Synthesis, biological evaluation, and molecular modelling studies of potent human neutrophil elastase (HNE) inhibitors

Abstract

We report the synthesis and biological evaluation of a new series of 3- or 4-(substituted)phenylisoxazolones as HNE inhibitors. Due to tautomerism of the isoxazolone nucleus, two isomers were obtained as final compounds (2-NCO and 5-OCO) and the 2-NCO derivatives were the most potent with IC50 values in the nanomolar range (20–70 nM). Kinetic experiments indicated that 2-NCO 7d and 5-OCO 8d are both competitive HNE inhibitors. Molecular modelling on 7d and 8d suggests for the latter a more crowded region about the site of the nucleophilic attack, which could explain its lowered activity. In addition molecular dynamics (MD) simulations showed that the isomer 8d appears more prone to form H-bond interactions which, however, keep the reactive sites quite distant for the attack by Ser195. By contrast the amide 7d appears more mobile within the active pocket, since it makes single H-bond interactions affording a favourable orientation for the nucleophilic attack.

New 3‐unsubstituted isoxazolones as potent human neutrophil elastase inhibitors: Synthesis and molecular dynamic simulation

“…Starting from 2‐aryl‐2‐formylethylacetate derivatives 1a‐i (Alves et al, ; Beccalli et al, ; Liu, Chen, Wu, & Tang, ; Rajput et al, ), we performed cyclization with hydroxylamine hydrochloride and piperidine in ethanol at reflux temperature (for compounds 2a‐g ; Beccalli et al, ; Snyder et al, ) or with hydroxylamine hydrochloride in a mixture MeOH/H 2 O 10:1 at reflux (for compounds 2h and 2i [Snyder et al, ]) to obtain the key intermediates with an isoxazolone core of Type 2 . Despite the possibility that this nucleus can exist in three different tautomers (Cencioni, Franchini, & Orienti, ; Franchini, ; Frolund et al, ), we confirmed in our previous work (Giovannoni et al, ) and here that acylation of intermediates 2a‐i (Beccalli et al, ; Snyder et al, ) resulted exclusively in the NCO derivatives 3a‐i and 4a‐i when used the appropriate acyl/aroyl chloride and sodium hydride in anhydrous tetrahydrofuran at room temperature. This result indicates that the NH form is the only product formed under these reaction conditions.…”

“…All the biological results are reported in Table , as compounds of Type 3 ( N ‐cyclopropylcarbonyl derivatives) and of Type 4 ( N ‐ m ‐tolylcarbonyl derivatives) containing the same substituents R in the phenyl ring at Position 4 ( R is shown in the central column). For comparison, we also include our previous reported compounds A and B , which belong to the series of the 3‐methylisoxazolones (Giovannoni et al, ). Our biological results verify that all compounds of Type 3 and 4 are potent HNE inhibitors, as their HNE inhibitory activity was in the low nanomolar range (IC 50 = 11–81 nM), with most compounds being more active than Sivelestat (IC 50 = 44 nM).…”

“…The experiments were performed in triplicate using N ‐methylsuccinyl‐Ala‐Ala‐Pro‐Val‐7‐amino‐4‐cumarin as substrate for HNE. *Giovannoni et al, .…”

“…In order to develop new compounds that modulate the HNE proteolytic activity, our research group has investigated several molecular scaffolds, such as indazoles (Crocetti et al, , ), indoles (Crocetti et al, ), azaindoles (Crocetti et al, ; Giovannoni et al, ), cinnolinones (Giovannoni et al, ), and thiazolones (Crocetti et al, ). Most recently, we focused on the synthesis isoxazol‐5(2 H )‐one derivatives (Giovannoni et al, ; Vergelli et al, ). These new isoxazolone‐based derivatives exhibited HNE inhibitory activity in the nanomolar range (IC 50 = 20–96 nM), and molecular modeling studies allowed us to determine that the endocyclic carbonyl at Position 5 of the isoxazolone was the group attacked by Ser195 of the catalytic triad.…”

“…The most potent isoxazolones were further evaluated for chemical stability, and the results showed that these derivatives were more stable than our previously described HNE inhibitors with a bicyclic scaffold. Finally, kinetic experiments demonstrated that these compounds acted as competitive HNE inhibitors, and structure–activity relationship (SAR) analyses established that the amide function at Position 2 was important for the activity, and the best substituents at this position were m‐ tolylcarbonyl and cyclopropylcarbonyl groups (Giovannoni et al, ). In order to complete SAR analysis for this class of HNE inhibitors, we designed and synthesized a new series of 4‐phenyl‐3‐unsubstituted derivatives (Figure ) bearing different substituents on the 4‐phenyl ring while maintaining the best fragments at Position 2 of the isoxazolone scaffold (i.e., m‐ tolylcarbonyl or cyclopropylcarbonyl groups).…”