Benzylhydroxamic Acids as IRAP Inhibitors: Sulfonamide Variations

Abstract

Small-molecule inhibitors of the zinc metalloprotease insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3), are a promising new class of potential cognitive enhancers for the treatment of neurodegenerative disorders such as Alzheimer’s disease. Previous studies identified a series of sulfonamide-substituted benzylhydroxamic acids with fair BBB permeability, no indication of efflux and subnanomolar potency. We herein report our further structural optimization of this compound series focusing on various alterations to the sulfonamide group. Notably, introduction of dihydropyridopyrimidine provided the most active small-molecule inhibitor compound reported to date, with an IC₅₀ value of 34 nM. Molecular modeling studies, based on free energy perturbation simulations, show that the accommodation of the explored substituents on the binding mode proposed explain the experimental SAR herein reported and provide a rationale for the enhanced affinity of the optimized compounds.

Key words

IRAP - cognitive enhancement - enzyme inhibitor - hydro­xamic acid - free energy perturbation (FEP)

Inhibition of insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3), a zinc metalloprotease belonging to the oxytocinase subfamily of the M1 family of metalloproteases,[1] has attracted significant attention over the last two decades as an approach to develop molecules with potential clinical applications as cognitive enhancers for the treatment of, e.g., Alzheimer’s disease. Hence, considerable efforts have been devoted to identifying inhibitors suitable for further optimizations into clinical candidates.[2] [3] [4]

According to one hypothesis, the cognitive enhancement observed after administration of IRAP inhibitors is due to a suppression of the enzyme-mediated degradation of the substrates vasopressin[5] and oxytocin,[6] endogenous cyclic peptides known to improve parameters linked to cognition.[7] [8] [9] [10] Newer findings suggest that IRAP inhibitors may also facilitate memory by increasing the hippocampal dendritic spine density occurring via a GLUT4-mediated mechanism.[11]

The first druglike small-molecule IRAP inhibitors were reported by Albiston et al. in 2008,[12] built around a benzopyran scaffold. Subsequently, as a result of rational design from substrate structures and by utilizing various screening approaches, numerous druglike IRAP inhibitors encompassing very different scaffolds have been developed.[2] Examples include aryl sulfonamides,[13] [14] diaminobenzoic acid derivatives,[15] pseudophosphinic peptide transition-state analogues,[16] spiro-oxindole dihydroquinazolinones,[17] amino acid derivatives of bestatin,[18] and imidazopyridines.[19]

We recently reported a high throughput screening of 400,000 small molecules towards IRAP inhibition.[20] From this investigation, a qualified hit list of potent IRAP inhibitors was constructed. The most potent of these compounds, QHL1 (Figure [1]), was selected for further investigation, and in a subsequent publication we reported several analogues and evaluated their IRAP inhibition, resulting in the chloro-substituted 1, with a 10-fold improvement in potency.[21]

Figure 1 Structure of compound QHL1, 1, and the general structure of compounds 2–20 explored in this paper

We herein report various alterations of the sulfonamide function based on compound 1. The synthesis and inhibitory capacity of 2–20 as well as proposed binding modes, as deduced from molecular docking and free energy perturbation (FEP) of selected IRAP inhibitors, are presented (Figure [2] and 3).

Figure 2 Synthesized analogues with inhibitory concentrations; IC₅₀ value of 1 from previous publication[21]

The three analogues of compound QHL1, 2–4, were synthesized via Fischer esterification of 2-(4-bromophenyl)acetic acid (1a) to the methyl ester 1b (Scheme S1). This was followed by regioselective chlorosulfonylation using chlorosulfonic acid. Due to its high reactivity, the sulfonyl chloride intermediate was carefully extracted and directly reacted with amines A2–A4 to achieve the sulfonamides 2.1–4.1. The sulfonamides were then debrominated using palladium catalysis (with simultaneous solvent-mediated transesterification to the butyl ester when n-butanol was used) resulting in 2.2–4.2. This was followed by installation of hydroxamic acid by treatment with an excess of hydroxylamine and KOH. Compounds 5–20 were synthesized using the same strategy, starting from 2-(4-chlorophenyl)acetic acid (2a), but with the omission of the dehalogenation step.

