The use of β-lactam (BL) and β-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-β-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

The design, syntheses and antibacterial evaluation of sulfone analogues of previously disclosed metallo-(3-lactamase inhibitors (MBLis) are described. The novel derivatives were overall more effective in gram-negative bacterial cell-based assays when combined with imipenem and relebactam. The major contributors to the improved anti-bacterial activity are enhanced enzyme-inhibitor interactions and reduced bacterial cell efflux monitored via an efflux assay involving isogenic Pseudomonas aeruginosa efflux + and efflux - tool strains.

DNA-encoded library (DEL) technology holds exciting potential for discovering novel therapeutic macrocyclic peptides (MPs). Herein, we describe the development of a DEL-compatible peptide macrocyclization method that proceeds via intramolecular click-condensation between 3-(2-cyano-4-pyridyl)-l-alanine (Cpa) and an N-terminal cysteine. Cyclization takes place spontaneously in a buffered aqueous solution and affords the cyclized products in excellent yields. The reaction exhibits a broad substrate scope and can be employed to generate MPs of variable ring size and amino acid composition.

Abstract Background Metallo-beta (β)-lactamases (MBLs) are in the class B group of β-lactamases due to zinc ions in the active site that are required for enzymatic activity. MK-3866 is a small molecule MBL inhibitor (MBLi) that restores antibacterial activity against resistant MBL-expressing gram-negative bacteria. Efflux is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa (Pa). Imipenem (IMI) is not subject to efflux and neither is relebactam (REL), a β-lactamase inhibitor (BLi) of class A and C β-lactamases that is approved in combination with IMI/cilastatin. An efflux assay was devised to characterize MBLis for potentiation of IMI or cefepime (FEP) in isogenic strain pairs of efflux wild-type and multiply efflux deleted (MED) strains of Pa. Our objective was to determine if MK-3866 and related analogs are subject to efflux in Pa. Methods Bacterial isolates were engineered to demonstrate the ability of MBLis to be effluxed by introducing IMI metallo-β-lactamase-1 (IMP-1) (by electroporation) or Verona integron-encoded metallo-β-lactamases (VIM-1, VIM-2) (utilizing the cloning vector pFlp2) into isogenic MED and wild-type (WT) Pa isolates. Susceptibility testing was performed with IMI or FEP at a fixed concentration equal to the Pa Clinical and Laboratory Standards Institute susceptibility breakpoint for each and including a fixed 4 µg/mL of REL to inhibit class A or C enzymes, referred to as the SLICE assay. The concentration of MK-3866 and analogues required to restore susceptibility to either antibiotic in WT and MED strains was assessed. Results MICs to the combination of MK-3866/REL/IMI or MK-3866/REL/FEP vary by ≤4-fold between the WT and MED strain of each isogenic strain pair (Table 1). In contrast, large differential MIC values (efflux ratios) can be seen for the MK-3866 analogues A and B, with more moderate efflux ratios observed for other analogues. The exact efflux ratio depended on the MBL studied and the antibiotic partner. Conclusion MK-3866 showed a low potential for efflux whether IMP-1, VIM-1, or VIM-2 was expressed, in contrast to analogues of MK-3866 which exhibited differentials from nominal to extreme (≥ 128-fold) between efflux WT versus MED isolates. Disclosures Katherine Young, M.S., Merck & Co., Inc.: Stocks/Bonds Asra Mirza, MS, Merck & Co., Inc.: Stocks/Bonds Carl Balibar, PhD, Merck & Co., Inc.: Stocks/Bonds Shuzhi Dong, PhD, Merck & Co., Inc.: Stocks/Bonds Frank Bennett, PhD, Merck & Co., Inc.: Stocks/Bonds Jinlong Jiang, PhD, Merck & Co., Inc.: Stocks/Bonds Haiqun Tang, PhD, Merck & Co., Inc.: Stocks/Bonds Claire Tudge, PhD, Merck & Co., Inc.: Stocks/Bonds Jack Scott, PhD, Merck & Co., Inc.: Stocks/Bonds Dexi Yang, PhD, Merck & Co., Inc.: Stocks/Bonds Alexander Pasternak, PhD, Merck & Co., Inc.: Stocks/Bonds.

Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3-sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.

