High Resolution Ion Mobility Enables the Structural Characterization of Atropisomers of GDC-6036, a KRAS G12C Covalent Inhibitor.

GDC-6036 is a covalent KRAS G12C inhibitor that demonstrates high potency and selectivity. Structurally, GDC-6036 consists of several motifs that make the analytical characterization of this molecule challenging, including a highly basic pyrrolidine motif bonded to a quinazoline ring via an ether bond and an atropisomeric carbon-carbon bond between functionalized pyridine and quinazoline groups. Structurally, the desired atropisomer was synthesized via an atroposelective Negishi coupling with very high yield. However, having a direct way to analyze and confirm the presence of the atropisomeric species remained challenging in routine analytical workflows. In this study, both variable temperature nuclear magnetic resonance (VT-NMR) and two different approaches of in-line ion mobility coupled to liquid chromatography mass spectrometry (LC-MS) workflows were evaluated for the characterization of GDC-6036 and its undesired atropisomer (Compound B) to support synthetic route development. Briefly, both VT-NMR and traveling wave ion mobility spectrometry (TWIMS) enabled by structures for lossless ion manipulation (SLIM) technology coupled to high resolution MS (HRMS) are able to elucidate the structures of the atropisomers in a complex mixture. Drift tube IMS (DTIMS) was also evaluated, but lacked the resolving power to demonstrate separation between the two species in a mixture, but did show slight differences in their arrival times when multiplexed and injected separately. The determined resolving power (Rp) by multiplexing the ions via DTIMS was 67.3 and 60.5 for GDC-6036 and Compound B, respectively, while the two peak resolving power (Rpp) was determined to be 0.41, indicating inadequate resolution between the two species. Alternatively, the SLIM-IM studies showed Rp of 103.8 and 99.4, with a Rpp of 2.64, indicating good separation between the atropisomers. Furthermore, the CCS/z for GDC-6036 and Compound B was determined to be 231.2 Å2/z and 235.0 Å2/z, respectively. Quantitative experiments demonstrate linearity (R2 >0.99) for both GDC-6036 and Compound B while maintaining separation via SLIM-IM. Spike recoveries of one atropisomer relative to the other yielded strong recoveries (98.7% to 102.5%) while maintaining reproducibility (<7% RSD). The study herein describes the analytical process for evaluating new technologies and strategies for implementation in routine biopharmaceutical characterization workflows.

The effect of tube quality on externally calibrated quantitative nuclear magnetic resonance analysis: How bad can it be?

Externally calibrated quantitative nuclear magnetic resonance (NMR) approaches offer practical means to simultaneously evaluate chemical identity and content without the addition of calibrants to the test sample. Despite continuous advances in external calibration over the last few decades, adoption of these approaches has been slower than expected. Variations in NMR tube geometry are a commonly overlooked factor that can have a substantial effect on externally calibrated quantitation methods. In this report, we investigate the extent to which tube-to-tube volume variability can affect quantitative NMR outcomes. The results highlight the importance of considering tube quality during the development stages of externally calibrated quantitative methods. In addition, we propose a simple, yet effective volume correction strategy using the residual protonated solvent signal that, based on experiments with mixed NMR tubes of varying quality, alleviates the effect of tube-to-tube variability.

Toward the Development of Rapid, Automated Identification Tests for Neat Organic Liquids Using Benchtop NMR Instrumentation.

The advent of benchtop nuclear magnetic resonance (NMR) instrumentation has paved the way for the use of this technology away from traditional NMR facility settings. Still, a wider adoption of benchtop NMR systems for routine identification testing has been hampered by inherent instrumental limitations (including low sensitivity and reduced signal dispersion) and workflow automation challenges. The present study summarizes the results of a cross-company collaboration aiming at the development of rapid, automated identification tests for incoming materials in liquid form intended for pharmaceutical manufacturing. Potential scenarios that analysts may encounter during the development of identification tests using benchtop NMR instrumentation are described, and suitable strategies for data collection and analysis are discussed. Challenges and opportunities for benchtop NMR implementation are illustrated using common organic solvents and laboratory reagents in a neat form, for which reference NMR data are provided.

Synthetic Sansanmycin Analogues as Potent Mycobacterium tuberculosis Translocase I Inhibitors.

Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

Synthesis and evaluation of analogues of the glycinocin family of calcium-dependent antibiotics.

The glycinocins are a class of calcium-dependent, acidic cyclolipopeptide antibiotics that are structurally related to the clinically approved antibiotic daptomycin. In this article, we describe the synthesis of a small library of glycinocin analogues that differ by variation in the exocyclic fatty acyl substituent. The glycinocin analogues were screened against a panel of Gram-positive bacteria (as well as Gram-negative P. aeruginosa). These analogues exhibited similar calcium-dependent activity to the parent natural products against Gram-positive bacteria but showed no activity against P. aeruginosa. The length of the fatty acid was shown to be important for optimal biological activity, while the hybridisation at the α,ß position and branching within the fatty acyl chain had only subtle effects on activity.

Marine Mammal Microbiota Yields Novel Antibiotic with Potent Activity Against Clostridium difficile.

The recent explosion of research on the microbiota has highlighted the important interplay between commensal microorganisms and the health of their cognate hosts. Metabolites isolated from commensal bacteria have been demonstrated to possess a range of antimicrobial activities, and it is widely believed that some of these metabolites modulate host behavior, affecting predisposition to disease and pathogen invasion. Our access to the local marine mammal stranding network and previous successes in mining the fish microbiota poised us to test the hypothesis that the marine mammal microbiota is a novel source of commensal bacteria-produced bioactive metabolites. Examination of intestinal contents from five marine mammals led to the identification of a Micromonospora strain with potent and selective activity against a panel of Gram-positive pathogens and no discernible human cytotoxicity. Compound isolation afforded a new complex glycosylated polyketide, phocoenamicin, with potent activity against the intestinal pathogen Clostridium difficile, an organism challenging to treat in hospital settings. Use of our activity-profiling platform, BioMAP, clustered this metabolite with other known ionophore antibiotics. Fluorescence imaging and flow cytometry confirmed that phocoenamicin is capable of shifting membrane potential without damaging membrane integrity. Thus, exploration of gut microbiota in hosts from diverse environments can serve as a powerful strategy for the discovery of novel antibiotics against human pathogens.

Total Synthesis of Glycinocins A-C.

The glycinocins are a class of calcium-dependent, acidic cyclolipopeptide antibiotics structurally related to the clinically approved daptomycin. Herein, we describe a divergent total synthesis of glycinocins A-C, which differ in the structure of a branched α,ß-unsaturated fatty acyl moiety. The three natural products exhibited calcium-dependent antimicrobial activity against Staphylococcus aureus and Bacillus subtilis with MICs ranging from 5.5 to 17 µM.

Total Synthesis of Teixobactin.

The first total synthesis of the cyclic depsipeptide natural product teixobactin is described. Synthesis was achieved by solid-phase peptide synthesis, incorporating the unusual l-allo-enduracididine as a suitably protected synthetic cassette and employing a key on-resin esterification and solution-phase macrolactamization. The synthetic natural product was shown to possess potent antibacterial activity against a range of Gram-positive pathogenic bacteria, including a virulent strain of Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus (MRSA).

Phenotype-Guided Natural Products Discovery Using Cytological Profiling.

Phenotype-guided natural products discovery is emerging as a useful new discovery tool that addresses challenges in early, unbiased natural product biological annotation. These high-content approaches yield screening results that report directly on the impact of test compounds on cellular processes in target organisms and can be used to predict the modes of action of bioactive constituents from primary screening data. In this study we explored the use of our recently implemented cytological profiling platform for the isolation of compounds with a specific, predefined mode of action, namely, induction of mitotic arrest. Screening of a microbially derived extract library revealed six extracts whose cytological profiles clustered closely with those of known antimitotic agents from the pure compound training set. Subsequent examination of one of these extracts revealed the presence of two separate bioactive constituents, each of which possessed a unique cytological profile. The first, diketopiperazine XR334 (3), recapitulated the observed antimitotic phenotype of the original extract, demonstrating that cytological profiling can be used for the targeted isolation of compounds with specific modes of action. The second, nocapyrone L (6), possessed a cytological profile that clustered with known calcium channel modulators, in line with previous published activities for this compound class, indicating that cytological profiling is a flexible and powerful platform for the de novo characterization of compound modes of action.