Next we designed substituents of the position

Next, we designed substituents of the 6-position. Edge-to-face aromatic interaction between aromatic ring of ligand and Phe162B needed to be retained because the interaction seems to be important for tight binding. 3′-Position of the 6-phenyl group of directs to a hydrophilic space where W1 exists. On the other hand, 4′-and 5′-positions of the 6-phenyl ring directs to a hydrophobic pocket surrounded by side-chains of residues Lys156B, Met157B and Phe162B. From these observations, we designed 7-azaindole whose 7-nitrogen faces the hydrophilic space and 5-membered ring occupies the hydrophobic pocket. The substituent on 1N-position, which directs to the solvent-accessible region and therefore may contribute to the solubility of inhibitors, should be explored. To understand the function of 7-azaindole, we prepared the corresponding indole derivatives and evaluated them in parallel. We did not employ the ionic interactions with Arg37A/Asn103A/Arg122B in our inhibitor design because introducing a carboxylate and/or an ammonium functional group to the -hydroxypyridone scaffold would result in inhibitors with poor cell permeability. The SAR of azaindole derivatives is shown in along with the corresponding indole derivatives. 1N-unsubstituted 7-azaindole () and indole () equally showed better activity than . 3Carbamoyl-benzyl () and methoxyalkyl groups ( and ) on 1N-position of 7-azaindole showed significant improvement in activity but the same substituents on indole () did not show such an effect. We solved a cocrystal structure of /GLO1 complex at a resolution of 1.5Å (, PDB code:). An asymmetric unit contains two active sites located at the interface of two peptide chains (A and B). The structure revealed that -hydroxypyridone of CI 976 australia coordinated a zinc cation ((NO⋯Zn)=2.4Å, (CO⋯Zn)=2.2Å). 4-Phenyl group, which made the edge-to-face interaction with Phe62A ((Phe62A⋯4-Ph π face)=4.3Å), was situated in the hydrophobic pocket. Azaindole was situated at the GSH binding pocket, forming an edge-to-face interaction with Phe162B ((Phe162B⋯7-azaindole π face)=4.0Å). Phe67A was near 7-azaindole π face ((Phe67A⋯7-azaindole π face)=4.5Å). Azaindole pyridine nitrogen made a hydrogen bond with a water ((N⋯O)=2.8Å) in the hydrogen bond network including W1. A methoxypropyl group directed to Phe162B ((Phe162B⋯HOMe)=3.9Å) and Trp170B ((Trp170B⋯HOMe)=3.5Å). Oxygen in the alkyl chain reached the solvent-accessible region. Thermodynamic parameters of inhibitor binding ( and ) to GLO1 were determined by isothermal titration calorimetry (). As expected from the hydrogen bond network including azaindole observed in the X-ray structure, the binding of was enthalpic driven (Δ=−11.1±1.2kcal/mol, −Δ=2.1±1.0kcal/mol). Such enthalpy-driven binding was not observed for indole (Δ=−6.3±1.7kcal/mol, −Δ=−2.0±1.4kcal/mol). Since the 1N-unsubstituted derivatives and did not show a difference in activity, the stabilization of the water network is affected by substituents at this position. The dependence of hydrogen bond strengths on microenvironment polarity is reported in stabilization of protein structure. We assume that the stabilization of the water network was achieved by , and resulted in improved activity. The effect of oxygen in the 1N-substituent was investigated by the comparison with carbon analogues (). Simple alkyl groups ( and ) were not effective in enhancing activity. Since oxygen in the alkyl chain of stayed in the solvent-accessible region, simple alkyl groups were unfavorable in such a hydrophilic environment. Preparation of was performed starting from 2,6-dichloropyridineoxide () via iridium-catalyzed bolyration, Suzuki coupling, de-chlorinating hydrolysis, and methoxymethyl (MOM) protection to give an intermediate . Suzuki coupling of with boronate followed by alkylation at 1N-position under basic conditions and subsequent deprotection afforded ().