Discovery of an effective anti-inflammatory agent for inhibiting the activation of NF-κB

Figures & data

Figure 1. Some reported NF-κB inhibitors.

Scheme 1. Synthesis of aryl amide (6–22). Reagents and conditions: (a) 1a (1.0 eq.), 2a (1.0 eq.), K₃PO₄ (1.5 eq.), Pd(PPh₃)₄ (0.05 eq.), 80 °C, 1,4-dioxane/H₂O (3:1), Ar₂ atmosphere, 12 h; (b) 5 (1.0 eq.), aniline (1.1 eq.), DIPEA (4.0 eq.), HATU (1.2 eq.), room temperature, DCM, 18 h.

Scheme 2. Synthesis of aryl amide (23–37). Reagents and conditions: (a) Acid (1.0 eq.), 3h (1.0 eq.), HATU (1.2 eq.), DIPEA (4.0 eq.), RT, DCM, 18 h; (b) Bromide (1.0 eq.), 2a (1.0 eq.), K₃PO₄ (1.5 equiv), Pd(PPh₃)₄ (0.05 eq.), 80 °C, Ar₂ atmosphere, 1,4-dioxane/H₂O (3:1), 12 h.

Scheme 3. Synthesis of aryl amide (28–52). Reagents and conditions: (a) 1a (1.0 eq.), 3H (1.0 eq.), HATU (1.2 eq.), DIPEA (4.0 eq.), RT, DCM, 18 h; (b) 4a (1.0 eq.), boric acid (1.0 eq.), K₃PO₄ (1.5 equiv), Pd(PPh₃)₄ (0.05 eq.), 1,4-dioxane/H₂O, Ar₂ atmosphere, 80 °C, 12 h.

Table 1. Structure-activity relationship study at position 3 of the pyridine core.

NO.	R^1	NO inhibition IC50 (μM)^a	NF-κB reporter IC50(nM)^b	Cytotoxicity IC50(μM)^c
6	H	19.7 ± 2.6	1619.7 ± 13.2	>25
7	4Me	26.7 ± 3.2	>2000	>25
8	3Me	15.2 ± 2.8	1328.6 ± 11.5	>25
9	2Me	30.2 ± 4.0	>2000	>25
10	4NH2	2.3 ± 0.9	419.7 ± 10.4	7.3 ± 1.2
11	3NH2	1.7 ± 0.9	95.8 ± 6.2	>25
12	2NH2	0.9 ± 0.5	570.9 ± 9.3	4.7 ± 1.0
13	4NHCOMe	10.2 ± 5.4	713.9 ± 4.2	>25
14	3NHCOMe	3.2 ± 1.1	167.4 ± 8.4	>25
15	2NHCOMe	1.1 ± 0.4	532.5 ± 13.5	3.2 ± 0.8
16	4F	>50	>2000	>25
17	4Cl	>50	>2000	>25
18	4OCF3	>50	>2000	>25
19	4Et	28.5 ± 2.1	>2000	>25
20	4OMe	22.0 ± 0.8	>2000	>25
21	4iPr	>50	>2000	>25
22	4CONH2	>50	>2000	>25
Bay11-7082	–	2.1 ± 0.7	91.5 ± 10.3	22.7 ± 3.4

^aNO inhibition IC₅₀ was detected by Griess Reagent; ^bNF-κB reporter assay: HEK293T cells were transfected with NF-κB reporter plasmid and stimulated by TNF-α. ^cThe toxicity of the compounds was detected by MTT assay.

Table 2. Structure-activity relationship study of the core.

NO.	X	NO inhibition IC50 (μM)	NF-κB reporter IC50(nM)	Cytotoxicity IC50(μM)
23		32.7 ± 3.4	>2000	>25
24		14.1 ± 1.7	763.2 ± 15.1	>25
25		25.4 ± 3.1	1329.5 ± 16.3	>25
26		19.5 ± 3.2	1962.3 ± 53.5	>25
27		21.5 ± 2.6	1109.7 ± 12.4	>25
Bay11-7082	–	2.1 ± 0.7	91.5 ± 10.3	22.7 ± 3.4

^aNO inhibition IC₅₀ was detected by Griess Reagent; ^bNF-κB reporter assay: HEK293T cells were transfected with NF-κB reporter plasmid and stimulated by TNF-α. ^cThe toxicity of the compounds was detected by MTT assay.

