Bicyclobutane Carboxylic Amide as a Cysteine-Directed Strained Electrophile for Selective Targeting of Proteins

Abstract

Expanding the repertoire of electrophiles with unique reactivity features would facilitate the development of covalent inhibitors with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]­butane (BCB) carboxylic amide as a new class of thiol-reactive electrophiles for selective and irreversible inhibition of targeted proteins. We first streamlined the synthetic routes to generate a variety of BCB amides. The strain-driven nucleophilic addition to BCB amides proceeded chemoselectively with cysteine thiols under neutral aqueous conditions, the rate of which was significantly slower than that of acrylamide. This reactivity profile of BCB amide was successfully exploited to develop covalent ligands targeting Bruton’s tyrosine kinase (BTK). By tuning BCB amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activity-based protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity profile.

Inhibition of Thiol‐Mediated Uptake with Irreversible Covalent Inhibitors

“…Other applications include further derivatizations of cysteines, [28,29] peptide Cys‐Cys and Cys‐Lys stapling, [30] functional terminators of CPDs, [31] and classical use as alkynylation reagents in organic synthesis [32,33] . The results from irreversible inhibition of thiol‐mediated uptake with hypervalent iodine reagents are then compared to classical and modern irreversible thiol‐reactive agents [34–45] . Hypervalent iodine reagents emerge top, together with Fukuyama ’s nosyl protecting group [34] and super‐cinnamaldehyde ligands of the pain receptor TRPA1, [35] all rivaling the best reversible inhibitors.…”

Discovery and biological evaluation of a small‐molecule inhibitor of CRM1 that suppresses the growth of triple‐negative breast cancer cells

“…The chemical reactions involving the relevant moieties of an LSF compound and the Cys539 of CRM1 are shown in Figure 1D. The electrophilic unsaturated carbonyl group of isothiocyanate acts as a Michael acceptor moiety, which allows Michael addition by the nucleophilic sulfhydryl group of cysteine residue 28,29 …”

“…Furthermore, we used the drug-likeness module to screen out of 2,207 analogues followed by absorption, distribution, metabolism, excretion/toxicity (ADME/T) module screening to identify 73 analogues molecules from the combinatorial library with group of cysteine residue. 28,29 Next, we sought to further investigate LFS-31 and CRM1 interactions using a BLI assay. 30 The following five drug concentrations were The nuclear retention of IκBα by LFS-31 and KPT-330 was also confirmed by western blotting (Figure 3E-H).…”

Synthesis of 1,3-disubstituted bicyclo[1.1.0]butanes via directed bridgehead functionalization

“…BCB Ibrutinib, 3). 10,20,21 A BCB-containing natural product (4) has even been isolated and synthesised. 22, 23 1,3-disubstituted BCBs are attractive precursors to bicyclo [1.1.1]pentanes (BCPs, 5), which are valuable motifs in drug design, [24][25][26] via a formal carbene insertion into the C1-C3 bond.…”