DELTA50: A Highly Accurate Database of Experimental 1H and 13C NMR Chemical Shifts Applied to DFT Benchmarking.

Density functional theory (DFT) benchmark studies of 1H and 13C NMR chemical shifts often yield differing conclusions, likely due to non-optimal test molecules and non-standardized data acquisition. To address this issue, we carefully selected and measured 1H and 13C NMR chemical shifts for 50 structurally diverse small organic molecules containing atoms from only the first two rows of the periodic table. Our NMR dataset, DELTA50, was used to calculate linear scaling factors and to evaluate the accuracy of 73 density functionals, 40 basis sets, 3 solvent models, and 3 gauge-referencing schemes. The best performing DFT methodologies for 1H and 13C NMR chemical shift predictions were WP04/6-311++G(2d,p) and ωB97X-D/def2-SVP, respectively, when combined with the polarizable continuum solvent model (PCM) and gauge-independent atomic orbital (GIAO) method. Geometries should be optimized at the B3LYP-D3/6-311G(d,p) level including the PCM solvent model for the best accuracy. Predictions of 20 organic compounds and natural products from a separate probe set had root-mean-square deviations (RMSD) of 0.07 to 0.19 for 1H and 0.5 to 2.9 for 13C. Maximum deviations were less than 0.5 and 6.5 ppm for 1H and 13C, respectively.

Deuterated Modifiers in Sub/Supercritical Fluid Chromatography for Streamlined NMR Structure Elucidation.

Isolation and chemical characterization of target components in fast-paced pharmaceutical laboratories can often be challenging, especially when dealing with mixtures of closely related, possibly unstable species. Traditionally, this process involves intense labor and manual intervention including chromatographic method development and optimization, fraction collection, and drying processes prior to NMR analyses for unambiguous structure elucidation. To circumvent these challenges, a foundational framework for the proper utilization of supercritical carbon dioxide (scCO2) and deuterated modifiers (CD3OD) in sub/supercritical fluid chromatography (SFC) is herein introduced. This facilitates a streamlined multicomponent isolation with minimized protic residues, further enabling immediate NMR analysis. In addition to bypassing tedious drying processes and minimizing analyte degradation, this approach (complementary to traditional reversed-phase liquid chromatography, RPLC) delivers highly efficient separations and automated fraction collection using readily available analytical/midscale SFC instrumentation. A series of diverse analytes across a wide spectrum of chemical properties (acid, basic, and neutral), combined with different stationary-phase columns in SFC are investigated using both a protic organic modifier (CH3OH) and its deuterated counterpart (CD3OD). The power of this framework is demonstrated with pharmaceutically relevant applications in the context of target characterization and analysis of complex multicomponent reaction mixtures from modern synthetic chemistry, demonstrating high isolation yields while reducing both the environmental footprint and manual intervention. This workflow enables unambiguous fast-paced structure elucidation on the analytical scale, providing results that are comparable to traditional, but time-consuming, RPLC purification approaches.

Driving to a Better Understanding of Acyl Glucuronide Transformations Using NMR and Molecular Modeling.

Acyl glucuronide (AG) metabolites of carboxylic acid-containing drugs and products of their transformations have long been implicated in drug-induced liver injury (DILI). To inform on the DILI risk arising from AG reactive intermediates, a comprehensive mechanistic study of enzyme-independent AG rearrangements using nuclear magnetic resonance (NMR) and density functional theory (DFT) was undertaken. NMR spectroscopy was utilized for structure elucidation and kinetics measurements of nine rearrangement and hydrolysis products of 1ß-O-acyl glucuronide of ibufenac. To extract rate constants of rearrangement, mutarotation, and hydrolysis from kinetic data, 11 different kinetic models were examined. Model selection and estimated rate constant verification were supported by measurements of H/D kinetic isotope effects. DFT calculations of ground and transition states supported the proposed kinetic mechanisms and helped to explain the unusually fast intramolecular transacylation rates found for some of the intermediates. The findings of the current study reinforce the notion that the short half-life of parent AG and slow hydrolysis rates of AG rearrangement products are the two key factors that can influence the in vivo toxicity of AGs.

One-Step Regio- and Stereoselective Electrochemical Synthesis of Orexin Receptor Antagonist Oxidative Metabolites.