The X-ray IRAP structure without inhibitor in the open-like conformation was retrieved from the PDB (code: 4PJ6)[22] and prepared for computational simulations with the Protein Preparation Wizard pipeline in Maestro (Schrödinger, version 2024-2). Preparation steps included proton addition and assignment of the most likely protonation state for titratable residues and rotameric assignment of planar residues (i.e., Asn, Gln, His). The docking pose obtained for compound 1 in our previous work[21] (Figure [3]A) was used as a basis to build an analogous docking pose for each compound considered (5–8, 11–13, and 15–20) with Maestro, followed by energy minimization of the ligand implemented with the OPLS4 force field[23] (Figure [3]B and supplementary Figure S1). The PDB files generated for each ligand, together with the prepared protein file, were the starting point for molecular dynamics (MD) simulations associated to relative binding free energy (RBFE) calculations using the dual-topology QligFEP automated workflow.[24] QligFEP allows the user to easily define each vertex comparing compound pairs and subsequently generate all free energy perturbation (FEP) input files and perform the ulterior analysis. For each RBFE calculation, the associated MD simulations in both protein and solvent environments were conducted on the MD software Q[25] under spherical boundary conditions, as described in the Supporting Information.

Figure 3 (A) Proposed binding mode of for compound 1. (B) Initial docking poses of all derivatives here considered for FEP simulations, following the same coloring scheme as in C. (C) Results from FEP simulations. The experimental relative binding free energy shift between each compound pair considered (gray) is compared with the FEP calculated value, colored according to the nature of the substituent explored (see legend and text for explanation), pentacyclic = 5-mebered heterocyclic. Error bars indicate the associated s.e.m. of the calculation estimated from replica simulations (see the Supporting information).

Initial studies to explore the impact of different sulfonamide functions were performed by modifying the original hit compound QHL1 from our previous screen.[20] In that work, we defined two possible binding modes for this scaffold, both showing bidentate coordination of the catalytic zinc ion through the hydroxamic acid moiety and a hydrogen bond with neighboring glutamic acid E431. A detailed exploration of the SAR generated therein followed on the basis of systematic FEP simulations, performed on each alternative binding mode.[20] The results of the FEP exploration allowed us to conclude that the dihydroisoquinoline ring of 1 would remain in a solvent accessible cavity, lining up with Y961 and further stabilized by hydrogen-bond interaction between the sulfonamide oxygen and Y549. We will herein discuss the SAR of the present series on the basis of such a binding mode, using our QligFEP approach for RBFE simulations (see Figure [3]).[24]

All three compounds lacking the chloro substituent in 1 (2–4) demonstrated no inhibitory activity below 10 μM (Figure [2]). In all subsequent studies, the crucial chloro group[21] was maintained while modifications to the sulfonamide moiety were explored. We initially explored the introduction of bromine in each of the four available positions, with the goal of probing available space in the enzyme and to evaluate potential electronic effects. Compound 5 with the electron-releasing halogen positioned para to the sulfonamide function, demonstrated an IC₅₀ of 0.117 μM, the best value in the bromo series. The potency as IRAP inhibitors was reduced twofold for compounds 6 and 7 (IC₅₀ = 0.234 μM and 0.251 μM, respectively) and more than 10-fold for 8 (IC₅₀ = 1.258 μM).

The RBFE exploration of the brominated derivatives partially captured these effects, effectively showing a detrimental effect of moving the halogen from the optimal para position in 5 to the alternative locations in 6, 7, and to a minor extent on 8 (Figure [3]C, yellow bars, Table S1). While the closure of the full cycle (i.e., 5 → 6 → 7 → 8 → 5) yields a low-associated error of 0.22 kcal/mol, the mean unassigned error (MUE) of the five possible pairwise transformations collected on Figure [3]C reached a considerable value of 0.73 kcal/mol, exceeding the average experimental RBFE values of the five pairs involved <ΔΔG_bind,exp> = 0.65 kcal/mol (brackets indicating an average value). This observation, together with the relatively high average standard error of the mean (s.e.m.) obtained for these calculations (<s.e.m.>) = 1.13 kcal/mol, error bars on Figure [3]C) weakens the statistical significance of the FEP results for the bromine analogues.

Opening of the aliphatic ring system of 1 to make the molecule more flexible resulted in a significant loss of activity: the N-ethyl-substituted sulfonamide 9 demonstrated an IC₅₀ value of 1.51 μM, while the corresponding value for the methyl sulfonamide 10 was 0.540 μM. These findings suggest that the locked aliphatic ring facilitates positioning of the aromatic ring in a favorable position. The position and orientation of the phenyl ring was further examined by alteration of the size of the nitrogen heterocycle. Extension of the ring system of 1 by incorporation of a methylene group to create 11 resulted in a more than 10-fold reduction of activity (IC₅₀ = 0.794 μM). The sulfonamide 12 comprising the smaller five-membered ring system and with the nitrogen atom connected to the phenyl ring resulted in a similar outcome (IC₅₀ = 0.676 μM).

