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    • In our preliminary evaluation of this series

    2021-07-21

    In our preliminary evaluation of this series, we were surprised to discover that Z-WEHD-FMK mg was found to bind CSF-1R in a classical DFG-in binding mode (a). Dramatic conformational differences in the juxtamembrane domain and activation loops are apparent when this DFG-in structure is compared with prior DFG-out CSF-1R structures, with some residues displaced by over 30Å. Indeed, superposing the DFG-out and -in structures shows that elements of the juxtamembrane domain in the former (especially residues 550–554) mimic elements of the activation loop in the latter (residues 797–804). This is the first example of a DFG-in binding mode for a CSF-1R kinase inhibitor., It superposes well on the structure of the active conformation of cKit bound to ADP (RCSB code 1PKG). Several known selective CSF-1R inhibitors bind the enzyme in a DFG-out mode. For example, is exquisitely selective for CSF-1R (b)., , An important feature of this co-crystal structure is the bidentate hydrogen bonding network made by the oxygen atoms of the methoxy and benzyloxy groups of the ‘DFG-out Tail’ to the backbone amide NH of D796. We hypothesized that this is an important feature for locking in the DFG-out binding mode revealing access to the deep pocket. We desired a chemical series with a DFG-out binding mode, which would lock the CSF-1R kinase into an inactive confirmation., DFG-out inhibition is expected to increase the biochemical efficiency, defined as how effectively the binding of an inhibitor to the target results in blocking a physiological response., Furthermore, the likelihood for kinase selectivity is enhanced by a DFG-out binding mode as only a subset of kinases can achieve DFG-out conformations. We hypothesized that we could transform this aminopyrazine class of inhibitors into a class of DFG-out binders. An overlay of the crystal structures of and (c) illustrates the overlap of the hinge binding group aminopyrazine in and the corresponding diaminopyrimidine group in . Since the basic amine head group of reaches out to solvent, apparently contributing little to binding affinity, we hypothesized that the head group could be truncated to a simple aromatic group (Step 1 in ). If this could be accomplished, then the DFG-out tail group found in could be incorporated (Step 2 in ) giving hydrid compound with a DFG-out binding mode, a hypothesis supported by molecular docking studies (, ). Compound and , while quite potent, have high molecular weights (MW) resulting in relatively poor ligand efficiencies (LE). In order to transform the dichlorobenzyl ether group into the higher MW DFG-out tail group, which was anticipated to be necessary to bind into the DFG-out deep pocket, we initiated an effort to truncate the aryl amide head group. In addition to reducing MW, we desired to remove the basic amine as it puts this series at risk for binding the hERG channel. Compounds – were prepared as shown in . Selective nucleophilic attack by benzylic alcohols or amines on dibromoaminopyrazine results in bromopyrazine intermediate which undergoes Suzuki coupling with aromatic boronic acids and esters such as pyrazole boronate to give final products –. Other aromatic and heteroaromatic boronic acids and esters were also prepared in a library format (data not shown). We were delighted to find that the entire aryl amide head group from compounds – could be truncated to a simple methyl pyrazole as shown in compounds – (). Although 9- and 30-fold less potent than the hits –, compounds – are significantly more ligand efficient (LE) than the screening hits due to their reduced MWs. Compound illustrates the second Cl atom was also unnecessary for CSF-1R potency. In terms of Log and lipophilic efficiency (LipE), compounds and are an improvement of approximately one order of magnitude. Furthermore, the hERG potential of this series was significantly reduced as evidenced by the reduction in inhibition of the binding of fluorescently tagged dofetilide to the hERG channel. As a result, compound was then utilized as the core pharmacophore onto which the DFG-out tail group was incorporated.