Synthesis of drug metabolites, which often have complex structures, is an integral step in the evaluation of drug candidate metabolism, pharmacokinetic (PK) properties, and safety profiles. Frequently, such synthetic endeavors entail arduous, multiple-step de novo synthetic routes. Herein, we present the one-step Shono-type electrochemical synthesis of milligrams of chiral α-hydroxyl amide metabolites of two orexin receptor antagonists, MK-8133 and MK-6096, as revealed by a small-scale (pico- to nano-mole level) reaction screening using a lab-built online electrochemistry (EC)/mass spectrometry (MS) (EC/MS) platform. The electrochemical oxidation of MK-8133 and MK-6096 was conducted in aqueous media and found to produce the corresponding α-piperidinols with exclusive regio- and stereoselectivity, as confirmed by high-resolution nuclear magnetic resonance (NMR) characterization of products. Based on density functional theory (DFT) calculations, the exceptional regio- and stereoselectivity for this electrochemical oxidation are governed by more favorable energetics of the transition state, leading to the preferred secondary carbon radical α to the amide group and subsequent steric hindrance associated with the U-shaped conformation of the cation derived from the secondary α-carbon radical, respectively.

Application of 1,1-ADEQUATE and DFT to correct 13 C misassignments of carbonyl chemical shifts for carbapenem antibiotics.

Prior to the development of sensitive proton-detected 2D NMR experiments, assigning 13 C signals could be a significant challenge, and mistakes have occurred even for prominent compound classes. In this study, 1,1-ADEQUATE data were used to unambiguously reassign the 13 C chemical shifts for the ß-lactam carbonyl at the C-7 position and the proximal carboxylate at the C-10 position of the carbapenems, meropenem and imipenem. Density functional theory (DFT) was then investigated to provide sufficiently accurate 13 C chemical shift predictions, allowing for the carbonyl signal reassignment of thienamycin.

Deep Dive into Machine Learning Models for Protein Engineering.

Protein redesign and engineering has become an important task in pharmaceutical research and development. Recent advances in technology have enabled efficient protein redesign by mimicking natural evolutionary mutation, selection, and amplification steps in the laboratory environment. For any given protein, the number of possible mutations is astronomical. It is impractical to synthesize all sequences or even to investigate all functionally interesting variants. Recently, there has been an increased interest in using machine learning to assist protein redesign, since prediction models can be used to virtually screen a large number of novel sequences. However, many state-of-the-art machine learning models, especially deep learning models, have not been extensively explored. Moreover, only a small selection of protein sequence descriptors has been considered. In this work, the performance of prediction models built using an array of machine learning methods and protein descriptor types, including two novel, single amino acid descriptors and one structure-based three-dimensional descriptor, is benchmarked. The predictions were evaluated on a diverse collection of public and proprietary data sets, using a variety of evaluation metrics. The results of this comparison suggest that Convolution Neural Network models built with amino acid property descriptors are the most widely applicable to the types of protein redesign problems faced in the pharmaceutical industry.

Denigrins and Dactylpyrroles, Arylpyrrole Alkaloids from a Dactylia sp. Marine Sponge.

Seven new arylpyrrole alkaloids (1-7), along with four known compounds, were isolated from an extract of a Dactylia sp. nov. marine sponge, and their structures were elucidated by interpretation of NMR and MS spectroscopic data. Denigrins D-G (1-4) have highly substituted pyrrole or pyrrolone rings in their core structures, while dactylpyrroles A-C (5-7) have tricyclic phenanthrene cores. Due to the proton-deficient nature of these scaffolds, key heteronuclear correlations from 1H-15N HMBC and LR-HSQMBC NMR experiments were used in the structure assignment of denigrin D (1). Dictyodendrin F (8), a previously described co-metabolite, inhibited transcription driven by the oncogenic PAX3-FOXO1 fusion gene with an IC50 value of 13 µM.

Optical Chirality Sensing with a Stereodynamic Aluminum Biphenolate Probe.

The determination of the enantiopurity and the concentration of chiral compounds by chiroptical sensing with molecular probes is increasingly attractive for high-throughput screening applications including streamlined asymmetric reaction development. In this study, we use stereodynamic aluminum biphenolate complexes for quantitative ee and concentration analysis of amino alcohols and α-hydroxy acids. An important feature of the tropos biphenolate ligand used is the presence of a phenylacetylene antenna for optimal chirality recognition and CD/UV responses at high wavelengths. The complexation-driven chirality amplification yields strong CD signals, which allows quantitative chiroptical sensing with good accuracy. We show that aluminate biphenolate sensors can exhibit linear and nonlinear correlations between the induced CD signals and the enantiomeric composition or concentration of the chiral substrate.

Highly Diastereoselective Synthesis of a HCV NS5B Nucleoside Polymerase Inhibitor.

An asymmetric synthesis of HCV NS5B nucleoside polymerase inhibitor (1) is described. This novel route features several remarkably diastereoselective and high-yielding transformations, including construction of the all-carbon quaternary stereogenic center at C-2 via a thermodynamic aldol reaction. A subsequent glycosylation reaction with activated uracil via C-1 phosphate and installation of the cyclic phosphate group using an achiral phosphorus(III) reagent followed by oxidation provides 1.

