Discovery and evaluation of novel synthetic 5-alkyl-4-oxo-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoxaline-1-carbox-amide derivatives as anti-inflammatory agents

Figures & data

Figure 1. Design of target compounds 6a–6t.

Scheme 1. Reagents and conditions: (a) oxalic acid, HCl/H₂O, 100 °C, 2 h; (b) Hydrazine hydrate, 100 °C, 2 h; (c) Diethyl oxalate, reflux, 3 h; (d) RX, K₂CO₃, DMF, 60 °C, 3 h; (e) NH₃·H₂O, methanol, r.t., 2 h.

Table 1. Effect of compounds 6a–6t on the viability of RAW264.7 cells.

Compound	R	Cell viability (%)
		30 μM	10 μM
blank	–	100	100
LPS	–	98.60 ± 2.12^a	NT^b
D1	–	96.30 ± 1.72	NT
6a	n-C4H9	92.33 ± 1.53	NT
6b	n-C5H11	97.95 ± 1.1	NT
6c	n-C6H13	99.11 ± 0.69	NT
6d	n-C7H15	81.00 ± 1.10	NT
6e	n-C8H17	99.51 ± 2.30	NT
6f	n-C9H19	60.00 ± 3.40	99.15 ± 1.65
6g	–CH2C6H5	85.02 ± 1.00	NT
6h	–CH2C6H4(o-F)	66.67 ± 1.33	98.49 ± 1.76
6i	–CH2C6H4(m-F)	59.33 ± 2.52	99.46 ± 4.17
6j	–CH2C6H4(p-F)	48.00 ± 3.00	73.45 ± 2.27
6k	–CH2C6H4(o-Cl)	46.33 ± 2.89	74.56 ± 1.38
6l	–CH2C6H4(m-Cl)	51.67 ± 2.12	87.92 ± 5.18
6m	–CH2C6H4(p-Cl)	44.67 ± 3.06	82.18 ± 1.74
6n	–CH2C6H4(m-OCH3)	93.33 ± 0.58	NT
6o	–CH2C6H4(p-OCH3)	80.12 ± 1.00	NT
6p	–CH2C6H2(3,4,5-(OCH3)3)	83.67 ± 1.53	NT
6q	–CH2C6H3(2,4-Cl2)	56.67 ± 1.34	84.69 ± 6.49
6r	–CH2C6H4(m-CF3)	99.30 ± 0.60	NT
6s	–CH2C6H4(p-NO2)	80.10 ± 1.34	NT
6t	–CH2C6H4(p-CN)	90.33 ± 1.21	NT

^a LPS (1 μg/mL).

^b No tested.

Figure 2. RAW264.7 cells were pre-cultured for 24 h, the cells were then treated with the indicated concentrations of compounds for 30 min, and then exposed to 1 μg/mL LPS for 24 h. The levels of NO in the culture medium were measured with NO assay kit. (A) Cells were treated with 10 μM compounds. Cells were pre-treated with different concentrations of compound 6p. NO, TNF-α and IL-6 levels in the medium were determined with an ELISA kit. (B, C and D). ### p < .01, vs. Control. *p < .05, **p < .01, ***p < .001 vs. LPS alone.

Figure 3. Compound 6p inhibited LPS-induced inflammatory response in RAW 264.7 cells. Cells were pre-treated with different concentrations of 6p (7.5–30 µM) and Celecoxib (7.5 µM) for 24 h. iNOS, COX-2 and GAPDH proteins expression were detected by Western blot analysis. Total cellular proteins were prepared and analysed by Western blotting. Results are the mean ± SD, n = 3. ### p < .001, vs. Control. *p < .05, **p < .01, ***p < .001 vs. LPS alone.

Figure 4. Compound 6p inhibited LPS-induced inflammatory response in RAW 264.7 cells. Cells were pre-treated with different concentrations of 6p (7.5–30 µM) and Celecoxib (7.5 µM) for 24 h. The levels of p-p38/P38, p-ERK/ERK, p-JNK/JNK and GAPDH proteins, and their phosphorylated forms were analysed using Western blotting. Total cellular proteins were prepared and analysed by Western blotting. Results are the mean ± SD, n = 3. ### p < .001, vs. Control. *p < .05, **p < .01, ***p < .001 vs. LPS alone.

Figure 5. The inhibited paw swelling actives of lead compound D1, 6p and positive control drugs (25 mg/kg) in a carrageenan-induced mouse paw oedema model after 3 h of oral administration. **p < .01, vs. D1; ##p < .01, vs. Ibuprofen; $ p < .05, vs. Celecoxib.

Table 2. The anti-inflammatory activity and ulcerogenic activity in vivo.

Compounds	% of oedema inhibition (% mean ± SD)^a			% ulceration
	1 h	3 h	5 h
D1	38.26 ± 2.82	42.81 ± 2.11	28.71 ± 2.92	20
6p	37.01 ± 8.45	50.83 ± 3.47	38.24 ± 6.04	20
Ibuprofen	31.92 ± 5.76	39.30 ± 4.86	25.29 ± 3.43	100
Celecoxib	48.09 ± 5.33	58.34 ± 4.08	41.92 ± 2.05	0

^a Values are expressed as the mean ± S.D (n = 4).

Control: 0.5% sodium CMC solution in distilled water (25 ml/kg, p.o.).

Compounds and positive drugs were administered at a dose of 25 mg/kg, p.o. in 0.5% sodium CMC solution.