Design and synthesis of pyridazinone-substituted benzenesulphonylurea derivatives as anti-hyperglycaemic agents and inhibitors of aldose reductase – an enzyme embroiled in diabetic complications

Abstract

Thirty new aryl-pyridazinone-substituted benzenesulphonylurea derivatives (I–XXX) were synthesized and evaluated for their anti-hyperglycaemic activity in glucose-fed hyperglycaemic normal rats. Twenty-three compounds (III–XI, XIV–XVII, XIX–XXIV, XXVI and XXVIII–XXX) showed more or comparable area under the curve (AUC) reduction percentage (ranging from 21.9% to 35.5%) as compared to the standard drug gliclazide (22.0%). On the basis of docking results, 18 compounds were screened for their in vitro ability to inhibit rat lens aldose reductase. Ten compounds (III–VI, XII, XVI–XVIII, XXI and XXVII) showed ARI activity with IC50 ranging from 34 to 242 μM. Out of these, two compounds IV and V showed best ARI activity which is comparable with that of quercetin. As a result, two compounds (IV and V) possessing significant dual action (anti-hyperglycaemic and aldose reductase inhibition) were identified and may be used as lead compounds for developing new drugs.

• Aldose reductase
• diabetes
• docking
• pyridazinone
• sulfonylurea

Introduction

Diabetes mellitus (DM) has become pandemic and according to reports, including a forecast by the World Health Organizations (WHO), there will be a sharp increase in the total number of cases world wide by 2030. In fact, India could be bracing itself for the dubious distinction of becoming the diabetes of capital of the world1. Alone, in India recently published report by ICMR-INDIAB national study stated that there are 62.4 million people with non-insulin-dependent diabetes mellitus (NIDDM) and 77 million people with prediabetes in India. NIDDM is the most common form of diabetes and has been found 90% of all diabetes2.

NIDDM is a multifactorial metabolic disease characterized by abnormalities at multiple organ sites. These defects include insulin resistance and insulin deficiency3,4. The former is primarily represented by decreased insulin-stimulated glucose uptake in skeletal muscle, augmented endogenous glucose production (predominately in the liver) and enhanced lipolytic activity in fat5. The latter is an apparent progressive process with both functional defects in islet cell function and, eventually, apparent loss of β-cell mass6,7. These defects are intimately linked with derangements in one system exacerbating those in the others8.

Patients with DM are at high risk of developing long-term complications including neuropathy, nephropathy, retinopathy and cataract9,10. Although strict glycaemic control is expected to prevent diabetic complications but perfect glycaemic control is not always possible. In hyperglycaemic conditions, aldose reductase catalyses an NADPH-dependent reduction of glucose to sorbitol, which in turn is oxidized to fructose by an NAD⁺-dependent sorbitol dehydrogenase (Figure 1). Once sorbitol is accumulated inside the cells, it cannot diffuse easily across the cell membrane as a result osmotic pressure increases causing cellular damage. Moreover, the depletion of NADPH and NAD⁺ cofactors compromises body’s antioxidant defence system. In addition, high blood levels of fructose may account for increased glycation and accelerating ageing11,12 (Figure 1). Thus, inhibition of AR is one of the important targets and its inhibition could be the key for the treatment of many diabetic complications13. Aldose reductase inhibitors (ARIs) have been shown to reduce tissue sorbitol accumulation in diabetic animals14 and there are evidences that blockage of AR can have beneficial effect in diabetic complications15,16.

Figure 1. Polyol metabolic pathway of glucose and its side effects.

Various structurally different compounds have been identified as potent in vitro ARIs and some of them are in clinical trials to test their efficacy in the prevention and treatment of peripheral neuropathy in diabetes10,17. They can be categorized into various groups based on their chemical structures: acetic acid derivatives (e.g. epalrestat, Imirestat), cyclic imides (especially spirohydantoins, e.g. sorbinil, fidarestat) etc. Despite being structurally different, all ARIs possess the following two peculiar pharmacophoric elements: (i) an acidic moiety that is able to interact with the “anion-binding site” of the catalytic site and (ii) a lipophilic scaffold that can bind to the highly flexible specificity pocket of the catalytic site18. Our interest in studying the molecular determinants for binding to the aldose reductase inhibitor site led us to study the aldose reductase inhibitory activity of target compounds possessing benzenesulphonylurea as carboxylic acid surrogates. Besides this benzenesulphonamide derivatives have been already reported as ARI19,20. The SO₂ group of the benzenesulphonamide forms the hydrogen bond with the amino acid residues of the anionic binding site of the AR enzyme21.

In curing, diabetes sulphonyl ureas have represented the backbone of oral therapy in NIDDM for more than 40 years22. Recently, compounds containing pyridazinone nucleus have been reported as AR inhibitors23–26. In the view of above facts and as part of our research programme (to develop new lead compounds that not only act as antihyperglycaemic agents, but at the same time prevent diabetic complications caused by the aldose reductase or the associated pathway), we herein report the synthesis of new pyridazinone-substituted benzenesulphonyl urea derivatives (I–XXX) incorporating with known bioactive moieties highlighted in Figure 2. We have introduced a diversity of aryl-substituted pyridazinones moiety at para position of benzenesulphonamide and other substitutions, such as isopropyl and 4-methylcyclohexyl groups, were made at N² position of the uriedo group, keeping the core structure intact (Figure 2).

Figure 2. Structures of clinically used antidiabetic drugs and pyridazinone derivatives reported as aldose reductase inhibitor. Rationally designed template for targeted compound (I–XXX).

The synthesized compounds (I–XXX) were evaluated for antihyperglycaemic effects in glucose-fed hyperglycaemic normal rats. In silico molecular docking studies of these compounds (I–XXX) were performed with respect to aldose reductase. Eighteen compounds displaying good docking results were evaluated for their ability to inhibit rat lens aldose reductase.

Experimental

Melting points were determined by open capillary tubes and are uncorrected. Purity of the compounds was checked on thin-layer chromatography (TLC) plates (silica gel G) that were visualized by exposing to iodine vapours. Infrared (IR) spectra were recorded (in KBr) on a BIO-RAD FTS-135 spectrophotometer and ¹HNMR spectra were recorded on a Bruker Spectrospin DPX 300 MHz spectrometer using deuterated dimethyl sulphoxide (DMSO) as solvent and tetramethyl silane (TMS) as an internal standard. Chemical shifts are given in δ (ppm) scale and coupling constants (J values) are expressed in hertz (Hz). Elemental analysis was carried out on CHNS Elementar (Vario EL III). Mass spectra (MS) were scanned by using ESI Bruker Esquire 3000 (Billerica, MA). The m/z values of the more intense peaks are mentioned.

General procedure for synthesis of sulfonylureas (I-XXX)

A solution of appropriate pyridazinone (1 mmol) in dry acetone was refluxed over anhydrous K₂CO₃ (2 mmol) for 1–1.5 h. At this temperature, a solution of the appropriate isocyanate (1.2 mmol) in dry acetone 5 ml was added in a dropwise manner. It was refluxed for 24–72 h. Acetone was removed under reduced pressure. The solid residue thus obtained was suspended in water and acidified with dilute acetic acid. It was stirred for 30 min and filtered. The residue was washed with plenty of distilled water in order to make the residue free from potassium acetate. It was dried and crystallized from methanol. The purity of the compounds was checked on TLC plate (Silica gel G) in the solvent system toluene/ethyl acetate/formic acid (5:4:1, TEF).

1-{4-[3–(2,3-Dihydro-1H-inden-5-yl)-6-oxopyridazin-1-yl]benzenesulphonyl}-3-isopropylurea (I)

Yellow crystals, yield  =  45%, m.p. 120–121 °C, R_f  = 0.65 (TEF). IR ν_max (KBr, in cm ⁻¹): 3335, 3037 and 2911 (NH-CO-NH), 1715 and 1525 (C=O) of urea, 1605 (C=N), 1320 and 1142 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.95 (6H, d, J = 6 Hz, -CH-(CH₃)₂), 1.90–2.07 (2H, m, H-3″′), 2.91 (4H, d, J = 4.2 Hz, H-2″′, H-4″′), 3.53–3.66 (1H, m, -NH-CH-(CH₃)₂), 5.49 (1H, brs, -NH-(CH₃)₂), 7.14–8.15 (9H, m, H-5, H-5′, H-6′, H-2′, H-3″, H-5″, H-2″, H-6″, H-4), 8.31 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 43.20 (CH-NH), 125.71 (C-4 pyridazinone), 137.60 (C=O uriedo group), 144.22 (C-5 pyridazinone), 152.10 (C=O pyridazinone), 155.62 (C=N pyridazinone). ESI-MS (m/z): 452 [M⁺], 453 [M + 1], 451 [M-1], 450 [M-2]. CHNS Analysis for C₂₃H₂₄N₄O₄S Found (Calculated): C: 61.10 (61.08), H: 5.34 (5.39), N: 12.38 (12.30), O: 14.12(14.15), S: 7.07 (7.13).

3-Isopropyl-1–(4-{3-[4–(2-methylpropyl)phenyl]-6-oxopyridazin-1-yl} benzenesulphonyl) urea (II)

Yellow crystals, yield  =  47%, m.p. 162–163 °C, R_f  = 0.67 (TEF). IR ν_max (KBr, in cm ⁻¹): 3360, 3025 and 2915 (NH-CO-NH), 1710 and 1510 (C=O) of urea, 1596 (C=N), 1325 and 1155 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.87 (6H, d, J = 6.6 Hz, Ph-CH₂-CH-(CH₃)₂), 0.98 (6H, d, J  =  8.1 Hz, -NH-CH-(CH₃)₂), 1.88 (1H, m, Ph-CH₂-CH-(CH₃)₂), 2.5 (2H, merged s, Ph-CH₂-CH-(CH₃)₂), 3.58–3.67 (1H, m, -NH-CH-(CH₃)₂), 5.46 (1H, d, J  =  4.5 Hz, -NH-CH-(CH₃)₂), 7.20 (1H, d, J  =  10.2 Hz, H-5), 7.29 (2H, d, J = 6.9 Hz, H-3′, H-5′), 7.62 (1H, d, J = 9.8 Hz, -SO2-NH-C=O), 7.85 (2H, d, J  =  7.2 Hz, H-2′, H-6′), 7.90 (2H, d, J = 7.8 Hz, H-3″, H-5″), 7.96 (2H, d, J = 8.4 Hz, H-2″, H-6″), 8.14 (1H, d, J = 9.9 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 43.25 (CH-NH), 125.74 (C-4 pyridazinone), 137.62 (C=O uriedo group), 144.35 (C-5 pyridazinone), 151.20 (C=O pyridazinone), 156.44 (C=N pyridazinone). ESI-MS (m/z): 468 [M⁺], 469 [M + 1], 467 [M-1], 466 [M-2].CHNS Analysis for C₂₄H₂₈N₄O₄S Found (Calculated): C: 61.55 (61.56), H: 6.05 (6.08) N: 12.10 (12.15), O: 13.54 (13.59), S: 6.85 (6.90).

1-{4-[3–(3,4-Difluorophenyl)-6-oxopyridazin-1-yl]benzenesulphonyl}-3-isopropylurea (III)

Light brown crystals, yield  =  51% m.p. 142–143 °C, R_f = 0.60 (TEF). IR ν_max (KBr, in cm^− 1): 3345, 3033 and 2940 (NH-CO-NH), 1698 and 1515 (C=O) of urea, 1613 (C=N), 1331 and 1151 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.93 (6H, d, J  =  6.5 Hz, -CH-(CH₃)₂), 3.75–3.84 (1H, m, -NH-CH-(CH₃)₂), 6.60 (1H, d, J = 5.4 Hz, -NH-CH-(CH₃)₂), 7.46 (1H, d, J  =  7.2 Hz, H-5), 7.78 (1H, dd, J = 7.8 Hz, H-5′), 8.04 (1H, d, J = 6 Hz, H-2′), 8.17 (2H, d, J = 6.3 Hz, H-3″, H-5″), 8.22–8.28 (3H, m, H-6′, H-2″, H-6″), 8.40 (1H, d, J = 7.5 Hz, H-4), 10.75 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 43.22 (CH-NH), 125.67 (C-4 pyridazinone), 138.60 (C=O uriedo group), 145.31 (C-5 pyridazinone), 151.17 (C=O pyridazinone), 155.54 (C=N pyridazinone). ESI-MS (m/z): 448 [M⁺], 449 [M + 1], 447 [M-1], 446 [M-2]. CHNS Analysis for C₂₀H₁₈F₂N₄O₄S Found (Calculated): C: 53.58 (53.59), H: 4.08 (4.09), F: 8.48 (8.49), N: 12.48 (12.49), O: 14.28 (14.29), S: 7.13 (7.14).

1-{4-[3–(4-Fluorophenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-3-isopropylurea (IV)

Yellow crystals, yield  =  41%, m.p. 102–103 °C, R_f = 0.60 (TEF). IR ν_max (KBr, in cm⁻¹): 3327, 3050 and 2970 (NH-CO-NH), 1705 and 1503 (C=O) of urea, 1597 (C=N), 1339 and 1157 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.99 (6H, d, J  =  6.3 Hz, -NH-CH-(CH₃)₂), 3.61–3.69 (1H, m, -NH-CH-(CH₃)₂), 4.10 (1H, d, J  =  5.7 Hz, -NH-CH-(CH₃)₂), 7.06 (1H, d, J = 9.6 Hz, H-5), 7.12 (2H, d, J  =  9 Hz, H-2′, H-6′), 7.74–7.96 (6H, m, H-3′, H-5′, H-3″, H-5″, H-2″, H-6″), 8.01 (1H, d, J  =  8.7 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 42.91 (CH-NH), 125.40 (C-4 pyridazinone), 137.20 (C=O uriedo group), 146.10 (C-5 pyridazinone), 152.10 (C=O pyridazinone), 155.31 (C=N pyridazinone). ESI-MS (m/z): 430 [M⁺], 431 [M + 1], 429 [M-1], 428 [M-2].CHNS Analysis for C₂₀H₁₉FN₄O₄S Found (Calculated): C: 55.75 (55.76), H: 4.43 (4.44), F: 4.39 (4.4), N: 13.10 (13.12), O: 14.87 (14.88), S: 7.46 (7.47).

3-Isopropyl-1-{4-[6-oxo-3–(5, 6, 7, 8-tetrahydronaphthalen-2-yl) pyridazin-1-yl] benzenesulphonyl} urea (V)

White crystals, yield  =  45%, m.p. 200–201 °C, R_f = 0.63 (TEF). IR ν_max (KBr, in cm⁻¹): 3356, 3071 and 2995 (NH-CO-NH), 1720 and 1518 (C=O) of urea, 1610 (C=N), 1345 and 1175 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 1.01 (6H, d, J = 6.3 Hz, -NH-CH-(CH₃)₂), 1.76 (4H, brs, H-3″′, H-4″′), 2.77 (4H, brs, H-2″′, H-5″′), 3.59–3.61 (1H, m, -NH-CH-(CH₃)₂), 5.50 (1H, d, J = 6.3 Hz, -NH-CH-(CH₃)₂), 6.27 (1H, d, J  =  15.6 Hz, SO₂-NH-C=O), 6.97–7.28 (2H, m, H-5, H-5′), 7.64–7.75 (2H, m, H-2′, H-6′), 7.90 (2H, d, J = 8.1 Hz, H-3″, H-5″), 8.01 (2H, d, J  =  6.9 Hz, H-2″, H-6″), 8.15 (1H, d, J  =  9.6 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 43.65 (CH-NH), 125.22 (C-4 pyridazinone), 138.52 (C=O uriedo group), 145.10 (C-5 pyridazinone), 151.37 (C=O pyridazinone), 155.72 (C=N pyridazinone). ESI-MS (m/z): 466 [M⁺], 467 [M + 1], 465 [M-1], 464 [M-2].CHNS Analysis for C₂₄H₂₆N₄O₄S Found (Calculated): C: 61.75 (61.74), H: 5.62 (5.60), N: 12.10 (12.11), O: 13.70 (13.72), S: 6.91(6.92).

1-{4-[3–(4-Benzylphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-3-isopropylurea (VI)

Light yellow crystals, yield  =  58%, m.p. 179–180 °C, R_f =0.61 (TEF). IR ν_max (KBr, in cm ⁻¹): 3340, 3049 and 2998 (NH-CO-NH), 1705 and 1522 (C=O) of urea, 1596 (C=N), 1355 and 1153 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 1.02 (6H, d, J = 8.4 Hz, -NH-CH-(CH₃)₂), 3.56–3.64 (1H, m, -NH-CH-(CH₃)₂), 4.01 (2H, s, Ph-CH₂-Ph), 6.39 (1H, s, -NH-CH-(CH₃)₂), 7.18–7.32 (6H, m, H-2″′, H-3″′, H-4″′, H-5″′, H-6″′, H-5), 7.37 (2H, d, J  =  6.3 Hz, H-3′, H-5′), 7.87 (2H, d, J  =  6 Hz, H-2′, H-6′), 7.95 (2H, d, J = 6.3 Hz, H-3″, H-5″), 8.03 (2H, d, J = 6.6 Hz, H-2″, H-6″), 8.15 (1H, d, J  =  7.2 Hz, H-4), 10.53 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm):43.72 (CH-NH), 125.82 (C-4 pyridazinone), 138.82 (C=O uriedo group), 145.70 (C-5 pyridazinone), 151.31 (C=O pyridazinone), 156.10 (C=N pyridazinone). ESI-MS (m/z): 502 [M⁺], 503 [M + 1], 501 [M-1], 500 [M-2]. CHNS Analysis for C₂₇H₂₆N₄O₄S Found (Calculated): C: 63.72 (63.73), H: 4.2 (4.1), N: 12.02 (12.03), O: 14.05 (14.06), S: 6.07 (6.08).

3-Isopropyl-1-{4-[6-oxo-3–(4- propylphenyl) pyridazin-1-yl] benzenesulphonyl} urea (VII)

Yellow crystals, yield  =  47%, m.p. 185–186 °C, R_f = 0.60 (TEF). IR ν_max (KBr, in cm^− 1): 3340, 3071 and 2975 (NH-CO-NH), 1700 and 1510 (C=O) of urea, 1589 (C=N), 1337 and 1121 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.90 (3H, t, Ph-CH₂CH₂CH₃), 1.001 (6H, d, J  =  6.3 Hz, -NH-CH-(CH₃)₂), 1.57–1.65 (2H, m, Ph-CH₂CH₂CH₃), 2.61 (2H, t, Ph-CH₂CH₂CH₃), 3.60–3.66 (1H, m, -NH-CH-(CH₃)₂), 5.48 (1H, d, J = 7.8 Hz, -NH-CH-(CH₃)₂), 6.35 (1H, brs, SO₂-NH-C=O), 7.22 (1H, d, J = 9.6 Hz, H-5), 7.32 (2H, d, J  =  8.1 Hz, H-3′, H-5′), 7.86 (2H, d, J = 7.8 Hz, H-2′, H-6′), 7.94 (2H, d, J  =  8.7 Hz, H-3″, H-5″), 8.02 (2H, d, J  =  8.4 Hz, H-2″, H-6″), 8.16 (1H, d, J = 9.6 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 42.27 (CH-NH), 124.36 (C-4 pyridazinone), 137.85 (C=O uriedo group), 146.19 (C-5 pyridazinone), 150.89 (C=O pyridazinone), 157.12 (C=N pyridazinone). ESI-MS (m/z):454 [M⁺], 455 [M + 1], 453 [M-1], 452 [M-2]. CHNS Analysis for C₂₃H₂₆N₄O₄S Found (Calculated): C: 60.52 (60.53), H: 5.78 (5.77), N: 12.43 (12.45), O: 14.18 (14.17), S: 7.15 (7.16).

1-{4-[3–(4-Ethoxyphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-3-isopropylurea (VIII)

Yellow crystals, yield = 50%, m.p. 148–149 °C, R_f = 0.62 (TEF). IR ν_max (KBr, in cm^− 1): 3317, 3090 and 2981 (NH-CO-NH), 1718 and 1520 (C=O) of urea, 1585 (C=N), 1331 and 1142 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 1.007 (6H, d, J = 6.3 Hz, -NH-CH-(CH₃)₂), 1.34 (3H, t, Ph-O-CH₂-CH₃), 3.61–3.67 (1H, m, -NH-CH-(CH₃)₂), 4.09 (2H, q, Ph-O-CH₂-CH₃), 5.40 (1H, brs, -NH-CH-(CH₃)₂), 7.03 (2H, d, J = 8.7 Hz, H-3′, H-5′), 7.15 (1H, d, J  =  11.1 Hz, H-5), 7.85–7.97 (6H, m, H-2″, H-6″, H-3″, H-5″, H-2′, H-6′), 8.02 (1H, d, J = 8.7 Hz, H-4), 8.27 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 42.22 (CH-NH), 126.11 (C-4 pyridazinone), 136.87 (C=O uriedo group), 146.13 (C-5 pyridazinone), 153.15 (C=O pyridazinone), 157.12 (C=N pyridazinone). ESI-MS (m/z): 456 [M⁺], 457 [M + 1], 455 [M-1], 454 [M-2]. CHNS Analysis for C₂₂H₂₄N₄O₅S Found (Calculated): C: 58.68 (58.67), H: 4.9 (5.01), N: 12.1 (12.09), O: 17.32 (17.31), S: 7.02 (7.01).

3-Isopropyl-1–(4-{6-oxo-3-[4–(2-phenylethyl) phenyl]pyridazin-1-yl}benzenesulphonyl) urea (IX)

White crystals, yield  =  51%, m.p. 190–191 °C, R_f = 0.62 (TEF). IR ν_max (KBr, in cm⁻¹): 3321, 3080 and 2983 (NH-CO-NH), 1729 and 1522 (C=O) of urea, 1582 (C=N), 1325 and 1144 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.98 (6H, d, J = 6.6 Hz, -NH-CH-(CH₃)₂), 2.92 (4H, s, Ph-CH₂-CH₂-Ph), 3.50–3.59 (1H, m, -NH-CH-(CH₃)₂), 7.16–7.26 (6H, m, H-5, H-2 ″′, H-3 ″′, H-4 ″′, H-5 ″′, H-6 ″′), 7.34 (2H, d, J  =  7.2 Hz, H-3′, H-5′), 7.72–8.13 (7H, m, H-4, H-2 ″, H-6 ″, H-3 ″, H-5 ″, H-2′, H-6′), 8.30 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 45.10 (CH-NH), 125.55 (C-4 pyridazinone), 137.67 (C=O uriedo group), 144.29 (C-5 pyridazinone), 152.19 (C=O pyridazinone), 155.81 (C=N pyridazinone).). ESI-MS (m/z): 516 [M⁺], 517 [M + 1], 515 [M-1], 514 [M-2]. CHNS Analysis for C₂₈H₂₈N₄O₄S Found (Calculated): C: 65.09 (65.1), H: 5.46 (5.44), N: 10.83 (10.84), O: 12.45 (12.46), S: 6.20 (6.22).

3-Isopropyl-1-{4-[6-oxo-3–(4-phenoxyphenyl) pyridazin-1-yl]benzenesulphonyl}urea (X)

White crystals, yield =  53%, m.p. 127–128 °C, R_f = 0.61 (TEF). IR ν_max (KBr, in cm ⁻¹): 3336, 3061 and 2981 (NH-CO-NH), 1732 and 1530 (C=O) of urea, 1591 (C=N), 1345 and 1165 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.97 (6H, d, J = 6.9 Hz, -NH-CH-(CH₃)₂), 3.03–3.64 (1H, m, -NH-CH-(CH₃)₂), 5.48 (1H, brs, -NH-CH-(CH₃)₂), 5.83 (1H, brs, SO₂-NH-C=O), 7.07–8.16 (15H, m, H-2′, H-6′, H-3′, H-5′, H-3″, H-5 ″, H-2 ″, H-6 ″, H-2 ″′, H-3 ″′, H-4 ″′, H-5 ″′, H-6 ″′, H-4, H-5), 8.30 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 42.72 (CH-NH), 125.81 (C-4 pyridazinone), 137.74 (C=O uriedo group), 144.67 (C-5 pyridazinone), 152.21 (C=O pyridazinone), 155.81 (C=N pyridazinone). ESI-MS (m/z): 504 [M⁺], 505 [M + 1], 503 [M-1], 502 [M-2]. CHNS Analysis for C₂₆H₂₄N₄O₅S Found (Calculated): C: 61.9 (61.89), H: 4.78 (4.79), N: 11.11 (11.12), O: 15.85 (15.84), S: 6.36 (6.37).

1-{4-[3–(4-Cyclohexylphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-3-isopropylurea (XI)

Yellow crystals, yield  =  60%, m.p. 188–189 °C, R_f  = 0.68 (TEF). IR ν_max (KBr, in cm^− 1): 3329, 3071 and 2977 (NH-CO-NH), 1740 and 1540 (C=O) of urea, 1605 (C=N), 1350 and 1168 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 1.06 (6H, d, J = 6.6 Hz, -NH-CH-(CH₃)₂), 1.22–1.97 (11H, m, cyclohexyl ring), 3.62–3.73 (1H, m, -NH-CH-(CH₃)₂), 5.56 (1H, d, J = 7.5 Hz, -NH-CH-(CH₃)₂), 6.28 (1H, brs, SO₂-NH-C=O), 7.27 (1H, d, J = 9.9 Hz, H-5), 7.41 (2H, d, J = 8.1 Hz, H-3′, H-5′), 7.90–8.01 (4H, m, H-3″, H-5 ″, H-2′, H-6′), 8.04 (2H, d, J = 8.7 Hz, H-2 ″, H-6 ″), 8.21 (1H, d, J  =  9.6 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 45.31 (CH-NH), 124.93 (C-4 pyridazinone), 138.20 (C=O uriedo group), 145.32 (C-5 pyridazinone), 153.10 (C=O pyridazinone), 154.92 (C=N pyridazinone). ESI-MS (m/z): 494 [M⁺], 495 [M + 1], 493 [M-1], 492 [M-2]. CHNS Analysis for C₂₆H₃₀N₄O₄S Found (Calculated): C: 63.17 (63.19), H: 6.25 (6.24), N: 11.3 (11.29), O: 12.87 (12.86), S: 6.45 (6.44).

1-{4-[3–(4-Bromophenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-3-isopropylurea (XII)

Dark yellow crystals, yield  =  43%, m.p. 168–169 °C, R_f = 0.70 (TEF). IR ν_max (KBr, in cm^− 1): 3341, 3102 and 2988 (NH-CO-NH), 1743 and 1538 (C=O) of urea, 1610 (C=N), 1341 and 1149 (SO₂N); ¹H NMR (300 MHz, DMSO-d₆, δ): 1.16 (6H, d, J  =  6.3 Hz, -NH-CH-(CH₃)₂), 4.12–4.14 (1H, m, -NH-CH-(CH₃)₂), 6.006 (1H, brs, -NH-CH-(CH₃)₂), 7.20–8.16 (11H, m, H-2′, H-3′, H-5′, H-6′, H-2″, H-3″, H-5″, H-6″, H-4, H-5, SO₂-NH C=O), ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 44.18 (CH-NH), 125.62 (C-4 pyridazinone), 136.71 (C=O uriedo group), 145.15 (C-5 pyridazinone), 152.24 (C=O pyridazinone) 154.31 (C=N pyridazinone). ESI-MS (m/z): 492 [M + 2], 491 [M + 1], 490 [M⁺], 489 [M-1]. CHNS Analysis for C₂₀H₁₉BrN₄O₄S Found (Calculated): C: 49.21 (49.19), H: 3.4 (3.38), Br: 16.17 (16.18), N: 11.45(11.44), O: 13.3(13.29), S: 6.54(6.55).

1-{4-[3–(4-Iodophenyl)-6-oxopyridazin-1-yl] benzenesulfonyl}-3-isopropylurea (XIII)

Yellow crystals, yield  =  41%, m.p. 171–172 °C, R_f = 0.66 (TEF). IR ν_max (KBr, in cm ⁻¹): 3307, 3175 and 2991 (NH-CO-NH), 1750 and 1541 (C=O) of urea, 1613 (C=N), 1345 and 1153 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 1.04 (6H, d, J = 7.2 Hz, -NH-CH-(CH₃)₂), 3.64–3.72 (1H, m, -NH-CH-(CH₃)₂), 5.56 (1H, d, J  =  7.5 Hz, -NH-CH-(CH₃)₂), 6.01 (1H, s, SO₂-NH-C=O), 7.27 (1H, d, J = 9.3 Hz, H-5), 7.59 (2H, d, J  =  19.8 Hz, H-2′, H-6′), 7.81 (2H, d, J  =  7.8 Hz, H-3′, H-5′), 7.91–8.01 (4H, m, H-2″, H-3″, H-5″, H-6″), 8.22 (1H, d, J  =  5.4 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 45.11 (CH-NH), 126.31 (C-4 pyridazinone), 135.13 (C=O uriedo group), 146.22 (C-5 pyridazinone), 153.10 (C=O pyridazinone), 156.10 (C=N pyridazinone). ESI-MS (m/z): 538 [M⁺], 539 [M + 1], 537 [M-1], 536 [M-2]. CHNS Analysis for C₂₀H₁₉IN₄O₄S Found (Calculated): C: 44.73 (44.71), H: 3.45 (3.47), I: 23.75 (23.74), N: 10.43 (10.44), O: 11.78 (11.77), S: 5.88 (5.87).

1-{4-[3–(4-Hexylphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-3-isopropylurea (XIV)

Yellow crystals, yield =  42%, m.p. 174–175 °C, R_f = 0.63 (TEF). IR ν_max (KBr, in cm⁻¹): 3319, 3091 and 2981 (NH-CO-NH), 1738 and 1529 (C=O) of urea, 1602 (C=N), 1336 and 1149 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.81 (3H, t, -Ph-(CH₂)₅-CH₃), 1.07 (6H, d, J  =  6.3 Hz, -NH-CH-(CH₃)₂), 1.26 (6H, s, -Ph-CH₂-CH₂-(CH₂)₃-CH₃), 1.55 (2H, q, -Ph-CH₂-CH₂-(CH₂)₃-CH₃), 2.57 (2H, t, -Ph-CH₂-(CH₂)₄-CH₃), 3.71–3.82 (1H, m, -NH-CH-(CH₃)₂), 4.27 (1H, d, J = 6.3 Hz, -NH-CH-(CH₃)₂), 6.19 (1H, d, J  =  9.6 Hz, SO₂-NH-C=O), 7.05 (1H, d, J  =  9.6 Hz, H-5), 7.20 (2H, d, J  =  7.8 Hz, H-3′, H-5′) 7.63–7.69 (3H, m, H-2′, H-6′, H-4′), 7.89 (2H, d, J = 8.4 Hz, H-3″, H-5″), 8.008 (2H, d, J  =  8.1 Hz, H-2″, H-6″). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 42.78 (CH-NH), 125.42 (C-4 pyridazinone), 135.81 (C=O uriedo group), 144.85 (C-5 pyridazinone), 153.14 (C=O pyridazinone), 155.41 (C=N pyridazinone). ESI-MS (m/z): 496 [M⁺], 497 [M + 1], 495 [M-1], 494 [M-2]. CHNS Analysis for C₂₆H₃₂N₄O₄S Found (Calculated): C: 62.76 (62.77), H: 6.52 (6.53), N: 11.31 (11.3), O: 12.92 (12.91), S: 6.51 (6.52).

3-Isopropyl-1-{4-[3–(4-isopropylphenyl)-6-oxopyridazin-1-yl]benzenesulphonyl}urea (XV)

Light yellow crystals, yield  =  50%, m.p. 157–158 °C, R_f = 0.65 (TEF). IR ν_max (KBr, in cm^− 1): 3362, 3055 and 2975 (NH-CO-NH), 1725 and 1521 (C=O) of urea, 1597 (C=N), 1340 and 1151 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 1.01 (12H, d, J = 6.3 Hz, -Ph-CH-(CH₃)₂, -NH-CH-(CH₃)₂), 2.96 (6H, d, J  =  6.9 Hz, Ph-CH-(CH₃)₂), 2.97 (1H, h, Ph-CH-(CH₃)₂), 3.61–3.68 (1H, m, -NH-CH-(CH₃)₂), 5.43 (1H, s, -NH-CH-(CH₃)₂), 7.17 (1H, d, J  =  7.8 Hz, H-5), 7.37 (2H, d, J  =  7.8 Hz, H-3′, H-5′), 7.75 (2H, d, J = 8.1 Hz, H-2′, H-6′), 7.84–7.98 (4H, m, H-2″, H-6″, H-3″, H-5″), 8.10 (1H, d, J = 7.2 Hz, H-4), 8.20 (1H, brs, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 42.95 (CH-NH), 125.54 (C-4 pyridazinone), 136.72 (C=O uriedo group), 145.45 (C-5 pyridazinone), 152.23 (C=O pyridazinone), 156.12 (C=N pyridazinone). ESI-MS (m/z): 454 [M⁺], 455 [M + 1], 453 [M-1], 452 [M-2]. CHNS Analysis for C₂₃H₂₆N₄O₄S Found (Calculated): C: 60.6 (60.59), H: 5.7 (5.71), N: 12.39 (12.41), O: 14.25 (14.26), S: 7.08 (7.1).

3-{4-[3–(2,3-Dihydro-1H-inden-5-yl)-6-oxopyridazin-1-yl]benzenesulphonyl}-1–(4-methylcyclohexyl) urea (XVI)

White crystals, yield  =  65%, m.p. 225–226 °C, R_f = 0.68 (TEF). IR ν_max (KBr, in cm ^− 1): 3331, 3028 and 2907 (NH-CO-NH), 1718 and 1531 (C=O) of urea, 1609 (C=N), 1329 and 1139 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.90 (3H, d, J = 6.3 Hz, -cyclohexyl-CH₃), 1.006–1.92 (9H, m, cyclohexyl ring protons), 2.08–2.13 (2H, m, H-3″′), 2.97 (4H, d, J = 5.1 Hz, H-2″′, H-4″′), 3.20 (1H, m, proton at C-1 of cyclohexyl), 5.50 (1H, brs, -NH-cyclohexyl ring), 7.23 (1H, d, J  =  9.9 Hz, H-5), 7.40 (1H, d, J  =  7.8 Hz, H-5′), 7.74 (1H, d, J = 8.1 Hz, H-6′), 7.83 (1H, s, H-2′), 7.95 (2H, d, J = 8.7 Hz, H-3″, H-5″), 8.02 (2H, d, J  =  8.4 Hz, H-2″, H-6″), 8.17 (1H, d, J  =  9.6 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 48.75 (CH-NH), 125.51 (C-4 pyridazinone), 145.42 (C-5 pyridazinone), 152.17 (C=O pyridazinone), 155.32 (C=N pyridazinone), 157.60 (C=O uriedo group). ESI-MS (m/z): 506 [M⁺], 507 [M + 1], 505 [M-1], 503 [M-2]. CHNS Analysis for C₂₇H₃₀N₄O₄S Found (Calculated): C: 65.91 (65.89), H: 5.85 (5.84), N: 11.9 (11.91), O: 10.88 (10.89), S: 5.54 (5.55).

1–(4-Methylcyclohexyl)-3–(4-{3-[4–(2-methylpropyl) phenyl]-6-oxopyridazin-1-yl} benzenesulphonyl) urea (XVII)

White crystal, yield  =  57%, m.p. 251–252 °C, R_f = 0.63 (TEF). IR ν_max (KBr, in cm^− 1): 3357, 3021 and 2913 (NH-CO-NH), 1713 and 1508 (C=O) of urea, 1597 (C=N), 1331 and 1142 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.89–0.98 (9H, m, cyclohexyl-CH₃, Ph-CH₂-CH-(CH₃)₂), 1.05–1.97 (9H, m, cyclohexyl ring), 3.26 (1H, m, proton at C-1 of cyclohexyl), 5.50 (1H, d, J  =  5.4 Hz, -NH-cyclohexyl ring), 7.25 (1H, d, J =  9.9 Hz, H-5), 7.35 (2H, d, J  =  7.5 Hz, H-3′, H-5′), 7.90 (2H, d, J = 6.9 Hz, H-2′, H-6′), 7.96 (2H, d, J  =  7.8 Hz, H-3″, H-5″), 8.02 (2H, d, J = 8.4 Hz, H-2″, H-6″), 8.022 (1H, d, J  =  9.6 Hz, H-4), 8.34 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 50.13 (CH-NH), 126.74 (C-4 pyridazinone), 145.35 (C-5 pyridazinone), 152.20 (C=O pyridazinone), 155.44 (C=N pyridazinone), 158.93 (C=O uriedo group). ESI-MS (m/z): 522 [M⁺], 523 [M + 1], 521 [M-1], 520 [M-2]. CHNS Analysis for C₂₈H₃₄N₄O₄S Found (Calculated): C: 64.43 (64.41), H: 6.5 (6.49), N: 10.82 (10.81), O: 12.15 (12.17), S: 6.10 (6.12).

3-{4-[3–(3,4-Difluorophenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4-methylcyclohexyl) urea (XVIII)

Light yellow crystals, yield =  61%, m.p. 212–213 °C, R_f = 0.62 (TEF). IR ν_max (KBr, in cm^− 1): 3341, 3027 and 2935 (NH-CO-NH), 1690 and 1510 (C=O) of urea, 1597 (C=N), 1329 and 1147 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.90 (3H, d, J = 6.3 Hz, -cyclohexyl-CH₃), 1.008–1.89 (9H, m, cyclohexyl ring protons), 3.31 (1H, m, proton at C-1 of cyclohexyl), 5.54 (1H, d, J  =  7.5 Hz, -NH-cyclohexyl ring),): 7.26–8.28 (10H, m, H-3′, H-5′, H-6′, H-4, H-5, H-2″, H-6″, H-3″, H-5″, SO₂-NHC=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 49.10 (CH-NH), 124.66 (C-4 pyridazinone), 144.73 (C-5 pyridazinone), 153.11 (C=O pyridazinone), 156.41 (C=N pyridazinone), 156.44 (C=O uriedo group).). ESI-MS (m/z): 502 [M⁺], 503 [M + 1], 501 [M-1], 500 [M-2]. CHNS Analysis for C₂₀H₁₈F₂N₄O₄S Found (Calculated): C: 57.33 (57.35), H: 4.91 (4.89), F: 7.71 (7.73), N: 11.22 (11.21), O: 12.52 (12.54), S: 6.35 (6.37).

3-{4-[3–(4-Fluorophenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4 methylcyclohexyl) urea (XIX)

Yellow crystals, yield  =  50%, m.p. 188–189 °C, R_f = 0.64 (TEF). IR ν_max (KBr, in cm^− 1): 3332, 3059 and 2981 (NH-CO-NH), 1715 and 1510 (C=O) of urea, 1603 (C=N), 1344 and 1162 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 091 (3H, d, J = 6.6 Hz, -cyclohexyl- CH₃), 0.95–2.15 (9H, m, cyclohexyl ring), 3.22 (1H, m, proton at C-1 of cyclohexyl), 7.30 (1H, d, J = 9.6 Hz, H-5), 7.42 (2H, d, J = 8.4 Hz, H-2′, H-6′), 7.97–8.07 (6H, m, H-3′, H-5′, H-2″, H-6″, H-3″, H-5″), 8.25 (1H, d, J  =  9.9 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 48.66 (CH-NH), 125.56 (C-4 pyridazinone), 145.63 (C-5 pyridazinone), 154.22 (C=O pyridazinone), 155.74 (C=O uriedo group), 156.32 (C=N pyridazinone). ESI (m/z): 484 [M⁺], 485 [M + 1], 483 [M-1], 482 [M-2]. CHNS Analysis for C₂₄H₂₅FN₄O₄S Found (Calculated): C: 59.47 (59.45), H: 5.28 (5.30), F: 3.88 (3.90), N: 11.62 (11.63), O: 13.21 (13.20), S: 6.58 (6.57).

1–(4-Methylcyclohexyl)-3-{4-[6-oxo-3–(5,6,7,8-tetrahydronaphthalen-2-yl) pyridazin-1-yl] benzenesulphonyl} urea (XX)

White crystals, yield =  45%, m.p. 240–241 °C, R_f = 0.67 (TEF). IR ν_max (KBr, in cm ^− 1): 3362, 3078 and 3003 (NH-CO-NH), 1729 and 1525 (C=O) of urea, 1621 (C=N), 1350 and 1188 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.89 (3H, d, J = 6.3 Hz, cyclohexyl-CH₃), 1.006–2.11 (9H, m, cyclohexyl ring), 1.79 (4H, s, H-3″′, H-4″′), 2.98 (4H, s, H-2″′, H-5″′), 3.26 (1H, m, proton at C-1of cyclohexyl), 5.57 (1H, brs, -NH-cyclohexyl ring), 7.24 (2H, m, H-5, H-5′), 7.67–7.74 (2H, m, H-2′, H-6′), 7.79–7.99 (4H, m, H-2″, H-6″, H-3″, H-5″), 8.17–8.34 (2H, m, H-4, SO₂-NH-C=O), ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 47.75 (CH-NH), 125.66 (C-4 pyridazinone), 145.77 (C-5 pyridazinone), 153.66 (C=O pyridazinone), 155.81 (C=O uriedo group), 156.32 (C=N pyridazinone). ESI-MS (m/z):520 [M⁺], 521 [M + 1], 519 [M-1], 518 [M-2]. CHNS Analysis for C₂₈H₃₂N₄O₄S Found (Calculated): C: 64.38 (64.40), H: 6.22 (6.20), N: 10.55 (10.56), O: 12.67 (12.68), S: 6.25 (6.24).

3-{4-[3–(4-Benzylphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4-methylcyclohexyl) urea (XXI)

White crystals, yield  =  68%, m.p. 239–240 °C, R_f = 0.7 (TEF). IR ν_max (KBr, in cm^− 1): 3347, 3053 and 3007 (NH-CO-NH), 1710 and 1529 (C=O) of urea, 1608 (C=N), 1361 and 1170 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.84 (3H, d, J = 6.3 Hz, cyclohexyl-CH₃), 0.94–1.90 (9H, m, cyclohexyl ring), 3.51 (1H, m, proton at C-1 of cyclohexyl), 3.99 (2H, s, Ph-CH₂-Ph),): 5.48 (1H, d, J  =  7.8 Hz, -NH-cyclohexyl ring), 5.71 (1H, brs, SO₂-NH-C=O), 7.16–7.38 (8H, m, H-5, H-2″′, H-3″′, H-4″′, H-5″′, H-6″′, H-3′, H-5′), 7.63–8.09 (6H, m, H-2″, H-6″, H-3″, H-5″, H-2′, H-6′), 8.12 (1H, d, J  =  7.2 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 47.80 (CH-NH), 125.44 (C-4 pyridazinone), 145.22 (C-5 pyridazinone), 155.33 (C=O uriedo group), 154.21 (C=O pyridazinone), 155.11 (C=N pyridazinone). ESI-MS (m/z): 556 [M⁺], 557 [M + 1], 555 [M-1], 554 [M-2]. CHNS Analysis for C₃₁H₃₂N₄O₄S Found (Calculated): C: 66.68 (66.69), H: 5.99 (5.97), N: 10.25 (10.26), O: 11.32 (11.31), S: 5.80 (5.79).

1–(4-Methylcyclohexyl)-3-{4-[6-oxo-3–(4-propylphenyl)pyridazin-1-yl] benzenesulphonyl} urea (XXII)

Light yellow crystals, yield =  57%, m.p. 246–247 °C, R_f = 0.66 (TEF). IR ν_max (KBr, in cm^− 1): 3335, 3063 and 2968 (NH-CO-NH), 1696 and 1507 (C=O) of urea, 1581 (C=N), 1332 and 1125 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.825 (3H, d, J  =  5.4 Hz, cyclohexyl-CH₃), 1.03–1.90 (9H, m, cyclohexyl ring), 3.20 (1H, m, proton at C-1 of cyclohexyl): 5.44 (1H, d, J = 8.1 Hz, -NH-cyclohexyl), 5.70 (1H, brs, SO₂-NH-C=O), 7.16–8.17 (10H, m, H-5, H-3′, H-5′, H-2′, H-6′, H-3″, H-5″, H-2″, H-6″, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 50.10 (CH-NH), 125.44 (C-4 pyridazinone), 145.22 (C-5 pyridazinone), 154.18 (C=O pyridazinone), 155.12 (C=N pyridazinone), 155.33 (C=O uriedo group). ESI-MS (m/z): 508 [M⁺], 509 [M + 1], 507 [M-1], 506 [M-2]. CHNS Analysis for C₂₇H₃₂N₄O₄S Found (Calculated): C: 63.36 (63.37), H: 6.74 (6.73), N: 10.97 (10.96), O: 12.58 (12.59), S: 6.35 (6.37).

3-{4-[3–(4-Ethoxyphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4-methylcyclohexyl) urea (XXIII)

Yellow crystals, yield  =  49%, m.p. 241–242 °C, R_f = 0.71 (TEF). IR ν_max (KBr, in cm ⁻¹): 3320, 3092 and 2986 (NH-CO-NH), 1721 and 1530 (C=O) of urea, 1590 (C=N), 1333 and 1146 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.96 (3H, d, J  =  8.7 Hz, cyclohexyl-CH₃), 1.002–1.24 (5H, m, cyclohexyl ring), 1.30 (3H, t, Ph-O-CH₂-CH₃), 1.37–1.73 (4H, m, cyclohexyl ring), 3.18 (1H, m, proton at C-1 of cyclohexyl), 4.09 (2H, q, Ph-O-CH₂-CH₃), 6.38 (1H, d, J = 5.1 Hz, -NH-cyclohexyl), 7.04 (2H, d, J = 6.6 Hz, H-3′, H-5′), 7.21 (1H, d, J  =  7.2 Hz, H-5), 7.90 (2H, d, J  =  6.6 Hz, H-2′, H-6′), 7.96 (2H, d, J  =  6.6 Hz, H-3″, H-5″), 8.03 (2H, d, J = 6.6 Hz, H-2″, H-6″), 8.16 (1H, d, J = 7.5 Hz, H-4), 10.51 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 46.91 (CH-NH), 125.33 (C-4 pyridazinone), 145.80 (C-5 pyridazinone), 153.42 (C=O pyridazinone), 155.35 (C=N pyridazinone), 156.81 (C=O uriedo group). ESI-MS (m/z):510 [M⁺], 511 [M + 1], 509 [M-1], 508 [M-2]. CHNS Analysis for C₂₆H₃₀N₄O₅S Found (Calculated): C: 61.35 (61.33), H: 5.80 (5.81), N: 10.86 (10.87), O: 15.75 (15.77), S: 6.25 (6.27).

1–(4-Methylcyclohexyl)-3–(4-{6-oxo-3-[4–(2-phenylethyl)phenyl]pyridazin-1-yl} benzenesulphonyl) urea (XXIV)

White crystals, yield  =  63%, m.p. 287–288 °C, R_f = 0.61 (TEF). IR ν_max (KBr, in cm^− 1): 3323, 3084 and 2987 (NH-CO-NH), 1733 and 1528 (C=O) of urea, 1589 (C=N), 1335 and 1151 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.84 (3H, d, J  =  6 Hz, cyclohexyl-CH₃), 0.89–1.24 (9H, m, cyclohexyl ring), 2.92 (4H, s, Ph-CH₂-CH₂-Ph), 3.12 (1H, m, proton at C-1 of cyclohexyl), 7.15–8.16 (17H, m, H-2′, H-3′, H-5′, H-6′, H-2″, H-3″, H-5″, H-6″, H-2″′, H-3″′, H-4″′, H-5″′, H-6″′, H-4, H-5, SO₂-NH-C=O, -NH-cyclohexyl ring), 0.84 (3H, d, J  =  6 Hz, cyclohexyl-CH₃). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 44.88 (CH-NH), 125.66 (C-4 pyridazinone), 145.17 (C-5 pyridazinone), 153.17 (C=O pyridazinone), 155.22 (C=N pyridazinone), 156.55 (C=O uriedo group). ESI-MS (m/z): 570 [M⁺], 571 [M + 1], 569 [M-1], 568 [M-2]. CHNS Analysis for C₃₂H₃₄N₄O₄S Found (Calculated): C: 67.41 (67.43), H: 5.81 (5.80), N: 9.81 (9.82), O: 11.30 (11.29), S: 5.70 (5.71).

1–(4-Methylcyclohexyl)-3-{4-[6-oxo-3–(4-phenoxyphenyl)pyridazin-1-yl]benzenesulphonyl}urea (XXV)

White crystals, yield =  51%, m.p. 236–237 °C, R_f = 0.72 (TEF). IR ν_max (KBr, in cm^− 1): 3340, 3062 and 2989 (NH-CO-NH), 1738 and 1545 (C=O) of urea, 1597 (C=N), 1358 and 1167 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.84 (1H, d, J  =  6.3 Hz, cyclohexyl-CH₃), 1.03–1.90 (9H, m, cyclohexyl ring), 3.27 (1H, m, proton at C-1 of cyclohexyl), 5.44 (1H, brs, -NH-cyclohexyl ring), 5.50 (1H, brs, SO₂-NH-C=O), 7.07–7.45 (10H, m, H-2′, H-3′, H-5′, H-6′, H-2″′, H-3″′, H-4″′, H-5″′, H-6′″, H-5), 7.88 (2H, d, J = 8.1 Hz, H-3″, H-5″), 7.95 (2H, d, J = 8.4 Hz, H-2″, H-6″), 8.11 (1H, d, J = 9.3 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 46.20 (CH-NH), 126.12 (C-4 pyridazinone), 146.40 (C-5 pyridazinone), 155.11 (C=O pyridazinone), 155.16 (C=N pyridazinone), 155.52 (C=O uriedo group). ESI-MS (m/z): 558 [M⁺], 559 [M + 1], 557 [M-1], 556 [M-2]. CHNS Analysis for C₃₀H₃₀N₄O₅S Found (Calculated): C: 64.55 (64.56), H: 5.26 (5.28), N: 10.15 (10.13), O: 14.37 (14.36), S: 5.75 (5.74).

3-{4-[3–(4-Cyclohexylphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4-methylcyclohexyl)urea (XXVI)

Yellow crystals, yield =  60%, m.p. 280–281 °C, R_f = 0.73 (TEF). IR ν_max (KBr, in cm^− 1): 3322, 3065 and 2971 (NH-CO-NH), 1736 and 1528 (C=O) of urea, 1601 (C=N), 1347 and 1161 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.90 (3H, d, J  =  6.3 Hz, cyclohexyl-CH₃), 0.96–2.08 (20H, m, cyclohexyl rings), 3.23 (1H, m, proton at C-1 of cyclohexyl),): 5.52 (1H, d, J  =  8.1 Hz, -NH-cyclohexyl ring), 6.13 (1H, brs, SO₂-NH-C=O), 7.25 (1H, d, J  =  9.6 Hz, H-5), 7.40 (2H, d, J  =  8.4 Hz, H-3′, H-5′), 7.90 (2H, d, J = 7.8 Hz, H-3″, H-5″), 8.01 (2H, d, J  =  8.1 Hz, H-2′, H-6′), 8.19 (1H, d, J  =  9.6 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 47.11 (CH-NH), 125.32 (C-4 pyridazinone), 145.43 (C-5 pyridazinone), 156.12 (C=O uriedo group), 156.20 (C=N pyridazinone), 156.71 (C=O pyridazinone). ESI-MS (m/z): 548 [M⁺], 549 [M + 1], 547 [M-1], 546 [M-2]. CHNS Analysis for C₃₀H₃₆N₄O₄S Found (Calculated): C: 65.81 (65.83), H: 6.53 (6.54), N: 10.30 (10.29), O: 11.59 (11.6), S: 5.80 (5.81).

3-{4-[3–(4-Bromophenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4-methylcyclohexyl)urea (XXVII)

Light yellow crystals, yield  =  55%, m.p. 231–232 °C, R_f = 0.62 (TEF). IR ν_max (KBr, in cm⁻¹): 3338, 3100 and 2982 (NH-CO-NH), 1740 and 1533 (C=O) of urea, 1608 (C=N), 1339 and 1142 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 1.17 (3H, d, J  =  8.1 Hz, cyclohexyl-CH₃), 1.22–1.92 (9H, m, cyclohexyl ring), 3.21 (1H, m, proton at C-1 of cyclohexyl), 6.50 (1H, brs, -NH-cyclohexyl ring), 7.45 (1H, d, J = 7.5 Hz, H-5), 7.91 (2H, d, J  =  6.3 Hz, H-2′, H-6′), 8.12 (2H, d, J = 6.3 Hz, H-3′, H-5′), 8.15 (2H, d, J = 6.6 Hz, H-3″, H-5″), 8.23 (2H, d, J = 6.3 Hz, H-2″, H-6″), 8.39 (2H, d, J  =  6.3 Hz, H-2″, H-6″), 8.39 (1H, d, J = 7.2 Hz, H-4), 10.72 (1H, s, SO₂-NH-C=O). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 49.10 (CH-NH), 125.70 (C-4 pyridazinone), 145.77 (C-5 pyridazinone), 154.35 (C=O pyridazinone), 155.66 (C=N pyridazinone), 156.35 (C=O uriedo group). ESI-MS (m/z):546 [M + 2], 544 [M⁺], 545 [M + 1]. CHNS Analysis for C₂₄H₂₅BrN₄O₄S Found (Calculated): C: 52.88 (52.86), H:4.7 (4.71), Br: 14.61 (14.60), N: 10.28 (10.29), O: 11.71 (11.73), S: 5.87 (5.88).

3-{4-[3–(4-Iodophenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4-methylcyclohexyl) urea (XXVIII)

Yellow crystals, yield  =  47%, m.p. 253–2545 °C, R_f = 0.63 (TEF). IR ν_max (KBr, in cm⁻¹): 3312, 3182 and 2997 (NH-CO-NH), 1752 and 1545 (C=O) of urea, 1617 (C=N), 1354 and 1166 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.90 (3H, d, J = 6.3 Hz, cyclohexyl-CH₃), 0.96–1.91 (9H, m, cyclohexyl ring), 3.2 (1H, m, proton at C-1 of cyclohexyl), 5.52 (1H, d, J = 7.8 Hz, -NH-cyclohexyl ring), 6.03 (1H, brs, SO₂-NH-C=O), 7.28 (1H, d, J = 6.9 Hz, H-5), 7.62 (2H, d, J = 9.6 Hz, H-2′, H-6′), 7.80 (2H, d, J = 8.4 Hz, H-3′, H-5′), 7.87–8.05 (4H, m, H-2″, H-3″, H-5″, H-6″), 8.23 (1H, d, J = 5.4 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 50.13 (CH-NH), 125.66 (C-4 pyridazinone), 146.55 (C-5 pyridazinone), 154.17 (C=O pyridazinone), 155.45 (C=O uriedo group), 156.22 (C=N pyridazinone). ESI-MS (m/z): 592 [M⁺], 593 [M + 1], 591 [M-1], 590 [M-2]. CHNS Analysis for C₂₄H₂₅IN₄O₄S Found (Calculated): C: 48.65 (48.63), H: 4.27 (4.26), I: 21.57 (21.55), N: 9.43 (9.41), O: 10.76 (10.75), S: 5.39 (5.41).

3-{4-[3–(4-Hexylphenyl)-6-oxopyridazin-1-yl] benzenesulphonyl}-1–(4-methylcyclohexyl) urea (XIXX)

White crystals, yield  =  53%, m.p. 281–282 °C, R_f = 0.65 (TEF). IR ν_max (KBr, in cm ⁻¹): 3325, 3101 and 2989 (NH-CO-NH), 1744 and 1538 (C=O) of urea, 1609 (C=N), 1341 and 1150 (SO₂N), ¹H NMR (300 MHz, DMSO-d₆, δ): 0.80 (3H, t, -Ph-(CH₂)₅-CH₃), 0.90 (3H, d, J = 6.3 Hz, cyclohexyl ring -CH₃), 0.96–1.33 (9H, m, cyclohexyl ring), 1.33 (6H, s, -Ph-CH₂-CH₂-(CH₂)₃-CH₃), 1.74 (2H, q, -Ph-CH₂-CH₂-(CH₂)₃-CH₃), 2.68 (2H, t, -Ph-CH₂-(CH₂)₄-CH₃), 3.24 (1H, m, proton at C-1 of cyclohexyl), 5.51 (1H, d, J = 7.8 Hz, -NH-cyclohexyl ring), 6.50 (1H, brs, SO₂-NH-C=O), 7.25 (1H, d, J  =  9.6 Hz, H-5), 7.37 (2H, d, J = 8.4 Hz, H-3′, H-5′), 7.88–8.05 (6H, m, H-2′, H-6′, H-3″, H-5″, H-2″, H-6″), 8.18 (1H, d, J = 9.6 Hz, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 48.35 (CH-NH), 125.64 (C-4 pyridazinone), 146.22 (C-5 pyridazinone), 155.25 (C=O uriedo group), 155.30 (C=N pyridazinone), 156.80 (C=O pyridazinone). ESI-MS (m/z): 550 [M⁺], 551 [M + 1], 549 [M-1], 548 [M-2]. CHNS Analysis for C₃₀H₃₈N₄O₄S Found (Calculated): C: 65.73 (65.75), H: 6.97 (6.96), N: 10.13 (10.11), O: 11.51 (11.50), S: 5.69 (5.71).

3-{4-[3–(4-Isopropylphenyl)-6-oxopyridazin-1-yl]benzenesulphonyl}-1–(4-methylcyclohexyl)urea (XXX)

White crystals, yield  =  57%, m.p. 248–249 °C, R_f = 0.65 (TEF). IR ν_max (KBr, in cm^− 1): 3364, 3056 and 2978 (NH-CO-NH), 1729 and 1523(C=O) of urea, 1607 (C=N), 1343 and 1157 (SO₂N). ¹H NMR (300 MHz, DMSO-d₆, δ): 0.79 (3H, d, J = 4.8 Hz, cyclohexyl ring -CH₃), 0.86–1.17 (9H, m, cyclohexyl ring), 1.23 (6H, d, J = 6 Hz, Ph-CH-(CH₃)₂), 2.86 (1H, h, Ph-CH-(CH₃)₂), 3.19 (1H, m, proton at C-1 of cyclohexyl), 3.20–3.31 (1H, m, -NH-CH-(CH₃)₂), 4.47 (1H, d, J  =  8.1 Hz, -NH-cyclohexyl ring), 7.08 (1H, d, J = 9.9 Hz, H-5), 7.27 (2H, d, J = 8.4 Hz, H-3′, H-5′), 7.52 (1H, s, SO₂-NH-C=O), 7.75 (2H, d, J  =  8.4 Hz, H-2′, H-6′), 7.86 (2H, d, J = 6.9 Hz, H-3″, H-5″), 7.93–8.04 (3H, m, H-2″, H-6″, H-4). ¹³C NMR (75 MHz, DMSO-d₆, δ, ppm): 47.80 (CH-NH), 125.44 (C-4 pyridazinone), 145.22 (C-5 pyridazinone), 154.21 (C=O pyridazinone), 155.11 (C=N pyridazinone), 155.33 (C=O uriedo group). ESI-MS (m/z): 508 [M⁺], 509[M + 1], 507 [M-1], 506 [M-2].CHNS Analysis for C₂₇H₃₂N₄O₄S Found (Calculated): C: 63.60 (63.59), H: 6.38 (6.39), N: 11.10 (11.09), O: 12.62 (12.63), S: 6.35 (6.33).

Effect of synthesized compounds on oral glucose tolerance in normal rats

All the experiments were performed utilizing albino rats of Wistar strain (either sex) weighing 210 ± 10 g which were provided by the Animal House of Department of Pharmacology, State Company for Drug Industries and Medical Appliances, Samarra/Baghdad (1871/SDI) and were housed in the same location under standardized conditions. Animals were fed commercial chaw and had free access to water ad libitum. The experiments were performed in congruence with the instructions for the care and use of laboratory animals, ordained by the State Company for Drug Industries and Medical Appliances SDI. Animals delineated as fasted were bereaved of food for at least 16 h but permitted free access to water. Carboxymethyl cellulose CMC (1%w/v) in distilled water was used as vehicle for dosing in all the experiments. All treatments were given orally using gauge. Fasted rats were divided into groups of six animals each. Animals of group I were fed with the vehicle (CMC 1% w/v in distilled water) in a volume of 10 ml/kg, while animals of group II were given gliclazide 0.05 mM/kg suspended in the vehicle. Animals of the experimental group (I–XXX) were administered the suspension of the test compounds at a dosage of 0.05 mM/kg b.w. A glucose load (3  g/kg) was given to each animal exactly after 30-min post-administration of the respective treatments. Blood samples were collected from retro-orbital plexus just prior to and 60 min after the glucose loading and blood glucose levels were measured with an autoanalyser (Accu Check Active glucose kit)27.

Docking

Docking studies were implemented utilizing Glide module of the Schrodinger-9 software on the aldose reductase (PDB id: 1EL3, 1USO, 2FZD and 2PDK). Receptor preparation was done using protein preparation wizard with defaults settings. The structures were sketched using maestro graphical user interface (GUI) and were energy minimized/cleaned up by Ligprep module of the same software using OPLS_2005 force field and proper protonation states were assigned with the ionizer subprogram at pH 7.2 ±0.228. A grid space of 20 Å around cocrystallized ligand was set for the docking calculations. Glide XP module was used for final docking studies29.

Preparation of rat lens aldose reductase

Crude AR was prepared from rat lens through reported method30. Institutional and national guidelines for the care and use of animals were followed and all experimental procedures involving animals were approved by the IAEC (institutional animal ethical committee) of the National Institute of Nutrition. Lenses were homogenized in nine volumes of 100 mM potassium phosphate buffer, pH 6.2. The homogenate was centrifuged at 15 000 × g for 30 min at 4 °C and the resulting supernatant was used as the source of ALR2.

Aldose reductase activity assay

Aldose reductase (AR) activity was assayed as described by Suryanarayana30. All the experiments were performed in triplicate. The assay mixture in 1 ml contained 50 mM potassium phosphate buffer, pH 6.2, 0.4 M lithium sulphate, 5 mM β-mercaptoethanol, 10 mM DL-glyceraldehyde, 0.1 mM NADPH and enzyme preparation. Appropriate blanks were employed for corrections. The assay mixture was incubated at 37 °C and the reaction was initiated by the addition of NADPH at 37 °C. The change in the absorbance at 340 nm due to NADPH oxidation was followed in a spectrophotometer.

Enzyme inhibition studies

For inhibition studies, a concentrated stock of pyridazinone-substituted benzenesulphonylurea derivatives (I–XXX) was prepared in DMSO. Aliquots drawn from a working solution were added to the enzyme assay mixture and incubated for 5 min before initiating the reaction by NADPH as described above. The per cent inhibition with test compound was calculated considering the enzyme activity in the absence of inhibitor as 100%. The concentration of each test sample giving 50% inhibition (IC₅₀) was determined by non-linear regression analysis of log concentration of compound versus percentage inhibition.

Results and discussion

Synthesis of compounds

The synthetic route used to synthesize title compounds (I–XXX) is outlined in Scheme 1. The 4-aryl-pyridazinone-substituted benzenesulphonamides derivatives (2a–o) synthesized through reported method31 were converted to corresponding sulphonyl urea derivatives by refluxing with appropriate isocyanate in dry acetone containing K₂CO₃.

Scheme 1. Synthesis of pyridazinone-based benzenesulphonylurea derivatives. Reagents and conditions: (a) maleic anhydride, 1,1,2,2-tetrachloroethane, AlCl₃; (b) Absolute alcohol, reflux 12–18 h; (c) Appropriate isocyanate, K₂CO₃, dry acetone, reflux 24–72 h.

The structure of synthesized pyridazinones-substituted benzenesulphonylurea derivatives (I–XXX) were determined on the basis of elemental analysis and various spectroscopic methods, such as IR, ¹H NMR, ¹³C NMR and MS. Elemental analysis (C, H, N and S) data were within ±0.5% of the theoretical values. All the peaks corresponding to IR, ¹H NMR and ