Discovery of new thymol-3,4-disubstituted thiazole hybrids as dual COX-2/5-LOX inhibitors with in vivo proof

Figures & data

Figure 1. Structures of compounds having COX-2, or 5-LOX inhibitory activities.

Figure 2. Design of thymol—3,4-disubstitutedthiazole hybrids as dual COX-2/5-LOX inhibitors.

Scheme 1. Synthesis of the target thymol—3,4-disubstitutedthiazole hybrids.

Table 1. In vitro COX-1, COX-2, and 5-LOX enzyme inhibitory activities, ^aIC₅₀ values and ^bSI of the tested compounds.

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Code	R	X	COX-1 IC50 (µM)	COX-2 IC50 (µM)	SI	5-LOX IC50 (µM)
Celecoxib	—	—	14.7 ± 0.07	0.045 ± 0.001	327	—
Diclofenac sodium	—	—	3.9 ± 0.04	0.8 ± 0.009	5	—
Quercetin	—	—	—	—	—	3.34 ± 0.05
6a	Phenyl	H	12.03 ± 0.12	0.077 ± 0.002	156	3.18 ± 0.04
6b	2-Chlorophenyl	H	14.01 ± 0.068	0.037 ± 0.007	379	2.51 ± 0.015
6c	2-Pyridyl	H	13.41 ± 0.068	0.068 ± 0.001	197	2.89 ± 0.026
6d	4-Methoxyphenyl	H	14.32 ± 0.15	0.042 ± 0.001	341	2.47 ± 0.06
6e	Phenyl	Br	13.84 ± 0.088	0.046 ± 0.0016	301	1.75 ± 0.06
6f	2-Chlorophenyl	Br	14.58 ± 0.04	0.039 ± 0.001	374	1.53 ± 0.02
6g	2-Pyridyl	Br	12.87 ± 0.15	0.065 ± 0.002	198	1.98 ± 0.06
6h	4-Methoxyphenyl	Br	11.78 ± 0.13	0.081 ± 0.0016	145	1.91 ± 0.026
6i	Phenyl	OCH3	10.47 ± 0.15	0.10 ± 0.007	105	2.98 ± 0.1
6j	2-Chlorophenyl	OCH3	9.87 ± 0.12	0.13 ± 0.007	76	2.63 ± 0.07
6k	2-Pyridyl	OCH3	8.97 ± 0.12	0.16 ± 0.01	56	2.87 ± 0.07
6l	4-Methoxyphenyl	OCH3	10.18 ± 0.055	0.18 ± 0.007	66	2.69 ± 0.027
7a	Phenyl	—	8.01 ± 0.1	0.22 ± 0.01	36	5.01 ± 0.12
7b	2-Chlorophenyl	—	9.01 ± 0.19	0.18 ± 0.007	50	4.31 ± 0.06
7c	4-Methoxyphenyl	—	10.22 ± 0.15	0.09 ± 0.007	114	3.87 ± 0.12

^aIC₅₀ is the concentration (µM) needed to cause 50% inhibition of COX-1 and COX-2 enzymatic activity. All values are expressed as a mean of three replicates with standard deviation less than 10% of the mean.

^bSI = (COX-1 IC₅₀/COX-2 IC₅₀).

Figure 3. % Inhibition of formalin-induced paw edoema.

Figure 4. Histological analysis of rats’ stomach sections stained with H&E (100x magnification).

Figure 5. Left: co-crystallized celecoxib, ligand-enzyme interaction (2D). Right: Overlay of co-crystallized celecoxib (yellow) and docked celecoxib (cyan) with RMSD = 0.422 (3D) inside COX-2 active site.

Figure 6. 6b, ligand–enzyme interaction 2D (left) and 3D (right) inside COX-2 active site.

Figure 7. 6f, ligand–enzyme interaction 2D (left) and 3D (right) inside COX-2 active site.

Figure 8. Overlay of 6b (green), 6f (magenta), and celecoxib (cyan) inside COX-2 active site.

Table 2. Induced fit docking results of the active compounds inside COX-2 active site.

