Development of a Gram-Scale Synthesis of PBRM, an Irreversible Inhibitor of 17beta-Hydroxysteroid Dehydrogenase Type 1

René Maltais and Donald Poirier
Organic Process Research & Development 2019
DOI: 10.1021/acs.oprd.8b00402

Efforts toward the development of a reliable gram scale synthesis of PBRM, a potent and selective steroidal covalent inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), are described. Among the three synthetic routes (C-E) developed herein, route E is the most efficient one with only 6 chemical steps from commercially available estrone, and an overall yield of 13% leading to PBRM with a high HPLC grade purity (99.7%) after recrystallization. Important improvements have been achieved in this sequence from previous reported routes (A and B). Notably, we used a palladium catalyzed Suzuki-Miyaura cross-coupling reaction to rapidly install the requested C3 chain on estrone. Also, catalytic hydrogenation of the C16-enone was shortened by half using Pearlman’s catalyst. Finally, we used a selective bromination through deoxygenation of alcohol at the last step of the sequence to provide PBRM without dehydration of its carboxamide functionality, a persistent problem observed in other routes. Crystals of PBRM were also obtained from recrystallization in acetonitrile and submitted to x-ray analysis, which confirmed the PBRM structure. This work now makes it possible to start a proof-of-principle in a non-human primate model for the treatment of endometriosis, while supporting its future pharmacological development.

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

Patrick R. A. Zanon, Lisa Lewald, Stephan M. Hacker
ChemRxiv, 2019
doi: 10.26434/chemrxiv.9853445.v1

Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Here, we describe isotopically labeled desthiobiotin azide (isoDTB) tags that are easily synthesized, shorten the chemoproteomic workflow and allow an increased coverage of cysteines in bacterial systems. We quantify 59% of all cysteines in essential proteins in Staphylococcus aureus and discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. We verify inhibition of HMG-CoA synthase, which will allow addressing the bacterial mevalonate pathway through a new target. Overall, a comprehensive map of the bacterial cysteinome is obtained, which will facilitate the development of antibiotics with novel modes-of-
action.

https://chemrxiv.org/articles/Isotopically_Labeled_Desthiobiotin_Azide_isoDTB_Tags_Enable_Global_Profiling_of_the_Bacterial_Cysteinome/9853445/1

SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase

Qinheng Zheng, Jordan L. Woehl, Seiya Kitamura, Diogo Santos-Martins, Christopher J. Smedley, Gencheng Li, Stefano Forli, John E. Moses, Dennis W. Wolan, and K. Barry Sharpless

PNAS,  2019 116 (38) 18808-18814
doi: 10.1073/pnas.1909972116

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC50 0.24 μM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.

Limitations of ligand-only approaches for predicting the reactivity of covalent inhibitors

Angus Voice, Gary Tresadern, Herman Van Vlijmen and Adrian J. Mulholland

J. Chem. Inf. Model. 2019
DOI:https://doi.org/10.1021/acs.jcim.9b00404

Abstract

Covalent inhibition has undergone a resurgence and is an important modern-day drug design and chemical biology approach. To avoid off-target interactions, and to fine tune reactivity, the ability to accurately predict reactivity is vitally important for the design and development of safer and more effective covalent drugs. Several ligand-only metrics have been proposed that promise quick and simple ways of determining covalent reactivity. In particular, we examine proton affinity and reaction energies calculated with the density functional B3LYP-D3/6-311+G**//B3LYP-D3/6-31G* method to assess the reactivity of a series of ,-unsaturated carbonyl compounds that form covalent adducts with cysteine. We demonstrate that, whilst these metrics correlate well with experiment for a diverse range of covalent fragments, these approaches fail for predicting the reactivity of drug-like compounds. We conclude that ligand-only metrics such as proton affinity and reaction energies do not capture determinants of reactivity in situ and fail to account for important factors such as conformation, solvation and intra-molecular interactions.

Quantum Chemical Methods for Modeling Covalent Modification of Biological Thiols

Ernest Awoonor‐Williams, William C. Isley III, Stephen G. Dale, Erin R. Johnson, Haibo Yu, Axel D. Becke, Benoît Roux, Christopher N. Rowley

J. Comput. Chem. 2019 doi: https://doi.org/10.1002/jcc.26064

Targeted covalent inhibitor drugs require computational methods that go beyond simple molecular‐mechanical force fields in order to model the chemical reactions that occur when they bind to their targets. Here, several semiempirical and density‐functional theory (DFT) methods are assessed for their ability to describe the potential energy surface and reaction energies of the covalent modification of a thiol by an electrophile. Functionals such as PBE and B3LYP fail to predict a stable enolate intermediate. This is largely due to delocalization error, which spuriously stabilizes the prereaction complex, in which excess electron density is transferred from the thiolate to the electrophile. Functionals with a high‐exact exchange component, range‐separated DFT functionals, and variationally optimized exact exchange (i.e., the LC‐B05minV functional) correct this issue to various degrees. The large gradient behavior of the exchange enhancement factor is also found to significantly affect the results, leading to the improved performance of PBE0. While ωB97X‐D and M06‐2X were reasonably accurate, no method provided quantitative accuracy for all three electrophiles, making this a very strenuous test of functional performance. Additionally, one drawback of M06‐2X was that molecular dynamics (MD) simulations using this functional were only stable if a fine integration grid was used. The low‐cost semiempirical methods, PM3, AM1, and PM7, provide a qualitatively correct description of the reaction mechanism, although the energetics is not quantitatively reliable. As a proof of concept, the potential of mean force for the addition of methylthiolate to methylvinyl ketone was calculated using quantum mechanical/molecular mechanical MD in an explicit polarizable aqueous solvent. 

Labeling and Natural Post-Translational Modification of Peptides and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine

Thomas Schlatzer, Julia Kriegesmann, Hilmar Schröder, Melanie Trobe, Christian Lembacher-Fadum, Simone Santner, Alexander V. Kravchuk, Christian F. W. Becker, and Rolf BreinbauerJ. Am. Chem. Soc. 2019

DOI: 10.1021/jacs.9b08279

The prenylation of peptides and proteins is an important post-translational modification observed in vivo. We report that the Pd-catalyzed Tsuji–Trost allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, peptides containing two cysteine residues can be stapled or cyclized using homobifunctional allylic carbonate reagents.