Development of clickable, covalent ligands for the adenosine A1 and metabotropic glutamate subtype 5 receptors

Author: Jun Weng, Daniel Chong

Date: 2021

Publisher: University of Otago

Type: Thesis

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The Adenosine A1 receptor (A1AR) and metabotropic glutamate subtype 5 (mGluR5) receptor are class A G protein-coupled receptors (GPCRs) which have been implicated in disease pathologies such as neurodegenerative disorders, inflammation, cardiovascular and pain disorders. Fluorescent ligands are invaluable tools for probing and understanding the expression, localisation and receptor-ligand interactions of A1AR and mGluR5. However, the development of fluorescent ligands with high affinity and selectivity for these receptors has been challenging. The development of covalent, clickable ligands for the A1AR and mGluR5 is an attractive concept whereby a ligand first covalently binds to the receptor via a chemoreactive group of the ligand and can then undergo a click reaction with a fluorophore, thus labelling the receptor. Two series of A1AR ligands were designed based on the xanthine scaffold with a bicyclic-ring at the C8 position which was guided by docking studies using the A1AR crystal structure. A covalent arm consisting a fluorosulfonyl group was placed at the xanthine N1 position. Corresponding sulfonic acid derivatives were also synthesised as reversible analogues. An azide or terminal alkyne ‘click’ handle connected via linker of varying length and type was installed from the bicyclic-ring, for ‘clicking’ with a fluorophore with a complementary clickable partner in situ by the azide-alkyne Huisgen cycloaddition to form a 1,2,3-triazole. Out of the 14 ligands biologically evaluated, the highest affinity covalent clickable A1AR ligand was 3.6 ((hA1AR pKi = 8.81 ± 0.24 (1.6 nM), chapter 3) containing a hexyne clickable linker, which was also found to act as A1AR antagonist. Covalent clickable ligands in chapter 2 and 3 were shown to bind irreversibly to hA1AR. Covalent clickable ligand 3.6 was docked in the A1AR crystal structure with the chemoreactive fluorosulfonyl group covalently binding to residue Y271. Assays of ligand 3.6 and other closely related analogues at other AR subtypes showed moderate hA1AR selectivity, which was suggested to be due to the covalent interaction of the fluorosulfonyl group with Y271 which is conserved in A1AR, A2AAR and A3AR. Covalent clickable A1AR ligands consisting of the clickable azide (2.24 and 3.1) and alkyne (3.6) linkers were ‘clicked’ with a Cy5.5 fluorophore consisting a complementary clickable group and demonstrated covalent binding and imaging capability. iii A library of compounds were designed based on the mGluR5 negative allosteric modulator (NAM) 4-(3-(6-methylpyridin-2-yl)prop-2-yn-1-ylidene)piperidin-1-yl)(phenyl)methanone (4.10). Molecular docking was carried out to investigate suitable sites for the introduction of a clickable linker and a chemoreactive group. Literature compounds (4.10 and 4.12) and non-covalent ligands consisting of a clickable alkyne (4.24) or azide (4.25) linker were successfully synthesised. These ligands will be sent for pharmacological evaluation in due course. Incorporation of the fluorosulfonyl group into the series of mGluR5 ligands was attempted using PdCl2(Amphos)2 catalyst, DABSO and NFSI. However, these reactions were found to only work for compounds without an alkyne or azide functional group. A different synthetic strategy avoiding the use of a palladium catalyst has been suggested for future work to synthesise the fluorosulfonyl derivatives designed in chapter 4.

Subjects: adenosinea1, receptor, adenosinereceptor, mglur5, metabotropic, a1ar, chemicalprobe, gpcr, gproteincoupledreceptor, ligands

Citation: ["Jun Weng, D. C. (2021). Development of clickable, covalent ligands for the adenosine A1 and metabotropic glutamate subtype 5 receptors (Thesis, Doctor of Philosophy). University of Otago. Retrieved from"]

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