The discovery of potent, kinome selective, brain penetrant LRRK2 inhibitors is the focus of extensive research seeking new, disease-modifying treatments for Parkinson's disease (PD). Herein, we describe the discovery and evolution of a picolinamide-derived lead series. Our initial optimization efforts aimed at improving the potency and CLK2 off-target selectivity of compound 1 by modifying the heteroaryl C-H hinge and linker regions. This resulted in compound 12 which advanced deep into our research operating plan (ROP) before heteroaryl aniline metabolite 14 was characterized as Ames mutagenic, halting its progression. Strategic modifications to our ROP were made to enable early de-risking of putative aniline metabolites or hydrolysis products for mutagenicity in Ames. This led to the discovery of 3,5-diaminopyridine 15 and 4,6-diaminopyrimidine 16 as low risk for mutagenicity (defined by a 3-strain Ames negative result). Analysis of key matched molecular pairs 17 and 18 led to the prioritization of the 3,5-diaminopyridine sub-series for further optimization due to enhanced rodent brain penetration. These efforts culminated in the discovery of ethyl trifluoromethyl pyrazole 23 with excellent LRRK2 potency and expanded selectivity versus off-target CLK2.

A robust palladium-catalyzed hydroxycarbonylation of aryl halides on DNA has been developed. Instead of Mo(CO)6 as a source of carbon monoxide as previously described in the literature, chloroform was used as a surrogate in this report for the purpose of avoiding to use a large excess of molybdenum reagent which is not totally soluble in aqueous reaction mixtures.

ABSTRACTAims/hypothesisBeta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is required for the production of toxic amyloid peptides and is highly expressed in the brain, but also to a lesser extent in major peripheral organs such as muscle and liver. In contrast, BACE2 is mainly expressed in peripheral tissues and is enriched in pancreatic beta cells, where it regulates beta- cell function and mass. Previous reports demonstrated that loss of BACE1 function decreases body weight, protects against diet-induced obesity and enhances insulin sensitivity in mice, whereas mice lacking Bace2 exhibit reduced blood glucose levels, improved intraperitoneal glucose tolerance and increased beta-cell mass. Impaired glucose homeostasis and insulin resistance are hallmarks of type 2 diabetes and have been implicated in Alzheimer’s disease. Therefore, we tested the contribution of the individual BACE isoforms to those metabolic phenotypes by placing Bace1 knockout (KO), Bace2 KO, Bace1/2 double knockout (dKO) and wild-type (WT) mice on a high-fat high-cholesterol diet (HFD) for 16 weeks.MethodsBace1 KO (n = 18), Bace2 KO (n = 18), Bace1/2 dKO (n = 18) and WT C57BL/6N mice (n = 54) were fed a HFD for 16 weeks (age 9–25 weeks). Body composition was measured before initiation of the HFD and after 11 weeks of HFD. Oral glucose tolerance and insulin sensitivity tests were performed after 12 and 13 weeks of HFD, respectively, and full blood chemistry was analyzed after 16 weeks of HFD. The effects of subchronic BACE1/2 inhibition were assessed by administration of 10 mg/kg/day of the dual BACE1/2 inhibitor MBi-3 in a HFD fed to C57BL/6N mice for 3 weeks.ResultsBace1 KO and Bace1/2 dKO mice showed decreased body weight and improved glucose tolerance and insulin resistance vs. WT mice. Conversely, Bace2 KO mice did not show any significant differences in body weight, glucose tolerance or insulin resistance under our experimental conditions. Finally, subchronic MBi-3–mediated BACE1/2 inhibition in mice in conjunction with a HFD resulted in a modest improvement of glucose tolerance.Conclusions/interpretationOur data indicate that lack of BACE1 – but not BACE2 – function contributes mainly to the metabolic phenotypic changes observed in Bace1/2 dKO mice, suggesting that inhibition of BACE1 has the greater role (vs. BACE2) in any potential improvements in metabolic homeostasis.HIGHLIGHTSInsulin resistance may develop in the brains of patients with Alzheimer’s disease (83/85 characters)BACE1 and BACE2 may play a role in glucose homeostasis and insulin sensitivity (80/85 characters)Body weight in mice decreased with Bace1 KO and Bace1/2 KO but not Bace2 KO alone (83/85 characters)Bace1 and Bace1/2, but not Bace2, KO improved glucose tolerance/insulin resistance (84/85 characters)Improved metabolic homeostasis may follow loss of BACE1 rather than BACE 2 activity (85/85 characters)

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson's disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.

β-Amyloid (Aβ) peptides are thought to be critically involved in the etiology of Alzheimer’s disease (AD). The aspartyl protease β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aβ, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aβ40, Aβ42, and sAPPβ (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aβ40, Aβ42, and sAPPβ in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aβ pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aβ levels in rats and nonhuman primates and CSF Aβ levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

A novel domino reaction for the preparation of non-racemic β-proline derivatives is reported. The addition of a methyl 2-(oxetan-3-yl)acetate titanium enolate to chiral tert-butanesulfinyl ketimines followed by an intramolecular oxetane ring-opening provides the highly-substituted pyrrolidine ring systems with three contiguous stereogenic centers.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.