Synthesis and anti-rheumatoid arthritis activities of 3-(4-aminophenyl)-coumarin derivatives

Figures & data

Scheme 1. Structural derivation of 3-(4-aminophenyl)-coumarins.

Scheme 2. The synthetic route of 3-(4-aminophenyl)-coumarins 3 and target product 4. Reagents and conditions: (a) acetic anhydride, Et₃N, 115 °C. (b) HCl ethanol. (c) COCl₂, DCM, reflux. (d) 3-(4-aminophenyl)-coumarin 3, acetone, pyridine, and RT.

Scheme 3. General synthetic route to compounds 5a–5h. Reagents and conditions: (e) 3-(4-aminophenyl)-coumarin 3, DCC, HOBt, toluene, and RT.

Table 1. Structures of compounds 4a–4q, 5a–5h.

Product	R1	R2	R3	R4	R5	R6	R7	Yield (%)
4a	H	OH	H	H	F	H	H	70
4b	H	OH	H	H	CH3	H	H	58
4c	H	OH	H	H	CH2Cl	H	H	77
4d	H	OH	H	CH3	H	H	H	85
4e	H	OH	H	Cl	H	H	H	81
4f	H	OH	F	H	H	H	H	72
4g	H	OH	H	H	Cl	H	H	80
4h	H	OH	H	CH3	H	CH3	H	72
4i	OCH3	H	H	H	H	F	H	75
4j	OCH3	H	H	H	F	H	H	78
4k	OCH3	H	H	H	CF3	H	H	68
4l	OCH3	H	H	H	CH2Cl	H	H	70
4m	OCH3	H	H	CH3	H	H	H	87
4n	OCH3	H	F	H	H	H	H	82
4o	OCH3	H	OCH3	H	H	H	H	77
4p	OCH3	H	Cl	Cl	H	H	H	62
4q	OH	OH	H	H	CH2Cl	H	H	58
5a	H	OH						70
5b	OCH3	H						75
5c	H	OH						78
5d	OCH3	H						82
5e	H	OH						65
5f	OCH3	H						72
5g	OCH3	H						65
5h	OCH3	H						72

Figure 1. The cell proliferation inhibitory activity of compounds 4e, 4p, and 5e.

Table 2. Effect of compounds on proliferation of RA-FLSs. (mean ± SD, n = 3)

Compound	IC50 value (µM)	Compound	IC50 value (µM)
	RA-FLSs cell proliferation inhibitory activity		RA-FLSs cell proliferation inhibitory activity
4a	10.55 ± 0.28	4n	>50
4b	13.93 ± 0.02	4o	16.57 ± 0.04
4c	>50	4p	7.61 ± 0.13
4d	>50	4q	17.39 ± 0.33
4e	5.60 ± 0.29	5a	>50
4f	18.59 ± 0.04	5b	>50
4g	9.13 ± 0.02	5c	13.07 ± 0.02
4h	>50	5d	19.83 ± 0.03
4i	8.79 ± 0.19	5e	1.78 ± 0.02
4j	>50	5f	10.40 ± 0.32
4k	>50	5g	24.72 ± 0.03
4l	15.71 ± 0.11	5h	11.74 ± 0.29
4m	>50	Methotrexate	5.00 ± 0.04

Figure 2. Effect of compounds 4e, 4p, and 5e on secretion of IL-1α, IL-1β, and TNF-α.

Figure 3. Effect of compounds 4e, 4p, and 5e on secretion of cytokine IL-10.

Figure 4. Inhibitory effect of compound 5e on luciferase activity of NF-κB. RA-FLS cells were cultured at 37 °C for 24 h, then NF-κB luciferase plasmid and control sea kidney plasmid were transfected for 48 h. Different concentrations of compound 5e and LPS (final concentration: 5 μg/mL) were added to stimulate the secretion of proinflammatory cytokines. After incubation for 48 h, the cells were collected and the luciferase activity of NF-κB pathway was detected by dual luciferase system. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with model group; ****p < 0.0001 compared with ctrl group.

Figure 5. Inhibitory effect of compound 5e on MAPK signal pathway. RA-FLS cells were cultured at 37 °C for 24 h. The cells were preincubated with compound 5e of different concentrations for 1 h, and then treated with LPS (final concentration: 5 μg/mL) for 72 h. Collected cells were used for Western blotting analysis of anti-ERK1/2, anti-pho-ERK1/2, anti-p38 and anti-pho-p38 antibodies. ^##p < 0.01, ^####p < 0.0001 compared with model group; ****p < 0.0001 compared with ctrl group.

Figure 6. Establishment and treatment of CFA rat model of RA. Normal control group (Ctrl, saline), model group (Model, saline), high dose compound 5e (60 mg/kg) group, low dose compound 5e (30 mg/kg) group, positive control MTX (30 mg/kg) group. (n = 5 per group).

Figure 7. The analgesic effect of compound 5e on rats was detected by the method of thermal pain threshold of rat claws. ^###p < 0.001, ***p < 0.001. (n = 5 per group).

Figure 8. Motor function test of SD rats. Place the rat in the plexiglass chamber of the treadmill (25 cm long and 5 cm wide), set the treadmill speed to 18 cm/s, the shutter speed to take pictures: 40 ms, and record 5 s for each animal. The motor function of RA rats was explained by the motor parameters of left stride length and hindlimb stance width. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with model group, ***p < 0.001 compared with ctrl group. (n = 5 per group).

Figure 9. Compound 5e recovers RA histopathological features of rats. (A) Normal control group (Ctrl, normal saline), (B-1, B-2) model group (model, normal saline), (C) low-dose compound 5e group (30 mg/kg), (D) high-dose compound 5e Group (60 mg/kg), and (E) positive control MTX (30 mg/kg). (n = 5 per group).