Inhibition of Insulin-Regulated Aminopeptidase by Imidazo [1,5-α]pyridines-Synthesis and Evaluation

Abstract

Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC₅₀ value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound's metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn²⁺ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.

Keywords: IRAP; enzyme inhibitor; imidazo [1,5-α]pyridine-based inhibitors; insulin-regulated aminopeptidase.

Figure 1

IRAP inhibitors HA08, HFI-419 and exemplified chemical cluster (A–C).

Figure 2

(A) Selectivity profiling of hit compound 6a through concentration–response experiments on human IRAP and APN. Data are presented as the average and standard deviation of three technical replicates for each concentration in experiments based on membrane preparations with overexpressed IRAP and APN, respectively. The symbols represent two independent test occasions for compound 6a on human IRAP (●,○) and aminopeptidase N (◼,◻). Also included are inhibition curves for human IRAP (▲) and APN (△) in the presence of a sulfonamide-based inhibitor that was used as a positive control (see compound 3 in ref [50]). Solid lines represent the best fits to a four-parameter inhibition curve within GraphPad Prism, with the average pIC₅₀ for human IRAP at 5.9 ± 0.05. (B) Rates of enzymatic processing by IRAP as a function of L-Leu-pNA concentration in the presence of 6a from 0 to 100 µM (2.5-fold dilutions between concentrations). Data are based on the average of four technical replicates in experiments conducted on IRAP in membrane preparations from CHO cells (for example, raw data at a low and a high substrate concentration are provided in Supplementary Materials Figure S1). The solid lines represent the best fit to a model of non-competitive inhibition, resulting in a K_m value of 0.48 ± 0.03 mM, a V_max of 3.6 ± 0.07 mAbs/min and a pK_i value of 5.64 ± 0.03. (C) Data from Figure 2A presented as a double-reciprocal Lineweaver Burk plot, demonstrating the characteristic behavior of non-competitive inhibitors with K_m unaffected and with V_max gradually reduced in the presence of increasing concentrations of inhibitor.

Scheme 1

Reagents and conditions: (a) i. Grignard reagent, toluene, 1h, ii. NaBH₄, 14 h, 67–95%; (b) i. aryl or alkyl bromide, Mg, I₂, THF, MW, 100 °C, 1 h [56], ii. nitrile, rt., 2 h, iii. NaBH₄, 14 h, 44–90%; (c) Boc-protected amino acid, EDC, HOBt, TEA, DCM, rt., 3 h, 70–95%; (d) POCl₃, pyridine, DCM, 0 °C to rt., 14 h, 70–85%; (e) HCl in dioxane, rt., 14 h, 99%; (f) acetyl chloride, MeOH, DCM, 0 °C, 2 h, 94–99%; (g) isocyanate or isothiocyanate, TEA, MeCN, rt., 20 min, 48–99%; (h) i. amine, triphosgene, TEA, DCM, 0 °C, 1 h, ii. 5a, TEA, DCM, rt., 30 min, 61–94%.

Scheme 2

Reagents and conditions: (a) i. SOCl₂, MeOH, rt., ii. Triphosgene, TEA, DCM, 0 °C, 1 h, iii. 5a, TEA, rt., iv. LiAlH₄, THF, 1 h, 69%.

Figure 3

Summary of SAR exploration.