A few DAO inhibitors have entered into clinical development. Currently, only SyneuRx is actively developing NaBen® (sodium salt of 1) in a phase II/III clinical trial for refractory schizophrenia in adults. 10 Sepracor was developing SEP-227900 for neuropathic pain around 2010. Takeda was developing TAK-831 (luvadaxistat, 8) in phase 2 clinical trials 11 for the treatment of schizophrenia, which was the subject of a license agreement with Neurocrine in 2020. 12 In March 2021, Neurocrine reported topline data from the Phase II INTERACT study in adults with negative symptoms of schizophrenia treated with luvadaxistat (NBI-1065844/TAK-831). Although luvadaxistat did not meet its primary endpoint in the study, as measured by the change from baseline on the PANSS NSFS at Day 84, Luvadaxistat met secondary endpoints of cognitive assessment, which merit further clinical evaluation. 13 The improvement of cognitive function for TAK-831 in schizophrenic patients is consistent with improvement of cognitive performance in rodent models. For example, another DAO inhibitor SEP-227900 increased D-serine in the cerebellum of rats in a dose dependent manner, and pretreatment of rats with this DAO inhibitor increased memory of the test object in the novel object recognition model in rats, suggesting improved cognitive function. 14 There are many published DAO co-crystal structures in the literature. Figure shows the co-crystal structure of human DAO enzyme with a hydroxy pyridazinone ligand , which was one of the most potent DAO inhibitors reported by both Takeda and Astellas. 7 Overall, the ligand adopts an L-shaped conformation in the binding site. The hydroxy pyridazinone head group is stacked between the flavin ring of FAD (flavin adenine dinucleotide) and Tyr224. The hydroxy-carbonyl moiety forms a salt bridge with Arg283, and the N-H forms an H-bond with Gly313. On the other side, the phenyl ring sticks into a relatively hydrophobic pocket and stacks with Tyr224 to form a πedge interaction. Intrigued by the target biology and therapeutic potential for treatment of cognitive impairment in schizophrenia or other neurological disorders, we initiated a program to identify novel DAO inhibitors with best-in-class properties. The program leveraged the Schrödinger physics-based modeling technology, specifically, a human DAO Free Energy Perturbation (FEP+) model which was developed on the basis of published SAR data. 15 Key protein-ligand interactions presented in the co-crystal structures were taken into account as novel ligands were designed by multiple internal medicinal and computational chemists. The designs were further evaluated with the hDAO FEP+ model, and the top ideas were prioritized for synthesis. Among them (Table ), 12, 13 and 14 16 showed good hDAO biochemical potency, which is consistent with the FEP+ model prediction. 17 Thanks to their low molecular weights, all three compounds have good ligand efficiency (LE) and lipophilic ligand efficiency (LLE). Initially SAR work indicated that various substituents can be tolerated on the phenyl ring of both dihydropyrazine dione (12, DHP dione) and N-hydroxyl pyrimidine dione (13, NHP dione) hit classes. For instance, the CF3 group of 12 can be replaced with a chlorine to yield 15 which shares similar DAO potency. Similarly, a chlorine can be incorporated at the para-position of 13 to afford 16, which is slightly more potent than 13. In order to understand the in vivo pharmacokinetic (PK) properties of the hits, especially their ability to cross the blood-brain barrier (BBB), compounds 14, 15, and 16 were dosed as a cassette in mice along with 17 18 (Table ) as a reference compound. To understand the binding interactions of the DHP dione chemical series, a co-crystal structure of 12 was obtained via a soaking experiment with the hDAO apo crystal. As shown in Figure , compound 12 binds to the hDAO enzyme in a fashion very similar to ligand 11. The dihydropyrazine dione head group is stacked between the flavin ring and Tyr224. The hydroxy-carbonyl moiety forms a salt bridge with Arg283, and the NH forms H-bond with Gly313. On the tail side, the 4-trifluoromethyl phenyl sticks into the hydrophobic pocket. Unlike the acidic hydroxy pyridazinone head group in compound 11, the pKa of 12 was measured at 9.7, 20 which would imply a pKa penalty in binding to DAO, as only the anionic form can actively bind to DAO. In addition, the head group of 12 is pseudosymmetric with two possible anionic tautomers, and substitution on the head group can impact the tautomer distribution. Fortunately, quantum mechanics (QM) calculations suggested that the active tautomer is strongly favored for 12, by 0.8 kcal/mol. Although the DHP dione chemical series is relatively weaker than the hydroxy pyridazinone 21 chemical series due to the higher pKa of the head group, it may benefit from other properties such as pharmacokinetics and brain penetration.