Table 3. Structure-activity relationship study at position 5 of the pyridine core.

NO.	R^5	NO inhibition IC50 (μM)^a	NF-κB reporter IC50(nM)^b	Cytotoxicity IC50(μM)^c
28	4Me	24.2 ± 4.1	>2000	>50
29	3Me	>50	>2000	>50
30	4SO2CH3	17.8 ± 2.7	1042.8 ± 17.5	>50
31	4OMe	>50	>2000	>50
32	3OMe	>50	>2000	>50
33	2OMe	>50	>2000	>50
34	2,3,4(OMe)3	>50	>2000	>50
35	4Cl	4.2 ± 0.8	206.5 ± 13.0	>50
36	3Cl	>50	>2000	>50
37	2Cl	>50	>2000	>50
38	4F	13.7 ± 1.8	827.3 ± 18.6	>50
39	3F	>50	>2000	>50
40	4CF3	26.5 ± 3.0	>2000	>50
41	3CF3	8.4 ± 1.7	306.6 ± 21.4	>50
42	2CF3	14.6 ± 2.3	1006.4 ± 18.4	>50
43	4OH	>50	>2000	>50
44	3OH	>50	>2000	>50
45	2OH	>50	>2000	>50
46	4CN	>50	>2000	>50
47	3CN	28.9 ± 2.1	>2000	>50
48	2CN	>50	>2000	>50
49	4-formyl-3-Me	>50	>2000	>50
50	4-F-3-Me	8.6 ± 1.9	577.1 ± 21.6	>50
51	4-NH2-2-Cl	3.1 ± 1.1	172.2 ± 11.4	>50
52	2-F-4-OMe	>50	1417.5 ± 24.7	>50
Bay11-7082	–	2.1 ± 0.7	91.5 ± 10.3	22.7 ± 3.4

^aNO inhibition IC₅₀ was detected by Griess Reagent; ^bNF-κB reporter assay: HEK293T cells were transfected with NF-κB reporter plasmid and stimulated by TNF-α. ^cThe toxicity of the compounds was detected by MTT assay.

Figure 2. Compound 51 suppressed the activation of NF-κB. (A–C) Compound 51 inhibited the phosphorylation of P65 and IκBα. RAW264.7 cells were treated with compound 51 for different concentrations and then stimulated by LPS. The cell lysates were analysed using WB. ^###p < 0.001 compared with control group. **p < 0.01, ***p < 0.001 compared to LPS group. (D–F) Compound 51 inhibited TNFα-induced nuclear translocation of p65 and p50. HEK293T were treated with compound 51 for different concentrations and then stimulated by TNF-α. The nuclear protein was analysed using WB. ^###p < 0.001 compared with control group. **p < 0.01, ***p  <  0.001 compared to TNF-α group.

Figure 3. Compound 51 inhibited the activation of MAPKs signalling. (A–D) Compound 51 inhibited the phosphorylation of MAPKs. RAW264.7 cells were treated with compound 51 for different concentrations and then stimulated by LPS. The cell lysates were analysed using WB. ^###p < 0.001 compared with control group. *p < 0.1, **p < 0.01, ***p < 0.001 compared to LPS group.

Figure 4. Compound 51 inhibited LPS-induced inflammatory response. (A–C) The expression of iNOS and COX-2. RAW264.7 cells were treated with compound 51 for 1 h, then stimulated by LPS for further 24 h. The samples were analysed by WB. (D). The level of IL-6. (E) The level of TNF-α. (F) The level of ROS. ^###p < 0.001 compared with control group. *p < 0.1, **p < 0.01, ***p < 0.001 compared to LPS group.

Figure 5. Compound 51 suppressed LPS-induced inflammatory response in vivo. (A-B) The picture of mice organs. (C) HE staining of spleen tissues. Scan bar:50 μM. (D-E) The levels of IL-6 and TNF-α in blood. The samples were analysed by ELISA kits. (F–G) The SOD and MDA activity in blood. ^###p < 0.001 compared with control group. *p < 0.1, **p < 0.01, ***p < 0.001 compared to LPS group. (H) The expression of NF-κB in splenic organ. The samples were analysed by WB.