Enantiospecific Recognition at the A_2B Adenosine Receptor by Alkyl 2-Cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylates

Abstract

A novel family of structurally simple, potent, and selective nonxanthine AAR ligands was identified, and its antagonistic behavior confirmed through functional experiments. The reported alkyl 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahy-dropyrimidine-5-carboxylates (16) were designed by bioisosteric replacement of the carbonyl group at position 2 in a series of 3,4-dihydropyrimidin-2-ones. The scaffold (16) documented herein contains a chiral center at the heterocycle. Accordingly, the most attractive ligand of the series [(±)16b, K = 24.3 nM] was resolved into its two enantiomers by chiral HPLC, and the absolute configuration was established by circular dichroism. The biological evaluation of both enantiomers demonstrated enantiospecific recognition at AAR, with the (S)-16b enantiomer retaining all the affinity (K = 15.1 nM), as predicted earlier by molecular modeling. This constitutes the first example of enantiospecific recognition at the A adenosine receptor and opens new possibilities in ligand design for this receptor.

Structure-Based Design of Potent and Selective Ligands at the Four Adenosine Receptors

“…In a later modification of the original scaffold, we created a series of 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylates [ 31 ]. This time the two enantiomers of the most attractive ligand ( 16b ) were separated by chiral HPLC and their absolute configurations established by circular dichroism ( Figure 4 ).…”

“…From this analysis, we identified a water molecule that mediated the interaction between the NH in position 1 of the parent 3,4-dihydropyrimidin-2(1 H )-ones and Glu169 EL2 , and a second water molecule in the region between the alkoxy substituent and the furan/thienyl ring, which is actually equivalent to a conserved water molecule observed in the crystal structures of A 2A AR with antagonist ZM241385 [ 23 ]. These pair of water molecules were maintained in successive docking runs for the expanded series of compounds, verifying their potential role in mediating this inter and intra molecular interactions [ 29 , 31 , 32 ]. We here provide further support for the existence of these structural water molecules, by means of an MD exploration of the solvent in the binding site (see methods).…”

“…During the last years, our labs have combined FEP and other computational methodologies with high-throughput synthetic methodologies and pharmacological characterization, to develop various series of structurally simple, novel AR ligands [ 23 ]. Our SBDD protocol includes homology modeling, protein–ligand docking, 3D-QSAR, MD and FEP simulations, and was used to assist the design of novel ligands as well as to predict or rationalize their pharmacological profile [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. The different series obtained with this strategy, represented in Figure 1 , have been optimized to yield potent and selective antagonists of the different ARs.…”

“…The first scaffold reported, a 2,4-diarylpyrimidine, was recently followed by a bioisosteric series of 2,4-diarylpyridines, both of them revealing high affinity and selectivity as A 3 AR antagonists [ 24 , 25 , 30 ]. For the A 2B receptor, we have documented the first non-planar, enantioselective antagonists, and disclosed the reasons of their enantiospecificity [ 31 , 32 ]. The A 2A AR emerges as the most attractive target for SBDD within the AR family, due to the existence of several crystal structures complemented with broad mutagenesis and SAR data.…”

Tritium-labeled agonists as tools for studying adenosine A2B receptors

“…However, functional assays are highly dependent on receptor expression levels and receptor reserve [14]. In contrast to the situation with agonists, many potent and selective A 2B AR antagonists have been developed [13,[15][16][17][18][19][20][21] and evaluated in preclinical and initial clinical trials [22]. PSB-603 is one of the most potent and selective A 2B antagonists, which is frequently used as a tool compound to study A 2B ARs [23][24][25][26].…”