Aicher, Peter L

Aicher, Peter L. 14 to 4-F-indazole, and incorporating a key hydroxyl group led to the discovery of 25, which possesses exquisite potency and selectivity, as well as an improved pharmacokinetic profile suitable for oral dosing. = 2 data differing by less than 3-fold; otherwise additional replicae were collected. bNA stands for not tested. This encouraging result prompted us to explore a variety of other amides. In general, analogs having bis-ortho substituents (4, 5) attached to the benzoyl moiety were preferred over their counterparts with mono-ortho or non-ortho substitution because of their superior potency and chemical stability to amide hydrolysis. Eventually 2-Cl-6-CF3 substituted analog 5 emerged not only by virtue of its potency but also due to its high stability under both basic and acidic conditions. At this point, gratifyingly we also were able to obtain the cocrystal structure of 5 (MRL-871),17 which not only revealed its unique allosteric binding mode but also provided useful structural insight into guide our subsequent SAR exploration and optimization. We next began to investigate the effect of substitution around the benzoic acid ring moiety. Since 4-F substitution on the indazole core was slightly more favorable for cellular activity (e.g., 6 vs 5), subsequent SAR exploration was carried out to the same context (7C13). Ortho-substituents next to the carboxylate moiety (F, Cl, or NH2) were generally detrimental for potency (7C9). In contrast, the presence of ortho-OH further improved both biochemical and cellular potency. Conversion of 10 into its MeO-analog 11 led to a significant drop in potency. Substitution at meta-position was also briefly explored (12, 13), with meta-fluoro substitution showing marginal benefit. With potent analogs such as 10 and 12 (MRL-299)17 in hand, one of our goals early on was to identify a tool compound with sufficient potency, metabolic stability, and favorable off-target profile suitable for preliminary and proof-of-biology studies. For this purpose, the compounds in Table Tarafenacin D-tartrate 2 were prepared. Incorporation of a nitrogen atom into either the 4 or 6 position of the core was tolerated with minimal loss of activity (14C16). However, nitrogen substitution at the 6 position of the indazole core (15) led to notable CYP inhibition (CYP3A4 IC50 = 4.8 M). Aza-indole analog 17 was also tolerated with a slight decrease in potency relative to 14. Aza-indazole analog 14 (MRL-248)21 was profiled in a Eurofins panel of counterscreening at a concentration of 10 M against a panel of 108 additional kinases, receptors, transporters, and nuclear receptors and showed only weak activity again one target in the panel (PPAR, IC50 = 2 uM). In contrast, compound 12 showed nine Vwf hits with >50% inhibition under the same conditions. Compound 14 also showed no appreciable activity against a panel of related nuclear hormone receptors.21 In addition, sufficient oral exposure could be achieved with 14 in a high dose mouse pharmacokinetic study. On the basis of its PK and selectivity profile, compound 14 was chosen as a tool compound and utilized extensively in various and studies.21,22 Table 2 Indazole Core Modification Open in a separate window aIC50 values are the mean of at least two runs. Our next Tarafenacin D-tartrate goal focused on improving the metabolic stability of 14 while maintaining its favorable potency, selectivity, and off-target profile. Met-ID studies of incubating 14 in hepatocytes revealed acyl-glucuronidation as one of its major clearance mechanisms. Unfortunately, several attempts to overcome this metabolism pathway by converting the carboxylate into other typical acid bioisosteres, such as tetrazole or acyl-sulfonamide groups, led to significant loss of potency.23 Another tactic to mitigate glucuronidation is to introduce steric hindrance next to the carboxylate moiety,24 but none of the pharmacokinetic profile in both rat and dog PK studies. In addition, 25 showed a clean off-target profile in Eurofins Panlabs panel (Supporting Information). Consistent with its lower clogP and higher Fsp3, 25 also shows improvement in physiochemical properties (e.g., solubility at pH = 2).26 Open in a separate window Figure 2 Overall profile of 25 vs 14. Gratifyingly, we also obtained an X-ray cocrystal structure of 25 bound to RORt ligand binding domain (LBD) (Figure ?Figure33). The carboxylate group forms several H-bond interactions with RORt backbone or residues, including Phe498, Ala497, and Gln329. Tarafenacin D-tartrate The 2-Cl-6-(cyclopropyl)phenyl.