Complex macrocycle exploration: parallel, heuristic, and constraint-based conformer generation using ForceGen.

ForceGen is a template-free, non-stochastic approach for 2D to 3D structure generation and conformational elaboration for small molecules, including both non-macrocycles and macrocycles. For conformational search of non-macrocycles, ForceGen is both faster and more accurate than the best of all tested methods on a very large, independently curated benchmark of 2859 PDB ligands. In this study, the primary results are on macrocycles, including results for 431 unique examples from four separate benchmarks. These include complex peptide and peptide-like cases that can form networks of internal hydrogen bonds. By making use of new physical movements ("flips" of near-linear sub-cycles and explicit formation of hydrogen bonds), ForceGen exhibited statistically significantly better performance for overall RMS deviation from experimental coordinates than all other approaches. The algorithmic approach offers natural parallelization across multiple computing-cores. On a modest multi-core workstation, for all but the most complex macrocycles, median wall-clock times were generally under a minute in fast search mode and under 2 min using thorough search. On the most complex cases (roughly cyclic decapeptides and larger) explicit exploration of likely hydrogen bonding networks yielded marked improvements, but with calculation times increasing to several minutes and in some cases to roughly an hour for fast search. In complex cases, utilization of NMR data to constrain conformational search produces accurate conformational ensembles representative of solution state macrocycle behavior. On macrocycles of typical complexity (up to 21 rotatable macrocyclic and exocyclic bonds), design-focused macrocycle optimization can be practically supported by computational chemistry at interactive time-scales, with conformational ensemble accuracy equaling what is seen with non-macrocyclic ligands. For more complex macrocycles, inclusion of sparse biophysical data is a helpful adjunct to computation.

Post-Synthetic Modification of Oligonucleotides via Orthogonal Amidation and Copper Catalyzed Cycloaddition Reactions.

An efficient multicomponent orthogonal protocol was developed for post-synthetic oligonucleotide modification using commercially available 2'- O-methyl ester and 2'- O-propargyl nucleoside scaffolds. Amidation of methyl esters with primary amines was achieved in the presence of 2'-propargyl groups which were utilized for subsequent copper catalyzed cycloaddition with GalNAc-azide. The methodology was applied to generate siRNA composed of multiple amide and triazole conjugates. Computational methods were used to illustrate the impact of substitution at the 2'-position. This a powerful post-oligomerization technique for rapidly introducing diversity to oligonucleotide design.

Modeling and predicting chiral stationary phase enantioselectivity: An efficient random forest classifier using an optimally balanced training dataset and an aggregation strategy.

Predicting whether a chiral column will be effective is a daily task for many analysts. Moreover, finding the best chiral column for separating a particular racemic compound is mostly a matter of trial and error that may take up to a week in some cases. In this study we have developed a novel prediction approach based on combining a random forest classifier and an optimized discretization method for dealing with enantioselectivity as a continuous variable. Using the optimization results, models were trained on data sets divided into four enantioselectivity classes. The best model performances were achieved by over-sampling the minority classes (α ≤ 1.10 and α ≥ 2.00), down-sampling the majority class (1.2 ≤ α < 2.0), and aggregating multicategory predictions into binary classifications. We tested our method on 41 chiral stationary phases using layered fingerprints as descriptors. Experimental results show that this learning methodology was successful in terms of average area under the Receiver Operating Characteristic curve, Kappa indices and F-measure for structure-based prediction of the enantioselective behavior of 34 chiral columns.

Model for the Enantioselectivity of Asymmetric Intramolecular Alkylations by Bis-Quaternized Cinchona Alkaloid-Derived Catalysts.

A model for the stereoselectivity of intramolecular alkylations by N,N'-disubstituted cinchona alkaloids reported by Xiang et al. was established using density functional theory (DFT) calculations. The stereocontrol is based on the minimal distortion of the transition state (TS) and catalyst required to achieve favorable electrostatic interactions in the favored TS. Counterions must be included in computational modeling of ion-paired catalysis in order to reproduce experimental enantioselectivity.

Asymmetric Formal Synthesis of the Long-Acting DPP-4 Inhibitor Omarigliptin.

A highly efficient asymmetric synthesis of the key tetrahydropyranol intermediate of DPP-4 inhibitor omarigliptin (1) is described. The successful development of a protecting-group- and precious-metal-free synthesis was achieved via the discovery of a practical asymmetric Henry reaction and the application of a one-pot nitro-Michael-lactolization-dehydration through-process. Other features of the synthesis include a highly efficient MsCl-mediated dehydration and a crystallization-induced dynamic resolution for exceptional ee and dr upgrade. The synthesis of this complex intermediate utilizes simple starting materials and proceeds in four linear steps.

