Enabling Deoxygenative C(sp2)-C(sp3) Cross-Coupling for Parallel Medicinal Chemistry.

Herein we report the development of an automated deoxygenative C(sp2)-C(sp3) coupling of aryl bromide with alcohols to enable parallel medicinal chemistry. Alcohols are among the most diverse and abundant building blocks, but their usage as alkyl precursors has been limited. Although metallaphotoredox deoxygenative coupling is becoming a promising strategy to form C(sp2)-C(sp3) bond, the reaction setup limits its widespread application in library synthesis. To achieve high throughput and consistency, an automated workflow involving solid-dosing and liquid-handling robots has been developed. We have successfully demonstrated this high-throughput protocol is robust and consistent across three automation platforms. Furthermore, guided by cheminformatic analysis, we examined alcohols with comprehensive chemical space coverage and established a meaningful scope for medicinal chemistry applications. By accessing the rich diversity of alcohols, this automated protocol has the potential to substantially increase the impact of C(sp2)-C(sp3) cross-coupling in drug discovery.

Direct-to-Biology Accelerates PROTAC Synthesis and the Evaluation of Linker Effects on Permeability and Degradation.

A platform to accelerate optimization of proteolysis targeting chimeras (PROTACs) has been developed using a direct-to-biology (D2B) approach with a focus on linker effects. A large number of linker analogs-with varying length, polarity, and rigidity-were rapidly prepared and characterized in four cell-based assays by streamlining time-consuming steps in synthesis and purification. The expansive dataset informs on linker structure-activity relationships (SAR) for in-cell E3 ligase target engagement, degradation, permeability, and cell toxicity. Unexpected aspects of linker SAR was discovered, consistent with literature reports on "linkerology", and the method dramatically speeds up empirical optimization. Physicochemical property trends emerged, and the platform has the potential to rapidly expand training sets for more complex prediction models. In-depth validation studies were carried out and confirm the D2B platform is a valuable tool to accelerate PROTAC design-make-test cycles.

Redefining the Histone Deacetylase Inhibitor Pharmacophore: High Potency with No Zinc Cofactor Interaction.

A novel series of histone deacetylase (HDAC) inhibitors lacking a zinc-binding moiety has been developed and described herein. HDAC isozyme profiling and kinetic studies indicate that these inhibitors display a selectivity preference for HDACs 1, 2, 3, 10, and 11 via a rapid equilibrium mechanism, and crystal structures with HDAC2 confirm that these inhibitors do not interact with the catalytic zinc. The compounds are nonmutagenic and devoid of electrophilic and mutagenic structural elements and exhibit off-target profiles that are promising for further optimization. The efficacy of this new class in biochemical and cell-based assays is comparable to the marketed HDAC inhibitors belinostat and vorinostat. These results demonstrate that the long-standing pharmacophore model of HDAC inhibitors requiring a metal binding motif should be revised and offers a distinct class of HDAC inhibitors.

Trends in Hit-to-Lead Optimization Following DNA-Encoded Library Screens.

DNA-encoded library (DEL) screens have emerged as a powerful hit-finding tool for a number of biological targets. In this Innovations article, we review published hit-to-lead optimization studies following DEL screens. Trends in molecular property changes from hit to lead are identified, and specific optimization tactics are exemplified in case studies. Across the studies, physicochemical property and structural changes post-DEL screening are similar to those which occur during hit-to-lead optimization following high throughputscreens (HTS). However, unique aspects of DEL-the combinatorial synthetic methods which enable DEL synthesis and the linker effects at the DNA attachment point-impact the strategies and outcomes of hit-to-lead optimizations.

Discovery of an Anion-Dependent Farnesyltransferase Inhibitor from a Phenotypic Screen.

By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.

Discovery of Highly Selective and Potent HDAC3 Inhibitors Based on a 2-Substituted Benzamide Zinc Binding Group.

The selectivity of histone deacetylase inhibitors (HDACis) is greatly impacted by the zinc binding groups. In an effort to search for novel zinc binding groups, we applied a parallel medicinal chemistry (PMC) strategy to quickly synthesize substituted benzamide libraries. We discovered a series containing 2-substituted benzamides as the zinc binding group which afforded highly selective and potent HDAC3 inhibitors, exemplified by compound 16 with a 2-methylthiobenzamide. Compound 16 inhibited HDAC3 with an IC50 of 30 nM and with unprecedented selectivity of >300-fold over all other HDAC isoforms. Interestingly, a subtle change of the 2-methylthio to a 2-hydroxy benzamide in 20 retains HDAC3 potency but loses all selectivity over HDAC 1 and 2. This significant difference in selectivity was rationalized by X-ray crystal structures of HDACis 16 and 20 bound to HDAC2, revealing different binding modes to the catalytic zinc ion. This series of HDAC3 selective inhibitors served as tool compounds for investigating the minimal set of HDAC isoforms that must be inhibited for the HIV latency activation in a Jurkat 2C4 cell model and potentially as leads for selective HDAC3 inhibitors for other indications.

