Design, biological evaluation, and molecular modelling insights of cupressic acid derivatives as promising anti-inflammatory agents

Figures & data

Figure 1. Inflammatory cascade includes the role of phospholipase A2 (PLA2) and targeting its inhibition by PLA2 inhibitors.

Figure 2. The chemical structure of isolated diterpenes from Araucaria heterophylla (Salisb.) Franco.

Figure 3. Summarised schemes (i-v) of the general preparation procedures of cupressic acid derivatives (1–10). (i) 1.5 equiv. of acetic anhydride and 2 equiv. of pyridine at room temperature for 24h; (ii) in DCM, reflux for 3h with 1.5 equiv. thionyl chloride (SOCl₂) or for 6h with an excess of SOCl₂; (ii-a) in DCM, reflux with 2 equiv. triethylamine (TEA) and 1.5 equiv. ethanolamine; (ii-b) in DCM, reflux with 2 equiv. 2-phenyl ethylamine at 45 °C for 48h; (iii) reflux in dry acetone, excess potassium carbonate, and a catalytic amount of DMAP; (iv) Stirring with excess selenium dioxide solution (SeO₂) in anhydrous dioxane.

Table 1. Primer sequence and their reference in NCBI.

Gene	Primer sequence	Reference sequence
COX-2	F: 5′-CCACTTCAAGGGAGTCTGGA	NM_011198.4
	R: 5′-AGTCATCTGCTACGGGAGGA-3′
β-Actin	F:5-GGG AAA TCG TGC GTG ACA T-3	NM_007393.5
	R:5-GCG GCA GTG GCC ATC TC-3

Figure 4. Modulation of COX-2 expression in RAW264.7 cells pre-treated with tested compounds. The fold of change of COX-2 gene was calculated after normalisation to β-actin. The statistical analysis was performed by One-Way ANOVA by using GraphPad Prism Version 8.0. *** means p values =0.0009 while ** means p values = 0.003.

Figure 5. Attenuation of the IL-6 level by the pre-treatment of Raw264.7 cells with the compounds 1, 2 or 3. The level of IL-6 has been statistically calculated by using GraphPad Prism Version 8.0 in comparison to the LPS-treated cells. * Means p values <0.05.

Table 2. IL-6 level measured in the medium of LPS-induced RAW264.7 cells.

Treatment	IL-6 (pg/mL)	SD*
DMSO	19.6	0.72
LPS	27.6	0.81
LPS + DEXA	19.5	0.45
1	20.73	0.723
2	19.3	0.7920
3	23.2	0.4425
4	26.3	0.369
5	26.3	0.35
6	25.1	0.19
7	27.2	0.20
8	21.4	1.20
9	26.3	0.30
10	25.7	0.86

*SD is the standard deviation from three independent replicates.

Table 3. Docking interactions of the prepared derivatives (1–10) against phospholipase A2 (PLA2) enzyme.

Derivative code	Phospholipase A2
	Docking scores (kcal/mol)	RMSD	Types of interactions
			Metal complex	Amino acid residues
1	−7.87	1.01	Ca:301	Gly31, Lys62
2	−7.86	1.73	Ca:301	Gly31
3	−7.05	2.08	–	Leu2
4	−7.00	1.88	Ca:301	Gly29
5	−7.25	1.28	Ca:301	Gly29, Gly31, Lys62
6	−7.37	1.19	Ca:301	–
7	−6.34	1.50	–	–
8	−7.36	1.33	Ca:301	Cys44
9	−6.72	1.61	Ca:301	Gly31, Lys52
10	−8.97	1.61	Ca:301 (x 2)	Gly29, Lys62
BR4^a	−8.01	1.42	Ca:301	Gly29, Gly31, His47
Dexamethasone	−5.81	1.42	–	–

^aBR4: co-crystallized ligand; 6-phenyl-4(R)-(7-phenyl-heptanoylamino)-hexanoic acid in the complex with the active site of phospholipase A2 (PLA2), PDB code: 1KQU¹¹.

Figure 6. 2D binding modes of interaction of; a) compound 1, (b) compound 2, (c) compound 5, and (d) compound 10 with the active site of phospholipase A2 (PDB code: 1KQU).

Figure 7. Molecular surface interactions of the docked structures of; a) compound 1, (b) compound 2, (c) compound 5, and (d) compound 10 with the active site of phospholipase A2 (PDB code: 1KQU).

Hansford KA, Reid RC, Clark CI, Tyndall JDA, Whitehouse MW, Guthrie T, McGeary RP, Schafer K, Martin JL, Fairlie DP. Martin JL, Fairlie DP. D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A2 (IIa) with antiinflammatory activity. Chembiochem. 2003;4(2–3):181–185.

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