibitors. A crystal structure of 13 suggests that the thioether moiety may bind to the S3 pocket of the enzyme. Additional optimization led to the discovery of aminoethylthioethers with improved enzymatic activity and submicromolar cellular potency.

Selective inhibition of peripheral cathepsin S reverses tactile allodynia following peripheral nerve injury in mouse.

Cathepsin S (CatS) is a cysteine protease found in lysosomes of hematopoietic and microglial cells and in secreted form in the extracellular space. While CatS has been shown to contribute significantly to neuropathic pain, the precise mechanisms remain unclear. In this report, we describe JNJ-39641160, a novel non-covalent, potent, selective and orally-available CatS inhibitor that is peripherally restricted (non-CNS penetrant) and may represent an innovative class of immunosuppressive and analgesic compounds and tools useful toward investigating peripheral mechanisms of CatS in neuropathic pain. In C57BL/6 mice, JNJ-39641160 dose-dependently blocked the proteolysis of the invariant chain, and inhibited both T-cell activation and antibody production to a vaccine antigen. In the spared nerve injury (SNI) model of chronic neuropathic pain, in which T-cell activation has previously been demonstrated to be a prerequisite for the development of pain hypersensitivity, JNJ-39641160 fully reversed tactile allodynia in wild-type mice but was completely ineffective in the same model in CatS knockout mice (which exhibited a delayed onset in allodynia). By contrast, in the acute mild thermal injury (MTI) model, JNJ-39641160 only weakly attenuated allodynia at the highest dose tested. These findings support the hypothesis that blockade of peripheral CatS alone is sufficient to fully reverse allodynia following peripheral nerve injury and suggest that the mechanism of action likely involves interruption of T-cell activation and peripheral cytokine release. In addition, they provide important insights toward the development of selective CatS inhibitors for the treatment of neuropathic pain in humans.

Pyrazole-based arylalkyne cathepsin S inhibitors. Part II: optimization of cellular potency.

Basic lipophilic substituents dramatically improved the cellular potency of a previously disclosed series of pyrazole-based arylalkyne cathepsin S inhibitors. The incorporation of substituted benzylamines in the para position of the arylalkyne maintained enzymatic activity (hCatS IC50=80-420 nM) and imparted cellular potency (IC50=0.8-4.0 microM). Further refinement of the morpholine portion of the pharmacophore enabled the identification of bicyclic piperidines with enhanced affinity for CatS (IC50=10-30 nM) and sub-micromolar cellular potency (JY Ii IC50=200-720 nM).

Pyrazole-based cathepsin S inhibitors with arylalkynes as P1 binding elements.

A crystal structure of 1 bound to a Cys25Ser mutant of cathepsin S helped to elucidate the binding mode of a previously disclosed series of pyrazole-based CatS inhibitors and facilitated the design of a new class of arylalkyne analogs. Optimization of the alkyne and tetrahydropyridine portions of the pharmacophore provided potent CatS inhibitors (IC50=40-300 nM), and an X-ray structure of 32 revealed that the arylalkyne moiety binds in the S1 pocket of the enzyme.

Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-c]pyrimidines as TARP γ-8 Selective AMPAR Negative Modulators.

This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure-activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

Lead Optimization of 5-Aryl Benzimidazolone- and Oxindole-Based AMPA Receptor Modulators Selective for TARP γ-8.

Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. High-throughput screen lead 4 was optimized for potency and brain penetration to provide benzimidazolone 3, JNJ-55511118.1 Replacement of the benzimidazolone core in 3 with an oxindole mitigated reactive metabolite formation and led to the identification of 18 (GluA1/γ-8 pIC50 = 9.7). Following oral dosing in rats, 18 demonstrated robust target engagement in hippocampus as assessed by ex vivo autoradiography (ED50 = 0.6 mg/kg, plasma EC50 = 9 ng/mL).

A novel radioligand for the ATP-gated ion channel P2X7: [3H] JNJ-54232334.

The ATP-gated ion channel P2X7 has emerged as a potential central nervous system (CNS) drug target based on the hypotheses that pro-inflammatory cytokines such as IL-1ß that are released by microglia, may contribute to the etiology of various disorders of the CNS including depression. In this study, we identified two closely related P2X7 antagonists, JNJ-54232334 and JNJ-54140515, and then tritium labeled the former to produce a new radioligand for P2X7. JNJ-54232334 is a high affinity ligand for the rat P2X7 with a pKi of 9.3±0.1. In rat cortical membranes, [3H] JNJ-54232334 reached saturable binding with equilibrium dissociation (Kd) constant of 4.9±1.3 nM. The compound displayed monophasic association and dissociation kinetics with fast on and off rates. In rat brain sections, specific binding of [3H] JNJ-54232334 was markedly improved compared to the previously described P2X7 radioligand, [3H] A-804598. In P2X7 knockout mouse brain sections, [3H] A-804598 bound to non-P2X7 binding sites in contrast to [3H] JNJ-54232334. In rat or wild type mouse brain sections [3H] JNJ-54232334 bound in a more homogenous and region independent manner. The ubiquitous expression of P2X7 receptors was confirmed with immunohistochemistry in rat brain sections. The partial displacement of [3H] A-804598 binding resulted in the underestimation of the level of ex vivo P2X7 occupancy for JNJ-54140515. Higher levels of P2X7 ex vivo occupancy were measured using [3H] JNJ-54232334 due to less non-specific binding. In summary, we describe [3H] JNJ-54232334 as a novel P2X7 radioligand, with improved properties over [3H] A-804598.

Pd-catalyzed carbonylative lactamization: a novel synthetic approach to FR900482.

An asymmetric synthesis of the benzazocine core of FR900482 has been achieved in 15 steps from 3,5-dinitro-p-toluic acid. Key features of the synthesis include an enantioselective N-methylephedrine-mediated zinc acetylide addition to a highly enolizable arylacetaldehyde and a novel Pd-catalyzed carbonylative lactamization to form an eight-membered ring. [reaction--see text]

Phosphoinositide 3-kinase gamma (PI3Kgamma) inhibitors for the treatment of inflammation and autoimmune disease.

Phosphoinositide 3-kinase gamma (PI3Kgamma) is a lipid kinase in leukocytes that generates phosphatidylinositol 3,4,5-trisphosphate to recruit and activate downstream signaling molecules. Distinct from other members in the PI3K family, PI3Kgamma is activated by G-protein coupled-receptors responding to chemotactic ligands. PI3Kgamma plays an important role in migration of both myeloid and lymphoid cells. It is also required for other leukocyte functions such as neutrophil oxidative burst, T cell proliferation and mast degranulation. Mice with PI3Kgamma inactivated by genetic or pharmacological approaches are protected from disease development in a number of inflammation and autoimmune disease models. The function of PI3Kgamma depends on its kinase activity and therefore it has been suggested by many reports that small molecules inhibiting its kinase activity could be promising for the treatment of inflammation and autoimmune diseases. Over the last five years, a number of pharmaceutical companies have reported a wide variety of PI3Kgamma inhibitors, of which several x-ray crystal structures with PI3Kgamma have been elucidated. The structural characteristics and selectivity profiles of these inhibitors, in particular thiazolidinones and 2-aminoheterocycles, and those disclosed in related patent applications are summarized in this review.