New NSAID Conjugates as Potent and Selective COX-2 Inhibitors: Synthesis, Molecular Modeling and Biological Investigation

Abstract

New sets of ibuprofen and indomethacin conjugates comprising triazolyl heterocycle were synthesized via click chemistry, adopting an optimized protocol through the molecular hybridization approach affording the targeted agents in good yields. The new non-steroidal anti-inflammatory drug (NSAID) conjugates were designed and synthesized and could be considered as potential drug candidates for the treatment of pain and inflammation. The anti-inflammatory properties were investigated for all the synthesized conjugates. Among 14 synthesized conjugates, four (5a, 5b, 5d, and 5e) were found to have significant anti-inflammatory properties potency 117.6%, 116.5%, 93.8%, and 109.1% in comparison to reference drugs ibuprofen (97.2%) and indomethacin (100%) in the rat paw edema carrageenan test without any ulcerogenic liability. The suppression effect of cytokines IL-6, TNF-α, and iNOS in addition to NO in the LPS-induced RAW264.7 cells supports the promising anti-inflammatory properties observed in the ibuprofen conjugates. In addition, several conjugates showed promising peripheral and central analgesic activity. The selectivity index (SI) of compound 5a (23.096) indicates the significant efficacy and selectivity for COX-2 over COX-1. Molecular modeling (docking and QSAR) studies described the observed biological properties.

Keywords: COX; analgesic; anti-inflammatory; ibuprofen; indomethacin; molecular modeling.

Figure 1

Clinically approved NSAIDs.

Figure 2

The targeted designed molecules: NSAID conjugates.

Scheme 1

Synthesis of designed Ibu conjugates 5a–g and Indo conjugates 8a–g.

Figure 3

% Inhibition of edema thickness by the tested compounds.

Figure 4

Evaluation of NO production and cell viability in LPS-stimulated RAW264.7 macrophages of Ibuprofen conjugates. (A) A concentration of 40 μg/mL of ibuprofen conjugates (5a, 5b, 5d, and 5e) and indomethacin (positive control) was used for the treatment of LPS-stimulated RAW264.7 macrophages. The Griess method was used to evaluate the nitrite content in cell supernatants. Significant differences indicated in the graphs are all in comparison to LPS-stimulated cells only. (B) A concentration of 40 μg/mL of ibuprofen conjugates and indomethacin (positive control) was used for the treatment of LPS-stimulated RAW264.7 macrophages. Cytotoxicity of the macrophages was evaluated using an MTT assay. No significant cytotoxicity was observed for LPS-stimulated cells only (designated as ##)., Ibuprofen conjugates and indomethacin (positive control) in comparison to control. Values are mean ± SD (n = 3). * p < 0.05, *** p < 0.001.

Figure 5

Expression levels of IL-6 (A), TNF-α (B), and iNOS mRNA (C) in LPS-stimulated RAW264.7 macrophages. A concentration of 40 μg/mL of indomethacin (positive control), 5a, 5b, 5d, and 5e was used for the treatment of LPS-stimulated RAW264.7 macrophages (10 ng/mL). The RT-qPCR was used to measure mRNA levels of IL-6, TNF-α, and iNOS using the comparative method (2^−ΔΔCT). Significant differences indicated in the graphs are all in comparison to LPS-stimulated cells only (designated as ##). Values are means ± S.D. (n = 3). * p < 0.05, ** p < 0.001, *** p < 0.001.

Figure 6

Binding interactions and docked pose of compound 5e in the COX-1 crystal structure, PDB: 3N8W. Critical interactions with Arg120, Leu531, and Val349 are shown.

Figure 7

Binding interactions and docked pose of compound 5a in the COX-1 crystal structure, PDB: 3N8W. There is a loss of interactions with Leu531 and Val349, whereas interaction with Ser530 is observed. The choro group is oriented on the other side of the binding pocket and interacts with Ile523.

Figure 8

Binding interactions and docked pose of compound 5a in the COX-2 crystal structure, PDB: 6COX. The choro group interacts with Pro86 and Val89, conferring 5a COX-2 selectivity.

Figure 9

Overlay of docked (XP) poses of indomethacin (colored green) and ibuprofen (colored pink) in the COX-2 crystal structure, PDB: 6COX. The carboxylic acid moiety of indomethacin was used as a seed group for conjugation. Enhanced COX-2 potency was achieved by targeting Pro86 and Val89.