Novel sulindac derivatives: synthesis, characterisation, evaluation of antioxidant, analgesic, anti-inflammatory, ulcerogenic and COX-2 inhibition activity

Figures & data

Scheme 1. Synthetic route of compounds (1‒25).

Figure 1. Comparison of DPPH scavenging activity of compounds (1–25) and BHT. All values are means of three replicates ± SD.

Table 1. Antioxidant activity of compounds (1–25) by DPPH method.

Compound	Scavenging effect % (mean ± SD)^a
1	67.40 ± 9.25
2	39.86 ± 10.80
3	85.10 ± 6.80
4	42.20 ± 13.20
5	45.06 ± 22.57
6	39.73 ± 15.65
7	71.03 ± 5.31
8	18.53 ± 4.86
9	49.36 ± 5.57
10	19.26 ± 5.32
11	33.10 ± 8.41
12	38.10 ± 10.92
13	62.93 ± 6.72
14	23.07 ± 6.95
15	40.60 ± 14.40
16	27.66 ± 2.12
17	51.40 ± 16.66
18	35.50 ± 9.05
19	37.90 ± 10.35
20	29.66 ± 16.93
21	30.06 ± 3.30
22	63.56 ± 0.47
23	78.97 ± 9.36
24	30.80 ± 2.50
25	42.33 ± 18.38
BHT	90.6 ± 3.83

Compound number 3 having highest Scavenging effect represented in bold.

^aAll values are means of three replicates ± SD.

Table 2. Analgesic effect of drugs by Tail flick method in mice.

			Reaction time (s) post drug
Treatment (n = 6)	Dose (mg/kg)	Reaction time (s) pre drug	30 m	% Inhibition	60 m	% Inhibition	120 m	% Inhibition
Compound 3	150	5.00 ± 0.36	4.83 ± 0.30	3.33	7.33 ± 0.42**	46.66	7.16 ± 0.30***	63.33
Sulindac	100	4.83 ± 0.40	5.50 ± 0.34	13.79	8.83 ± 0.79***	82.75	11.00 ± 0.57***	127.58

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

**p < 0.01. ***p < 0.001.

Table 3. Analgesic effect of drugs by Hot Plate method in mice.

			Reaction Time (seconds) post drug
Treatment (n = 6)	Dose (mg/kg)	Reaction Time (seconds) pre drug	30 m	% Inhibition	60 m	% Inhibition	120 m	% Inhibition
Compound 3	150	6.66 ± 0.33	7.66 ± 0.42	15	9.83 ± 0.30***	47.50	11.16 ± 0.30***	67.50
Sulindac	100	7.66 ± 0.33	10.83 ± 0.47***	41.30	11.83 ± 0.47***	54.34	14.33 ± 0.42***	86.95

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

***p < 0.001.

Table 4. Analgesic effect of drugs by acetic acid-induced writhing in mice.

Treatments (n = 6)	Dose (mg/kg)	Number of writhing in 20 min	% Inhibition
Compound 3	150	14.66 ± 0.95***	56.86
Sulindac	100	8.83 ± 0.70***	74.01
Control (acetic acid)	0.1 ml of 20%	34.00 ± 1.23	—

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

***p < 0.001.

Table 5. Effect of compound 3 and sulindac on yeast-induced hyperthermia in mice.

			Rectal temperature after yeast	Rectal temperature °C post drug administration
Treatment (n = 6)	Dose (mg/kg)	Normal rectal temperature	administration 20 mL/kg of 20%	30 m	60 m	120 m
Compound 3	150	35.16 ± 0.10	38.38 ± 0.19***	38.15 ± 0.10	37.83 ± 0.15*	37.33 ± 0.12***
Sulindac	100	35.21 ± 0.11	38.21 ± 0.31***	37.46 ± 0.14	37.16 ± 0.09**	36.23 ± 0.08***

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

*p < 0.05, **p < 0.01, ***p < 0.001.

Table 6. Anti-inflammatory activity of drugs by carrageenan-induced paw edoema method in albino rats.

		Before Carrageenan	Increase paw volume after 3 h			Increase paw volume after 5 h
Group (n = 6)	Dose (mg/kg)	Mean ± SE	Mean ± SE	Net	% Inhibition	Mean ± SE	Net	% Inhibition
Only carrageenan		1.04 ± 0.02	1.68 ± 0.01***	0.63 ± 0.01		1.65 ± 0.01***	0.61 ± 0.02	Compound 3
Compound 3	150	1.02 ± 0.02	1.34 ± 0.02***	0.31 ± 0.03***	50.52	1.33 ± 0.01***	0.30 ± 0.02***	50.54
Sulindac	100	1.03 ± 0.03	1.26 ± 0.03**	0.22 ± 0.01***	65.18	1.25 ± 0.03***	1.25 ± 0.03***	65.02

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

**p < 0.01, ***p < 0.001.

Table 7. Ulcer study of drugs using 80% Ethanol.