Discovery of selective, orally bioavailable, N-linked arylsulfonamide Nav1.7 inhibitors with pain efficacy in mice.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for the treatment of pain with gain-of-function mutations in man eliciting a variety of painful disorders and loss-of-function mutations affording insensitivity to pain. Unfortunately, drugs thought to garner efficacy via Nav1 inhibition have undesirable side effect profiles due to their lack of selectivity over channel isoforms. Herein we report the discovery of a novel series of orally bioavailable arylsulfonamide Nav1.7 inhibitors with high levels of selectivity over Nav1.5, the Nav isoform responsible for cardiovascular side effects, through judicious use of parallel medicinal chemistry and physicochemical property optimization. This effort produced inhibitors such as compound 5 with excellent potency, selectivity, behavioral efficacy in a rodent pain model, and efficacy in a mouse itch model suggestive of target modulation.

The evolution of drug design at Merck Research Laboratories.

On October 5, 1981, Fortune magazine published a cover article entitled the "Next Industrial Revolution: Designing Drugs by Computer at Merck". With a 40+ year investment, we have been in the drug design business longer than most. During its history, the Merck drug design group has had several names, but it has always been in the "design" business, with the ultimate goal to provide an actionable hypothesis that could be tested experimentally. Often the result was a small molecule but it could just as easily be a peptide, biologic, predictive model, reaction, process, etc. To this end, the concept of design is now front and center in all aspects of discovery, safety assessment and early clinical development. At present, the Merck design group includes computational chemistry, protein structure determination, and cheminformatics. By bringing these groups together under one umbrella, we were able to align activities and capabilities across multiple research sites and departments. This alignment from 2010 to 2016 resulted in an 80% expansion in the size of the department, reflecting the increase in impact due to a significant emphasis across the organization to "design first" along the entire drug discovery path from lead identification (LID) to first in human (FIH) dosing. One of the major advantages of this alignment has been the ability to access all of the data and create an adaptive approach to the overall LID to FIH pathway for any modality, significantly increasing the quality of candidates and their probability of success. In this perspective, we will discuss how we crafted a new strategy, defined the appropriate phenotype for group members, developed the right skillsets, and identified metrics for success in order to drive continuous improvement. We will not focus on the tactical implementation, only giving specific examples as appropriate.

Absolute configuration assignment of (+)-fluralaner using vibrational circular dichroism.

The absolute configurations of the separated enantiomers of fluralaner, a racemic animal health product used to prevent fleas and ticks, have been assigned using vibrational circular dichroism (VCD). The crystallographic structure of the active enantiomer (+)-fluralaner has previously been shown to have the (S) configuration using small molecule crystallography. We sought a faster analytical method to determine the absolute configuration of the separated enantiomers. When comparing the measured IR (infrared) and VCD spectra, it is apparent that the amide carbonyl groups appear in the IR but are nearly absent in the VCD. Computational work to calculate the VCD and IR using in vacuo models, implicit solvation, and explicitly solvated complexes has implicated conformational averaging of the carbonyl VCD intensities.

Antenna Biphenols: Development of Extended Wavelength Chiroptical Reporters.

Molecular hosts capable of chiroptical sensing of complexed guest molecules offer an attractive alternative to conventional methods for the analysis of the absolute configuration and enantiopurity. Sensors based on the Pfeiffer effect rely on complexation-driven asymmetric transformation of the first kind and can produce a chiroptical signal against an otherwise null background. To be most effective, the wavelength of the induced chiroptical sensor readout should be free and clear of interfering signals coming from the sample under investigation. In this study, we report the introduction of stereodynamic zinc complexes of antenna biphenols, a new class of sensors bearing antenna-like appendages that can extend the wavelength of the chiroptical signal while also improving enantioselective guest recognition.

Systematic chemical modifications of single stranded siRNAs significantly improved CTNNB1 mRNA silencing.

Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.

Discovery and development of benzo-[1,2,4]-triazolo-[1,4]-oxazepine GPR142 agonists for the treatment of diabetes.

A novel series of benzo-[1,2,4]-triazolo-[1,4]-oxazepine GPR142 agonists are described. The series was designed to address the suboptimal PK (pharmacokinetic) and off-target profile of a class of N-aryl-benzo-[1,4]-oxazepine-4-carboxamides, represented by 1, that were identified from a high-throughput screen of the Merck compound collection for GPR142 agonists. This work led to the discovery of 3-phenoxy-benzo-[1,2,4]-triazolo-[1,4]-oxazepine 47, a potent GPR142 agonist with an off-target and PK profile suitable for in vivo studies. This compound and a related analogue 40 were shown to be active in mouse oral glucose tolerance tests (OGTTs). Furthermore, a GPR142 knock-out mouse OGTT study with compound 40 provides evidence that its glucose-lowering effect is mediated by GPR142.