Identification of new 4-(6-oxopyridazin-1-yl)benzenesulfonamides as multi-target anti-inflammatory agents targeting carbonic anhydrase, COX-2 and 5-LOX enzymes: synthesis, biological evaluations and modelling insights

Figures & data

Figure 1. Examples of clinically used sulphonamide-based drugs.

Figure 2. Chemical structures of the dual CA/COX-2 inhibitors Polmacoxib and Celecoxib, as well as the target pyridazinones 5a-c, and 7a-f.

Scheme 1. General Synthesis of pyridazine derivatives (2 and 5a-c); Reagents and conditions: (i) H₂O, reflux, overnight; (ii) Potassium carbonate, DMF, stirring at 5 °C, 3 h.

Scheme 2. General Synthesis of pyridazine derivatives (7a-f); Reagents and conditions: (i) Pyridine, stirring at 5 °C, 1 h.

Table 1. In vitro inhibition data of hCA I, II, IX and XII with pyridazine derivatives (3, 5a-c and 7a-f) by the stopped flow kinetic assay using AAZ as a reference drug.

Compd.	R	KI (nM)^a
		CA I	CA II	CA IX	CA XII
3	H	23.5	55.8	45.1	5.3
5a		98.3	5.3	14.8	32.2
5b		116.3	37.1	12.3	26.1
5c		221.5	106.4	4.9	18.4
7a		48.3	42.2	52.3	13.3
7b		52.6	79.1	58.1	17.2
7c		185.9	34.2	19.4	49.7
7d		123.5	19.2	22.8	42.6
7e		362.8	88.2	30.1	35.9
7f		165.8	31.6	6.4	8.7
AAZ	–	250	12.5	25.0	5.7

^aMeans from three different assays, by a stopped flow technique.

Table 2. IC₅₀ values for the in vitro COX-1/2 and LOX inhibition, as well as COX SI values of the pyridazine-based sulphonamide derivatives 3, 5a-c and 7a-f.

Compd.	R	IC50 µM^a		SI COX-1/ COX-2^b
		COX-1	COX-2
3	H	11.5	0.07	167.7
5a		5	0.11	46
5b		9.5	0.14	67.9
5c		6.5	0.07	92.9
7a		10.4	0.05	208
7b		12.6	0.06	210
7c		8.4	0.08	106
7d		8.4	0.08	105.9
7e		6.5	0.09	73.1
7f		8.5	0.11	81
Celecoxib	–	14.5	0.05	313
Indomethacin	–	0.099	0.08	1.3

^aIC₅₀ values are expressed as the mean of three replicates with a standard deviation less than 10% of the mean. ^bSelectivity index (SI)= IC₅₀ (COX-1)/IC₅₀ (COX-2).

Table 3. Inhibition data of LOX enzyme with pyridazine-based sulphonamide derivatives 3, 5a-c and 7a-f.

Comp.	IC50 (μM)^a
3	2
5a	7
5b	5
5c	7
7a	3
7b	2.5
7c	4
7d	3.5
7e	5
7f	6.5
Zileuton	3.5

^aMean from 3 different assays.

Table 4. Analgesic impact of the tested methanesulfonate and ethanesulfonate pyridazines 7a and 7b by the use of acetic acid writhing test in mice.

Comp.	Number of writhing (20 min) (Mean ± SD)	Analgesic activity (% protection)
7a	13 ± 0.5*	67.5%
7b	14 ± 1.3*	65.0%
Diclofenac	12 ± 1.9*	70.0%
Celecoxib	16 ± 0.9*	60.0%
Control	40 ± 2.9	---

The value is expressed as mean ± SD (n = 5). *Significantly different from the control group.

Table 5. Results of the carrageen-induced paw edoema assay that was used to investigate the effects of the pyridazine sulphonates 7a and 7b on paw height.

Comp.	FIF% change in paw height (Mean ± SD)				Anti-inflammatory activity (% inhibition of edoema)
	0 h	1 h	2 h	3 h	3 h
7a	0.52 ± 0.04	0.46 ± 0.09	0.44 ± 0.05	0.34 ± 0.05*	56%
7b	0.5 ± 0.07	0.44 ± 0.09	0.58 ± 0.04	0.48 ± 0.03*	38.5%
Diclofenac	0.52 ± 0.04	0.28 ± 0.04	0.32 ± 0.04	0.32 ± 0.04*	59%
Celecoxib	0.5 ± 0.07	0.44 ± 0.09	0.52 ± 0.04	0.48 ± 0.04*	38.5%
Control	0.56 ± 0.05	0.68 ± 0.04	0.64 ± 0.05	0.78 ± 0.08*	---

The value is expressed as mean ± SD (n = 5). *Significantly different from the control group.

Figure 3. Serum level of the inflammatory mediators (TNF-α and IL-1β) after paw edoema test. The value is expressed as mean ± SD (n = 5). *Significantly different from the positive control group. A) Tumour necrosis factor – alpha (TNF-α). B) Interleukin one beta (IL-1β).

Figure 4. Ulcerogenic effects of the tested pyridazine sulphonates (7a and 7b), diclofenac and celecoxib on the gastric tissue of rats. Histopathological examination was performed using H & E stain and the magnification power of the images was 40X. The red arrows indicated the normal or damaged parts.

Figure 5. Predicted binding mode of 5c (purple) and 7f (cyan) within A) CA I, B) CA II, C) CA IX and D) CA XII active site.