Code	Score in Kcal/mol	Hydrogen bonds Interaction amino acids	Hydrophobic Interaction amino acids	Polar Interaction amino acids
Co-crystallized ligand (celecoxib) (Figure 5)	−9.26	Three H-bonds: two bonds between N of SO2NH2 and Ser339, Leu338; O of SO2 with Arg499	Val335, Leu370, Phe367, Leu517, Leu345, Trp373, Met508, Ala502, Phe504, Val509 Ala513, Ile503	His75, Tyr341, Ser516, Tyr371, Gly340, Arg106, Gln178, Gly512
6b (Figure 6)	−7.71	Two H-bonds between CL and Val335*, and between N and Arg106	Val335, Leu517, Leu338, Trp373, Val102, Met508, Ala513, Phe504, Val174, Pro71, Leu78	His75, Tyr341, Tyr371, Arg559, Glu510, Gly512, Ser105, Ser339, Tyr334, Tyr101. Two pi-H bonds between 2-Chlorophenyl and Val509, and between phenyl and Ser516
6f (Figure 7)	−7.91	H-bond between N and Arg106	Val335, Leu338, Trp373, Val102, Met508, Ala513, Phe504, Val74, Pro71, Leu78, Leu370, Val509	His75, Tyr341, Tyr371, Arg559, Glu510, Gly512, Ser339, Ser516, Ser105, Tyr101.

*Amino acids interacted with celecoxib were marketed in bold format.

Figure 9. Mode of binding of NDGA inside 5-LOX active site 2D (left) and 3D (right).

Figure 10. Overlay of NDGA (cyan) and docked NDGA (yellow) inside 5-LOX active site with RMSD = 1.2.

Figure 11. Mode of binding of 6b inside 5-LOX active site 2D (left) and 3D (right).

Figure 12. Mode of binding of 6f inside 5-LOX active site 2D (left) and 3D (right).

Table 3. Induced fit docking results of the active compounds in 5-LOX active site.

Code	Score in Kcal/mol	Hydrophobic interaction amino acids	Polar interaction amino acids
NDGA (Figures 9, 10)	−6.27	Phe359, Leu368, Leu373, Ala410, Leu414, Pro569, Trp599, Ala603, Val604, Leu607	Gln363, His367, His372, Asn407, Arg411, Arg596, His600
6b (Figure 11)	−8.06	Phe359*, Leu368, Leu373, Ile406, Ala410, Leu414, Pro569, Trp599, Ala603, Val604, Leu607, Ile673	His360, Gln363, His367, His372, Asn407, Arg411, His432, Arg596, His600, FE701
6f (Figure 12)	−7.86	Phe359, Leu368, Leu373, Ile406, Ala410, Leu414, Pro569, Trp599, Ala603, Val604, Leu607, Ile673	His360, Gln363, His372, Glu376, Asn407, Arg411, His432, Arg596, His600, FE701

*Amino acids interacted with NDGA were marked in bold format.

Figure 14. RMSD of COX-2 apo (Grey), 6b (Blue), and 6f (red) complex structures.

Figure 13. Overlay of docked NDGA (yellow), 6b (green), and 6f (magenta) inside 5-LOX active site.

Figure 15. RMSF of COX-2 -ligand interaction residues, Apo (Grey), 6b (Blue), and 6f (red): (A) Part I and II and (B) Part III.

Figure 16. Rg plot of Apo (Grey), 6b (Blue), and 6f (red).

Figure 17. Hydrogen bonds between 6b (Blue) and 6f (red) ligands and protein.

Figure 18. RMSD of 5-LOX apo (Grey), 6b (Blue), and 6f (red) complex structures.

Figure 19. RMSF of 5-LOX -ligand interaction residues, Apo (Grey), 6b (Blue), and 6f (red): (A) Part I and II and (B) Part III.

Figure 20. Rg plot of Apo (Grey), 6b (Blue), and 6f (red) complexes.

Figure 21. Hydrogen bonds between 6b (Blue) and 6f (red) ligands and protein.