Compound 17 was also included in Table as a reference compound, which was measured 17 nM in the hDAO biochemical assay. It is slightly right shifted in the hDAO cell assay, but about 4-fold left shifted in the mouse DAO cell assay. In the DHP dione chemical series, para-substitution on the phenyl ring is beneficial to potency, as the unsubstituted analog 18 is much less active. At the para-position, CN substitution (19) can also be tolerated in addition to Cl, while the methoxy analog 20 is less potent. From the mono Cl-substituted analogs 15, 21, and 22, para-substitution is the most preferred, while ortho-substitution is not tolerated. Compound 19 can be substituted with a fluorine ortho to the cyano group as in analog 23, while 3,5-dichloro substituted analog 24 is less active when compared to the mono-substituted analog 21. The tail region tolerates other hetero aromatic rings such as pyridine (25) and bicyclic aromatic rings such as quinoline (26) with some loss of potency. Polar groups can also be tolerated in this region as exemplified by compound 27 and 28. It is worth noting that both compound 27 and 28 were designed by the AutoDesigner algorithm featuring uncommon yet drug-like functionalities. In terms of hDAO FEP+ model performance, the majority of the prediction is within 1 log unit of the experimental IC50 value. Compared to 17, most analogs showed lower but moderate cell permeability and low efflux ratio in the MDCK-MDR1 assay, which may partially account for the near 10-fold shift in the hDAO cell assay. To ensure that the compound activity is not an artifact from their redox potential, the horseradish peroxidase assay (HRP) was developed as a counter screen. All compounds in Table were shown to be clean up to 10 µM in the HRP assay. SAR of the linker region was also explored (Table ). In terms of the linker length, the 2-carbon linker ( ) is superior to the 3-carbon linker (33) according to the FEP+ predictions. 22 It is also better than the 1-carbon linker based on the matched pair of 15 and 34. Analogs with fluorinated linker (i.e. 35) are also interesting, as fluorinesubstituted linkers were predicted to lower the pKa of the head group. However, compound 35 failed in the synthesis due to chemical stability issues. Linkers with hetero atoms were also explored. Although analogs with an oxygen linker failed in synthesis, the sulfur-linked analogs are stable enough for further SAR development. Encouragingly, both 36 and 37 are about 3-fold more potent than 12 in the hDAO biochemical assay. The enhancement in biochemical potency may partly be attributed to lower pKa's of the head groups in 36 and 37. Compound 36 has a measured pKa of 8.5, and compound 37 9.2. Compared to 12 with a pKa of 9.7, 36 and 37 are more favored to form the bio-active anionic structures. In addition, they both show a lower cell shift when compared to 12, possibly due to moderately higher cell permeability as measured in the MDCK-MDR1 assay. The tail SAR of the sulfur-linked analogs 36 and 37 largely resembles that of 12 (Table ). Further exploration of the tail SAR of sulfur-linked analogs led to significant potency improvement in the hDAO biochemical and cell assays. In order to further enhance compound inhibitory potency against hDAO, fused ring designs in the linker region were also assessed by the FEP+ model. One of the ideas that stood out was 54, which was designed by cyclizing the linker of 37 to form a fused 1,4-oxathiane ring. It was predicted that cyclization would lead to a gain in potency resulting at least in part from stabilization of the linker and tail piece. FEP+ predicted this compound to be a 3 nM inhibitor in the hDAO assay (Figure ). To our delight, the compound showed an IC50 of 25 nM in the assay, a 3fold improvement from 37. Thus, cyclized analogs with the best substituents from the chemical series were prepared. Most of these analogs showed significant improvement when compared to their acyclic counterparts in the hDAO biochemical assay. However, there was less improvement in the human and mouse cell DAO assays due to larger cell shift for the cyclized analogs. The binding mode of the cyclized analogs was confirmed by X-ray cocrystal structure of 59 (Figure ), which very much resembles the FEP+ snapshot of compound 54 binding to hDAO. The biggest changes are in the tail region due to different substituents at the para-position. Another interesting design on the cyclized analogs is quaternary methyl adduct 61. While this methyl addition was not initially predicted by our FEP+ model to lead to any gain in potency when compared to the des-methyl analog 55, the racemic quaternary methyl 61-rac 23 was tested to be about 5-fold more potent than 55-rac. That is because we did not have more closely related starting references for our FEP+ model at the time of the original prediction for 61. Subsequently we troubleshooted the FEP+ model by using the more closely related des-methyl analog 55 as the starting reference and observed that the methyl group displaces a high-energy water molecule concurrent with a predicted gain of potency (Figure ). The quaternary methyl group was incorporated into other analogs and resulted in roughly 2-fold improvement in DAO biochemical and cellular assays. Notably, compound 63 showed inhibition potency near 100 nM in both human and mouse DAO cell assays. In an effort to explore new opportunities for potency enhancement, careful examination of the DAO catalytic site revealed a subpocket just beyond the tail region, which was not explored by other groups (Figure ). In order to design into this subpocket, we employed our AutoDesigner algorithm to enumerate novel design ideas using compound 54 as a template. Initially over 198 million design ideas were generated by the algorithm, which were filtered by an array of criteria such as molecular properties, CNS and drug-like MPO's, and synthetic complexity.