Discovery of N-(1,4-Benzoxazin-3-one) urea analogs as Mode-Selective TRPV1 antagonists.

A series of 1,4-benzoxazin-3-one analogs were investigated to discover mode-selective TRPV1 antagonists, since such antagonists are predicted to minimize target-based adverse effects. Using the high-affinity antagonist 2 as the lead structure, the structure activity relationship was studied by modifying the A-region through incorporation of a polar side chain on the benzoxazine and then by changing the C-region with a variety of substituted pyridine, pyrazole and thiazole moieties. The t-butyl pyrazole and thiazole C-region analogs provided high potency as well as mode-selectivity. Among them, antagonist 36 displayed potent and capsaicin-selective antagonism with IC50 = 2.31 nM for blocking capsaicin activation and only 47.5 % inhibition at 3 µM concentration toward proton activation, indicating that more than a 1000-fold higher concentration of 36 was required to inhibit proton activation than was required to inhibit capsaicin activation. The molecular modeling study of 36 with our homology model indicated that two π-π interactions with the Tyr511 and Phe591 residues by the A- and C-region and hydrogen bonding with the Thr550 residue by the B-region were critical for maintaining balanced and stable binding. Systemic optimization of antagonist 2, which has high-affinity but full antagonism for activators of all modes, led to the mode-selective antagonist 36 which represents a promising step in the development of clinical TRPV1 antagonists minimizing side effects such as hyperthermia and impaired heat sensation.

Discovery of potent indazole-based human glutaminyl cyclase (QC) inhibitors as Anti-Alzheimer's disease agents.

The toxic pyroglutamate form of amyloid-ß (pE-Aß) is important for the pathogenesis of early Alzheimer's disease (AD); therefore, reducing pE-Aß by inhibiting glutaminyl cyclase (QC) provides a promising strategy for developing disease-modifying AD drugs. In this study, potent and selective QC inhibitors with desirable drug-like properties were discovered by replacing the 3,4-dimethoxyphenyl group in a QC inhibitor with a bioisosteric indazole surrogate. Among them, 3-methylindazole-6-yl and 3-methylindazole-5-yl derivatives with an N-cyclohexylurea were identified as highly potent inhibitors with IC50 values of 3.2 nM and 2.3 nM, respectively, both of which were approximately 10-fold more potent than varoglutamstat. In addition, the three inhibitors significantly reduced pE-Aß3-40 levels in an acute animal model after intracerebroventricular (icv) injection and were selective for hQC. Further in vitro pharmacokinetic and toxicity studies, including those investigating cytotoxicity, hERG inhibition, blood-brain barrier (BBB) permeability and metabolic stability, indicated that N-(3-methylindazole-6-yl)-N'-(cyclohexyl)urea derivative exhibited the most promising efficacy, selectivity and drug-like profile; thus, it was evaluated for its in vivo efficacy in an AD model.

2-Amino-1,3,4-thiadiazoles as Glutaminyl Cyclases Inhibitors Increase Phagocytosis through Modification of CD47-SIRPα Checkpoint.

Glutaminyl cyclases (QC, isoQC) convert N-terminal glutamine or glutamate into pyroglutamate (pGlu) on substrates. IsoQC has recently been demonstrated to promote pGlu formation on the N-terminus of CD47, the SIRPα binding site, contributing to the "don't eat me" cancer immune signaling of CD47-SIRPα. We developed new QC inhibitors by applying a structure-based optimization approach starting from fragments identified through library screening. Screening of metal binding fragments identified 5-(1H-benzimidazol-5-yl)-1,3,4-thiadiazol-2-amine (9) as a potent fragment, and further modification provided 5-(1-(3-methoxy-4-(3-(piperidin-1-yl)propoxy)benzyl)-1H-benzo[d]imidazol-5-yl)-1,3,4-thiadiazol-2-amine (22b) as a potent QC inhibitor. Treatment with 22b in A549 and H1975 lung cancer cells decreased the CD47/αhCD47-CC2C6 interaction, indicative of the CD47/SIRPα interaction, and enhanced the increased phagocytic activity of both THP-1 and U937 macrophages.

Discovery of highly potent human glutaminyl cyclase (QC) inhibitors as anti-Alzheimer's agents by the combination of pharmacophore-based and structure-based design.

The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the treatment of early Alzheimer's disease (AD) by reducing the amount of the toxic pyroform of ß-amyloid (AßΝ3pE) in the brains of AD patients. In this work, we identified potent QC inhibitors with subnanomolar IC50 values that were up to 290-fold higher than that of PQ912, which is currently being tested in Phase II clinical trials. Among the tested compounds, the cyclopentylmethyl derivative (214) exhibited the most potent in vitro activity (IC50 = 0.1 nM), while benzimidazole (227) showed the most promising in vivo efficacy, selectivity and druggable profile. 227 significantly reduced the concentration of pyroform Aß and total Aß in the brain of an AD animal model and improved the alternation behavior of mice during Y-maze tests. The crystal structure of human QC (hQC) in complex with 214 indicated tight binding at the active site, supporting that the specific inhibition of QC results in potent in vitro and in vivo activity. Considering the recent clinical success of donanemab, which targets AßΝ3pE, small molecule-based QC inhibitors may also provide potential therapeutic options for early-stage AD treatment.

Structure-activity relationship investigation of Phe-Arg mimetic region of human glutaminyl cyclase inhibitors.

Glutamyl cyclase (QC) is a promising therapeutic target because of its involvement in the pathogenesis of Alzheimer's disease. In this study, we developed novel QC inhibitors that contain 3-aminoalkyloxy-4-methoxyphenyl and 4-aminoalkyloxyphenyl groups to replace the previously developed pharmacophore. Several potent inhibitors were identified, showing IC50 values in a low nanomolar range, and were further studied for in vitro toxicity and in vivo activity. Among these, inhibitors 51 and 53 displayed the most potent AßN3pE-40-lowering effects in in vivo acute model with reasonable BBB penetration, without showing cytotoxicity and hERG inhibition. The molecular modeling analysis of 53 indicated that the salt bridge interaction and the hydrogen bonding in the active site provided a high potency. Given the potent activity and favorable BBB penetration with low cytotoxicity, we believe that compound 53 may serve as a potential candidate for anti-Alzheimer's agents.