Further studies on 2-arylacetamide pyridazin-3(2H)-ones: Design, synthesis and evaluation of 4,6-disubstituted analogs as formyl peptide receptors (FPRs) agonists

Abstract

Formyl peptide receptors (FPRs) play an essential role in the regulation of endogenous inflammation and immunity. In the present studies, a large series of pyridazin-3(2H)-one derivatives bearing an arylacetamide chain at position 2 was synthesized and tested for FPR agonist activity. The pyridazin-3(2H)-one ring was confirmed to be an appropriate scaffold to support FPR agonist activity, and its modification at the 4 and 6 positions led to the identification of additional active agonists, which induced intracellular Ca2+ flux in HL-60 cells transfected with either FPR1, FPR2, or FPR3. Seven formyl peptide receptor 1 (FPR1)-specific and several mixed FPR1/FPR2 dual agonists were identified with low micromolar EC50 values. Furthermore, these agonists also activated human neutrophils, inducing intracellular Ca2+ flux and chemotaxis. Finally, molecular docking studies indicated that the most potent pyridazin-3(2H)-ones overlapped in their best docking poses with fMLF and WKYMVM peptides in the FPR1 and FPR2 ligand binding sites, respectively. Thus, pyridazinone-based compounds represent potential lead compounds for further development of selective and/or potent FPR agonists.

2-Arylacetamido-4-phenylamino-5-substituted pyridazinones as formyl peptide receptors agonists

“…These molecules have quite subtle differences in structure and are oriented in the binding site with brominated benzene rings directed deep into the binding site (Figure 3A). This orientation of the p -bromophenyl moiety is analogous to that observed previously for parent compounds [20]. Agonist 13a with an ethyl substituent at position 5 of the pyridazine ring is H-bonded with Thr177, while the m -methoxyphenyl substituent occupies a hydrophobic subpocket surrounded by Ala181, Gly264, Leu268, Tyr277, and Ile279 (Figure 3B).…”

“…Human neutrophils were suspended in HBSS containing 2% (v/v) fetal bovine serum (FBS) (2×10 6 cells/mL), and cell migration was analyzed in 96-well ChemoTx chemotaxis chambers (Neuroprobe, Gaithersburg, MD), as previously described [20]. Briefly, lower wells were loaded with 30 µL of HBSS containing 2% (v/v) FBS and the indicated concentrations of test compound, DMSO (negative control), or 1 nM f MLF as a positive control.…”

“…The products were then converted to the 4-bromophenylacetamide derivatives 5a–f as follows. Intermediates 2a and 2d,e were alkylated with ethylbromoacetate under standard conditions to generate 3a [26] and 3d,e , which were transformed into the corresponding carboxylic acids 4a [22] and 4d,e through alkaline hydrolysis. These were transformed into the final amides of type 5 by treatment with 4-bromoaniline, ethyl chloroformate and triethylamine in THF.…”

“…Key requirements for agonist activity of this class of compounds were the presence of a 4-bromophenylacetamide side chain at the N-2 position of the scaffold [17, 19] and the presence of a methyl group at C-6 [20]. Position 4 could be substituted with a benzyl or phenylamino group, resulting in compounds with micromolar activity (Figure 1, general structure A ).…”

“…In the present study, we further investigated pyridazinone derivatives belonging to the series of 4-phenylamino derivatives (structure A , X = NH) which until now was only poorly studied [20]. In particular, we inserted at position 5 of the pyridazinone scaffold a variety of substituents, such as alkyl or acyl groups, ester, unsaturated chains, and pyrazole rings, in order to evaluate how such modifications affected target specificity and compound potency.…”