In situ click chemistry generation of cyclooxygenase-2 inhibitors

Abstract

Cyclooxygenase-2 isozyme is a promising anti-inflammatory drug target, and overexpression of this enzyme is also associated with several cancers and neurodegenerative diseases. The amino-acid sequence and structural similarity between inducible cyclooxygenase-2 and housekeeping cyclooxygenase-1 isoforms present a significant challenge to design selective cyclooxygenase-2 inhibitors. Herein, we describe the use of the cyclooxygenase-2 active site as a reaction vessel for the in situ generation of its own highly specific inhibitors. Multi-component competitive-binding studies confirmed that the cyclooxygenase-2 isozyme can judiciously select most appropriate chemical building blocks from a pool of chemicals to build its own highly potent inhibitor. Herein, with the use of kinetic target-guided synthesis, also termed as in situ click chemistry, we describe the discovery of two highly potent and selective cyclooxygenase-2 isozyme inhibitors. The in vivo anti-inflammatory activity of these two novel small molecules is significantly higher than that of widely used selective cyclooxygenase-2 inhibitors.Traditional inflammation and pain relief drugs target both cyclooxygenase 1 and 2 (COX-1 and COX-2), causing severe side effects. Here, the authors use in situ click chemistry to develop COX-2 specific inhibitors with high in vivo anti-inflammatory activity.

Fig. 1

Design of building blocks for in situ click chemistry reaction in the COX-2 isozyme. Structure of selective COX-2 inhibitor celecoxib (1) and rofecoxib (2), target compounds (7–12, 16–25, 28, 29, 32, and 33) and illustration of in situ click chemistry reaction principle inside the COX-2-binding pocket

Fig. 2

Synthesis scheme of target compounds. Synthesis of various 5-azido-pyrazoles (5, 14, 27, and 31) and reference compounds (7–12, 16–25, 28, 29, 32, and 33)

Fig. 3

Monitoring of COX-2 isozyme-mediated in situ click chemistry reaction. In situ click chemistry formation of compounds 18 (a) and 21 (b) as determined by LC/MS-SIM

Fig. 4

Multicomponent in situ click chemistry reaction. Comparison of triazole compounds formed after the incubation of all azide and alkyne building blocks in the presence of the COX-2 isozyme

Fig. 5

Thermodynamic analyses for the binding of 5-azido-pyrazole (14) to COX-2. a Raw ITC data obtained upon titration of compound 14 with COX-2 protein at room temperature (298.15 K); b Plot of the integrated heat signal as a function of the molar ratio of ligand to the protein; c Thermodynamic parameters for the binding of compound 14 with COX-2 (n = 3, ±s.e.m.)

Fig. 6

In vivo anti-inflammatory activity profile of in situ click chemistry hits. a, b Effective dose (ED₅₀) calculated at 3 and 5 h time after treatment of compound 18 (n = 4, ±s.e.m.) and 21 (n = 5, ±s.e.m.); c, e Control; d, f Images showing the in vivo effect at 3 h after treatment with 0.3 mg kg⁻¹ p.o. of compounds 18 (left paw) and 21 (right paw), respectively

Fig. 7

Computational analysis of click chemistry building blocks in the COX-2 active site. Molecular docking of 5-azido-pyrazole compounds (5, 14, 27, and 31) and alkynes (6a–6f and 15a–15e) in the binding site of COX-2 isozyme (PDB ID: 6COX). Hydrogen atoms of amino-acid residues are omitted for clarity

Fig. 8

Comparison of the binding conformation of in situ click chemistry reaction hit compounds with crystalized SC558. a In COX-2 isozyme (PDB:6COX) the compound 18 (cyan carbon) and SC558 (green carbon) are displayed in stick mode and surface of compound 18 is highlighted (grey) color; b compound 21 (magenta carbon) and SC558 (green carbon) are displayed in stick mode and surface of compound 21 is highlighted (grey) color