Triazoloquinazolines: dual-target, a_2ar - pde 10a, anti-proliferative compounds

I. J. Winfield^1,2, D. Safitri¹, L. Kalash², S. Carvalho¹, A. Bender², G. Ladds¹. ¹Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom, ²Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.

Introduction: Activation of the Adenosine A_2A receptor (A_2AR), and inhibition of phosphodiesterases, has been show to be anti-proliferative [1]. Using in silico docking, we identified a series of known PDE 10A inhibitors, triazoloquinazolines [2], which displayed promising docking scores to the orthosteric agonist-binding site of the A_2AR. Here we present data pertaining to the characterisation of these compounds with respect to activation of the A_2AR, identifying their selectivity profiles amongst adenosine receptors, as well as identifying their dual-target action to also be anti-proliferative.

Methods: Test compounds were initially screened in yeast expressing either the A₁, A_2A or A_2B receptors [3]. cAMP accumulation/inhibition was measured using a LANCE cAMP kit (Perkin Elmer), for CHO-K1-A_2AR and CHO-K1-A₃R cells, as well as C6 and HEK 293S cells. All cell types were stimulated with agonist (10 mM - 10 pM) for 30 minutes prior to determining intracellular cAMP concentrations. For proliferation assays, cells were seeded onto 96 well plates at a density of 2000 cells per well, and treated with agonists after 24 hours, for a period of 72 hours. Cell number was then quantified using CCK-8 (Sigma Aldrich).

Results: Screening of test compounds (Cmpds) 1-5: in yeast (β-galactosidase), CHO-K1-A_2AR and CHO-K1-A₃R cells (cAMP accumulation/inhibition), identified the ability of all compounds to act as agonists against the A_2AR, with Cmpds 1-3 being selective for the A_2AR (Table 1). Further testing in cells endogenously expressing the A_2AR and PDE 10A, C6 and HEK 293S cells, identified their abilities to elevate intracellular cAMP levels (Figure 1A-B), as well as inhibit proliferation (Figure 1C-D). This anti-proliferative action is specific to the triazoloquinazoline compounds, as CGS21680 (a selective A_2AR agonist) had no effect upon proliferation in either cell type (Figure 1C-D). However, cAMP appears to be the key mediator of these compounds’ actions as forskolin was also found to be anti-proliferative (Figure 1C-D).

Conclusions: We have identified a series of dual action compounds, of the triazoloquinazoline chemical series, that are both A_2AR agonists and PDE 10A inhibitors. Testing these compounds in cells endogenously expressing both of these targets identified that our compounds appear to have an anti-proliferative effect, which is cAMP dependent; this been particularly apparent in a rat model of glioma (C6 cells).

References:

[1] Rickles et al (2010). Blood. 116:593-602.

[2] Kehler et al (2011). Bioorg. Med. Chem. Lett. 21:3738-42;

[3] Knight et al (2016). J. Med. Chem. 59:947-64.