Design, synthesis, in vitro and in vivo biological evaluation of pterostilbene derivatives for anti-inflammation therapy

Figures & data

Figure 1. Design of the title compounds to achieve potential anti-inflammatory activity.

Scheme 1. Synthesis of compounds D1-D17 and E1-E11. Reagents and conditions: (i) C₂H₃N, K₂CO₃, 60 °C, 15 h, R₁; (ii) C₂H₃N, DMF, POCl₃; (iii) CH₃NO₂, CH₃COONH₄, 100 °C; (iv) DMF, NaN₃, P-TSOH, 60 °C; (v)DCM, R₂, DMF, (CH₃CH₂)₃N, 2 h.

Figure 2. Single crystal structures of compound D11.

Figure 3. The cytotoxicity of the pterostilbene derivatives was tested by MTT assay. RAW264.7 cells were pre-incubated with compound (20 μM) for 24 h and then were detected by MTT assay. All data are expressed as mean ± standard deviation.

Figure 4. Inhibitory effects of the synthetic compounds (D1-D17) on LPS-induced NO production. ***P < 0.001 Compared with LPS treated group. Cel: celecoxib; as a positive control.

Table 1. Compounds E1-E13 inhibitory activity against NO release.

			Cytotoxicity IC50 (µM)
Compd	10 µM (inhibition rate % ±SD)	IC50 ±SD (µM)	With LPS	Without LPS
D4	75.14 ± 5.26	8.07 ± 1.40	> 50	> 50
E1	71.26 ± 3.12	10.58 ± 1.69	> 50	> 50
E2	94.21 ± 4.19	0.70 ± 0.15	> 50	> 50
E3	78.67 ± 3.08	5.20 ± 1.15	> 50	> 50
E4	46.32 ± 4.15	12.93 ± 1.32	> 50	> 50
E5	50.42 ± 4.17	11.65 ± 1.75	> 50	> 50
D5	77.78 ± 3.62	5.43 ± 0.96	> 50	> 50
E6	78.67 ± 5.02	2.90 ± 0.98	>50	>50
E7	83.49 ± 3.16	2.23 ± 0.75	> 50	> 50
E8	77.46 ± 6.07	4.20 ± 1.24	> 50	> 50
E9	78.67 ± 4.69	5.50 ± 1.39	> 50	> 50
E10	76.26 ± 3.28	5.21 ± 0.98	> 50	> 50
E11	82.26 ± 4.26	6.10 ± 1.26	> 50	> 50
E12	74.22 ± 3.48	8.21 ± 1.15	> 50	> 50
E13	69.29 ± 5.51	10.62 ± 2.98	> 50	> 50
Cel	35.16 ± 5.19	> 20	> 50	> 50

Figure 5. Inhibition of inflammatory protein expression by compound E2. RAW264.7 was pre-treatment with compounds (2 µM, 1 µM, 0.5 µM) for 1 h, and then exposed to LPS (500 ng/mL) for 24 h. Bay11-7082 (1 µM) is an NF-κB signalling pathway inhibitor. *** P < 0.001 compared with LPS treated group.

Figure 6. Inhibition of NF-κB inflammatory signalling pathway by compound 8. RAW264.7 was pre-treatment with compound 8 (2 µM, 1 µM, 0.5 µM) for 1 h, and then exposed to LPS (500 ng/mL) for 30 min. Bay11-7082 (1 µM) is an NF-κB signalling pathway inhibitor. *** P < 0.001 compared with LPS treated group.

Figure 7. Inhibition of MAPKs inflammatory signalling pathway by compound E2. RAW264.7 was pre-treatment with compound E2 (2 µM, 1 µM, 0.5 µM) for 1 h, and then exposed to LPS (500 ng/mL) for 30 min. *** P < 0.001 compared with LPS treated group.

Figure 8. Compound E2 alleviates DSS-induced colitis in mice. (a) Mice show bloody stools (b) Weight change; (c) DAI was calculated; (d, e) Measuring the length of the colon; (f) Histopathological analysis of mouse colon. Data are represented as mean and SEM. Sulfasalazine (SASP) was used as a positive control drug. Compared with the Normal group, ^### P < 0.001; Compared with DSS treated group, *** P < 0.001.

Figure 9. Compound E2 inhibits pro-inflammatory factor expression and MPO levels. (a) Detection of MPO activity in colon tissue. (b − d) ELISA determined the cytokine levels of TNF-α, IL-1β, and IL-6. Values are expressed as a mean ± SEM. ^### P < 0.001 compared with the Normal group; *** P < 0.001 Compared with DSS treated group.

Figure 10. Compound E2 treatment downregulated the Inflammatory proteins and signalling pathway in acute DSS-induced colitis. (A-F) TNF-α, IL-1β, IL-6, iNOS, COX-2 and TLR4 mRNA levels in colon tissues. (G-J) Representative Western blot images of colonic COX-2, p-NF-κB, p-Stat3, and β-actin proteins. Compared with Normal group, ^### P < 0.001; Compared with DSS treated group, *** P < 0.001.