Discovery of ethyl ketone-based HDACs 1, 2, and 3 selective inhibitors for HIV latency reactivation.

A novel series of ethyl ketone based HDACs 1, 2, and 3 selective inhibitors have been identified with good enzymatic and cellular activity and high selectivity over HDACs 6 and 8. These inhibitors contain a spirobicyclic group in the amide region. Compound 13 stands out as a lead due to its good potency, high selectivity, and reasonable rat and dog PK. Compounds 33 and 34 show good potency and rat PK profiles as well.

Reducing Limitation in Probe Design: The Development of a Diazirine-Compatible Suzuki-Miyaura Cross Coupling Reaction.

Access to high quality photoaffinity probe molecules is often constrained by synthetic limitations related to diazirine installation. A survey of recently published photoaffinity probe syntheses identified the Suzuki-Miyaura (S-M) coupling reaction, ubiquitous in drug discovery, as being underutilized to incorporate diazirines. To test whether advances in modern cross-coupling catalysis might enable efficient S-M couplings tolerant of the diazirine moiety, a fragment-based screening approach was employed. A model S-M coupling reaction was screened under various conditions in the presence of an aromatic diazirine fragment. This screen identified reaction conditions that gave good yields of S-M coupling product while minimally perturbing the diazirine reporter fragment. These conditions were found to be highly scalable and exhibited broad scope when applied to a chemistry informer library of 24 pharmaceutically relevant aryl boron pinacol esters. Furthermore, these conditions were used to synthesize a known diazirine-containing probe molecule with improved synthetic efficiency.

Benzoxazolinone aryl sulfonamides as potent, selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model.

Studies on human genetics have suggested that inhibitors of the Nav1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Nav1.7 inhibitors with excellent selectivity against the Nav1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Nav1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.

Discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity and pharmacokinetics.

Selective inhibition of Kv1.5, which underlies the ultra-rapid delayed rectifier current, IKur, has been pursued as a treatment for atrial fibrillation. Here we describe the discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity versus the off-target current IKs, whose inhibition has been associated with ventricular proarrhythmia. MK-1832 exhibits improved selectivity for IKur over IKs (>3000-fold versus 70-fold for MK-0448), consistent with an observed larger window between atrial and ventricular effects in vivo (>1800-fold versus 210-fold for MK-0448). MK-1832 also exhibits an improved preclinical pharmacokinetic profile consistent with projected once daily dosing in humans.

Synthesis and optimization of N-heterocyclic pyridinones as catechol-O-methyltransferase (COMT) inhibitors.

A series of N-heterocyclic pyridinone catechol-O-methyltransferase (COMT) inhibitors were synthesized. Physicochemical properties, including ligand lipophilic efficiency (LLE) and clogP, were used to guide compound design and attempt to improve inhibitor pharmacokinetics. Incorporation of heterocyclic central rings provided improvements in physicochemical parameters but did not significantly reduce in vitro or in vivo clearance. Nevertheless, compound 11 was identified as a potent inhibitor with sufficient in vivo exposure to significantly affect the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and indicate central COMT inhibition.

Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT).

3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

[18F]Fluoroazabenzoxazoles as potential amyloid plaque PET tracers: synthesis and in vivo evaluation in rhesus monkey.

INTRODUCTION: An (18)F-labeled positron emission tomography (PET) tracer for amyloid plaque is desirable for early diagnosis of Alzheimer's disease, particularly to enable preventative treatment once effective therapeutics are available. Similarly, such a tracer would be useful as a biomarker for enrollment of patients in clinical trials for evaluation of antiamyloid therapeutics. Furthermore, changes in the level of plaque burden as quantified by an amyloid plaque PET tracer may provide valuable insights into the effectiveness of amyloid-targeted therapeutics. This work describes our approach to evaluate and select a candidate PET tracer for in vivo quantification of human amyloid plaque. METHODS: Ligands were evaluated for their in vitro binding to human amyloid plaques, lipophilicity and predicted blood-brain barrier permeability. Candidates with favorable in vitro properties were radiolabeled with (18)F and evaluated in vivo. Baseline PET scans in rhesus monkey were conducted to evaluate the regional distribution and kinetics of each tracer using tracer kinetic modeling methods. High binding potential in cerebral white matter and cortical grey matter was considered an unfavorable feature of the candidate tracers. RESULTS: [(18)F]MK-3328 showed the most favorable combination of low in vivo binding potential in white matter and cortical grey matter in rhesus monkeys, low lipophilicity (Log D=2.91) and high affinity for human amyloid plaques (IC(50)=10.5±1.3 nM). CONCLUSIONS: [(18)F]MK-3328 was identified as a promising PET tracer for in vivo quantification of amyloid plaques, and further evaluation in humans is warranted.

