Design, synthesis, anti-inflammatory evaluation, and molecular modelling of new coumarin-based analogs combined curcumin and other heterocycles as potential TNF-α production inhibitors via upregulating Nrf2/HO-1, downregulating AKT/mTOR signalling pathways and downregulating NF-κB in LPS induced macrophages

Figures & data

Figure 1. Design of coumarin-based analogs combined with curcumin derivatives and other different nitrogenous heterocyclic cores as anti-inflammatory candidates.

Scheme 1. Synthetic pathways for preparation of starting materials 3 and 4a–c. Reagents: a) ethyl chloroacetate, anhydrous K₂CO₃, acetone, reflux 24 h; b) hydrazine hydrate, absolute ethanol, reflux 2 h; c) 30% NaOH, ethanol, stirring r.t., 2 h.

Scheme 2. Synthetic pathways for preparation of target coumarin derivatives 5a-c and 6–9. Reagents: d) chalcone 4a-c, NaOH, absolute ethanol, reflux 72 h; e) malononitrle, absolute, ethanol, reflux 8 h; f) ethylcyanoacetate, absolute ethanol, reflux 9 h; g) acetylacetone, absolute ethanol, reflux 15 h; h) 3-methyl-1-phenyl-2-pyrazoline-5-one, dioxane, reflux 12 h.

Scheme 3. Synthetic pathways for preparation of target coumarin derivatives 10–12, 13a,b and 14a,b. Reagents: i) 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole, absolute ethanol, reflux 8 h; j) tosyl chloride, dioxane, reflux 8 h; k) isatin, absolute ethanol, glacial acetic acid reflux 8 h. l) appropriate aryl aldehyde, absolute ethanol, glacial acetic acid, reflux 9–16 h; m) appropriate aryl ketone, absolute ethanol, glacial acetic acid reflux 10–16 h.

Table 1. The calculated EC₅₀ and IC₅₀ of the tested compounds on normal and LPS-treated Macrophages.

Compd.	^aMW	Normalized IC50 on normal macrophages	Normalized EC50 on LPS-treated macrophages
5a	452	21.8	10.51
5b	470	27.52	42.59
5c	500	36.69	23.4
6	328	26.45	18.23
7	329	20.68	13.27
8	418	69.48	17.44
9	418	16.37	10.96
10	425	39.31	8.38
11	570	32.2	23.24
12	391	50.05	22.56
13a	393	26.56	16.16
13b	410	130	21.16
14a	379	25.12	19.64
14b	424	33.61	5.32

^aMW: molecular weight.

Figure 2. Represents the impact of coumarin molecule bearing 3,4-dimethoxybenzylidene hydrazinyl 14b compared to negative control and macrophage cells treated with Dexa on the levels of pro-inflammatory cytokines. (A) Effect on IL-6 level. (B) Effect on IL-1β level. (C) Effect on TNF-α level. (D) Effect on NF-kβ level.

Figure 3. Expression of mTOR and Nrf2 protein in LPS-macrophage cells by Immunofluorescence assay.

Figure 4. Represents the calculation of H-Score for m-TOR (A) and Nrf-2 (B) protein expression in the tested groups induced by coumarin molecule bearing 3,4-dimethoxybenzylidene hydrazinyl 14b compared to negative control and macrophage cells treated with Dexa.

Figure 5. Represents the impact of coumarin molecule bearing 3,4-dimethoxybenzylidene hydrazinyl 14b compared to negative control and macrophage cells treated with Dexa on the gene expression levels of HO-1 and Akt in the tested groups.

Figure 6. The main proposed action of coumarin derivative 14b to promote anti-inflammatory activity.

Figure 7. Docked pose of 14b in the active site of TNF-α (3D left panel and 2D right panel).

Figure 8. Docked pose of Dexa in the active site of TNF-α (3D left panel and 2D right panel).

Table 2. Binding score, H-bonding, and Hydrophobic interactions of the target compound 14b, Dexa and co-lig. 307 in the active site of TNF-α.

Compd.	CDOCKER ENERGY (kcal/mol)	Amino acid residues	Type of Interactions
14b	−41.3	Tyr119	Pi-Pi stacking
		Tyr151	Pi-allkyl
		Ser60	H-Bond
		GLy121	Carbon H-Bond
Dexa	−34.3	Tyr119	Pi-alkyl
		Ser60	H-Bond
Co-lig. 307	−42.56	Tyr119	Pi-Pi stacking
		Gly121	Halogen (F)