An enlargement of the ring system in 12 by addition of a methylene group was essentially deleterious for the activity and 13 demonstrated an IC₅₀ value of 3.16 μM. A comparison of compound 1 and its isomer 13 demonstrates that a correct location and direction of the bicyclic system is crucial for efficient inhibition as 13 is 50-times less efficient at IRAP inhibition than 1. Thanks to the dual topology approach implemented in QligFEP, we could simulate such effects on ring-size variation with high accuracy, with the transformations 11 → 12 → 13 → 11 showing qualitative and quantitative agreement with experimental data (Figure [3]C, light-green bars, Table S1). In other words, based on the binding mode proposed, the FEP simulations can accurately explain the relative differences in affinity, with a MUE = 0.13 kcal/mol, in this case significantly smaller than the average experimental RBFE values of this compound series (<ΔΔG_bind,exp> = 0.62 kcal/mol). Following these insights, the aliphatic ring was left intact, while the phenyl ring of 1 was replaced with various heteroaromatic systems. The indole and pyrazole derivatives 14 and 15, both possessing hydrogen-bond-donating capacities, were inefficient inhibitors (IC₅₀ = 6.92 μM and 1.38 μM, respectively). Conversely, the thiophene and oxazole compounds 16 and 17 with hydrogen-bond-accepting abilities resulted in better outcomes, inhibiting IRAP with IC₅₀ values of 0.117 μM and 0.480 μM respectively. Within this series, we restricted the FEP exploration to 5-membered heterocycles, including the transformations 15 → 16 → 17 → 15 (Figure [3]C, dark green bars, Table S1). As in the previous case, the accuracy of the calculations was remarkable (MUE = 0.25 kcal/mol), showing enough sensitivity of the proposed binding mode to quantitively assess the SAR of these compounds, which on average resulted in experimental RBFE variations of <ΔΔG_bind,exp> = 1 kcal/mol.

The exploration continued with other hydrogen-accepting heterocycles and replacement of the phenyl ring with pyridine to provide 18 resulted in a compound with an IC₅₀ value of 0.100 μM, while its regioisomer 19 with the nitrogen atom located at approximately the same position as the nitrogen in the oxazole derivative 17 exhibited an IC₅₀ value of 0.044 μM. Hence, 19 acts as a better IRAP inhibitor than the lead compound 1. The pyridine derivative 19 also has a more than 10-fold better ligand-lipophilicity efficiency (LLE) than that calculated for 1, the latter devoid of the nitrogen atom (LLE = 6.42 vs 5.00, pIC₅₀ – logD) and a better LLE than its pyridine regioisomer 18 (LLE ) 5.91 pIC₅₀ – logD). The LLE is a parameter-linking potency and lipophilicity and is applied in efforts to estimate druglikeness.[26] An incorporation of an additional nitrogen atom in the ring system to provide the dihydropyridopyrimidine hydroxamic acid derivative 20 was productive and resulted in the best IRAP inhibitor in the series with an IC₅₀ value of 0.034 μM.

These bioisosteric effects were again well captured by our structural model of IRAP inhibitor binding, with the RBFE simulations 18 → 19 → 20 → 18 showing the gradual improvement in affinity described above (from 18 to 20, Figure [3]C, blue bars, Table S1) with very high sensitivity (MUE = 0.19, significantly under the average value of experimental RBFE values, <ΔΔG_bind,exp> = 0.65 kcal/mol).

In summary a series of 16 new chloro-substituted benzylhydroxamic acids were synthesized and their capacity to inhibit IRAP assessed. We report that replacement of the dihydroisoquinoline moiety in 1, with a dihydropyridopyrimidine, leads to a twofold improvement of the inhibition, and 20 exhibited an IC₅₀ value of 34 nM. The binding mode proposed was challenged with extensive FEP simulations, strongly supporting the observed structure–activity relationship (SAR) in this series of benzylhydroxamic acids. Further evaluations of this series of new IRAP inhibitors are ongoing.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

We thank Professor em Anders Hallberg for constructive discussions and the SciLifeLab Drug Discovery and Development Platform. We gratefully acknowledge support from the Kjell and Märta Beijer Foundation.

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Publication History

Received: 31 January 2025

Accepted: 19 March 2025

Accepted Manuscript online:
19 March 2025

Article published online:
15 April 2025