Treatments	Dose mg/kg	Ulcer Index	% Inhibition
80% Ethanol only	1 mL/200 g Rat	7.33 ± 0.33
80% Ethanol only + Sulindac	100	6.83 ± 0.40	6.81
80% Ethanol only + Compound 3	150	4.33 ± 0.4***	40.90
Sulindac only	100	1.33 ± 0.49
Compound 3 only	150	—

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

***p < 0.001.

Table 8. Ulcer study of drugs compared with Indomethacin

Treatments	Dose mg/kg	Ulcer Index	% Inhibition
Indomethacin	30	37.00 ± 1.59
Sulindac only	100	32.00 ± 3.86
Compound 3 only	150	22.16 ± 1.10***	40.09

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

***p < 0.001.

Table 9. Anti-oxidant activity, MDA, NP-SH and total protein of drugs in stomach tissue of rat.

Treatments	Dose mg/dL	MDA (nmol/g)	NP-SH (nmol/g)	Total protein (g/L)
Normal saline	1 mL	1.03 ± 0.02	8.29 ± 0.53	113.37 ± 2.94
80% Ethanol only	1 mL	6.53 ± 0.56***	3.04 ± 0.39***	47.90 ± 2.89***
80% Ethanol only + Sulindac	1 mL + 100	2.28 ± 0.11***	5.08 ± 0.28**	93.81 ± 3.05***
80% Ethanol only + Compd. 3	1 mL + 150	1.59 ± 0.06***	6.62 ± 0.26***	100.99 ± 2.42***
Sulindac only	100	1.31 ± 0.04***	7.00 ± 0.032***	105.78 ± 1.43***
Compound 3 only	150	1.06 ± 0.02***	7.52 ± 0.31***	114.57 ± 1.89***

All values represent mean ± SEM. ANOVA, followed by Dunnett’s multiple comparison test.

***p < 0.001.

Table 10. LD₅₀ determination of compound 3.

Compound 3	Group	Dose mg/kg	D. F (a)	Dead	M.M (b)	Pro. (a*b)
	1	100		0
	2	200	100	0
	3	400	200	1	0.5	100
	4	800	400	3	2	800
	5	1600	800	5	4	3200
	6	2000	400	9	7	2800
						6900
						1310
		Exp. Dose	131 mg/kg

Figure 2. Quantitative real-time PCR analysis for COX-2 mRNA expression in hepatocytes of treated and untreated rats with actin as housekeeping gene. Data analysed by one-way analysis of variance using graph pad prism 8. Asterisks **** indicate p < 0.0001 significance from control and vehicle group, ⁺⁺⁺⁺ indicates p < 0.0001 significance from cisplatin (CP 12 mg/kg) treated group and ^## indicate p < 0.01 significance difference between CP + T (20 mg/kg) and CP + T (40 mg/kg) treatment groups. T stands for compound 3 treatment.

Figure 3. Effect of compound 3 treatment in regulating COX-2 protein expression in liver tissues of rats inoculated with cisplatin (12 mg/kg dose). Immunoblot analysis of COX-2 and β-actin in rat hepatocytes (n = 7). CP: cisplatin, T: compound 3 treatments.

Figure 4. The orientation of docked compounds with Cox-2. The Sulindac is shown in blue colour while the compound 3 is shown in green colour. (A) The orientation of docked molecules is shown in sticks format. (B) The orientation of docked molecules is shown in spheres format.

Figure 5. The orientation of Cox-2 residues making interactions with ligands (green) in 3 D confirmation. (A) The binding of drug Sulindac with Cox-2 protein. The amino acids residues are making hydrophobic interaction with drug. (B) The binding of compound 3 with Cox-2 protein. The amino acids residues are making two hydrogen bonds and hydrophobic interaction with the compound. (C) The Cys-32 and Tyr-116 making hydrogen bonds with compound 3.

Figure 6. 2D map of interactions of Cox-2 protein with Sulindac (A) and compound 3 (B).

Table 11. Docking energies of compounds (1–25) and sulindac.

Compound	Docking energies	Compound	Docking energies
1	−81.19	14	−247.54
2	−257.54	15	−298.94
3	−440.29	16	−252.92
4	−417.10	17	−266.51
5	−413.27	18	−227.18
6	−96.75	19	−220.75
7	−392.71	20	−150.57
8	−317.14	21	−220.38
9	−388.61	22	−457.75
10	−345.55	23	−421.83
11	−242.36	24	−466.48
12	−428.94	25	−77.10
13	−275.16	Sulindac	−325.99

Data Availability

Samples of the compounds (1‒25) in pure form are available from authors.