Design, synthesis, and evaluation of novel 3,6-diaryl-4-aminoalkoxyquinolines as selective agonists of somatostatin receptor subtype 2.

Agonists of somatostatin receptor subtype 2 (sst(2)) have been proposed as therapeutics for the treatment of proliferative diabetic retinopathy and exudative age-related macular degeneration. An HTS screen identified 2-quinolones as weak agonists of sst(2), and these were optimized to provide small molecules with sst(2) binding and functional potency comparable to peptide agonists. Agonist 21 was shown to inhibit rat growth hormone secretion following systemic administration and to inhibit ocular neovascular lesion formation after local administration.

High concentration electrophysiology-based fragment screen: discovery of novel acid-sensing ion channel 3 (ASIC3) inhibitors.

The Merck Fragment Library was screened versus acid-sensing ion channel 3 (ASIC3), a novel target for the treatment of pain. Fragment hits were optimized using two strategies, and potency was improved from 0.7 mM to 3 µM with retention of good ligand efficiency and incorporation of reasonable physical properties, off-target profile, and rat pharmacokinetics.

Synthesis and Evaluation of 5-Fluoro-2-aryloxazolo[5,4-b]pyridines as ß-Amyloid PET Ligands and Identification of MK-3328.

5-Fluoro-2-aryloxazolo[5,4-b]pyridines were synthesized and investigated as potential (18)F containing ß-amyloid PET ligands. In competition binding assays using human AD brain homogenates, compounds 14b, 16b, and 17b were identified as having favorable potency versus human ß-amyloid plaque and were radiolabeled for further evaluation in in vitro binding and in vivo PET imaging experiments. These studies led to the identification of 17b (MK-3328) as a candidate PET ligand for the clinical assessment of ß-amyloid plaque load.

The synthesis and preclinical evaluation in rhesus monkey of [¹8F]MK-6577 and [¹¹C]CMPyPB glycine transporter 1 positron emission tomography radiotracers.

Two positron emission tomography radiotracers for the glycine transporter 1 (GlyT1) are reported here. Each radiotracer is a propylsulfonamide-containing benzamide and was labeled with either carbon-11 or fluorine-18. [¹¹C]CMPyPB was synthesized by the alkylation of a 3-hydroxypyridine precursor using [¹¹C]MeI, and [¹8F]MK-6577 was synthesized by a nucleophilic aromatic substitution reaction using a 2-chloropyridine precursor. Each tracer shows good uptake into rhesus monkey brain with the expected distribution of highest uptake in the pons, thalamus, and cerebellum and lower uptake in the striatum and gray matter of the frontal cortex. In vivo blockade and chase studies of [¹8F]MK-6577 showed a large specific signal and reversible binding. In vitro autoradiographic studies with [¹8F]MK-6577 showed a large specific signal in both rhesus monkey and human brain slices and a distribution consistent with the in vivo results and those reported in the literature. In vivo metabolism studies in rhesus monkeys demonstrated that only more-polar metabolites are formed for each tracer. Of these two tracers, [¹8F]MK-6577 was more extensively characterized and is a promising clinical positron emission tomography tracer for imaging GlyT1 and for measuring GlyT1 occupancy of therapeutic compounds.

Recent progress in the discovery of non-sarcosine based GlyT1 inhibitors.

The simple amino acid glycine is implicated in both inhibitory and excitatory neurotransmission in mammalian central nervous system, and it modulates excitatory neurotransmission through its role as a necessary co-agonist for glutamatergic N-methyl-D-aspartate (NMDA) receptors. Given the involvement of NMDA receptor-mediated neurotransmission in complex cerebral processes such as cognition, pharmacological manipulation of extracellular synaptic glycine biology is an active area of pharmaceutical research to develop novel treatments for neuropsychiatric disorders. A key component of cerebral glycine metabolism is the glycine transporter type 1 (GlyT1) and elevation of extracellular synaptic glycine concentration by blockade of GlyT1 has been hypothesized to potentiate NMDA receptor function in vivo and to represent a rational approach for the treatment of schizophrenia and cognitive disorders. The present article will review the wealth of scientific evidence supporting that hypothesis and the medicinal chemistry effort by many pharmaceutical companies and academic institutions to develop potent and selective GlyT1 inhibitors.

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties.

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.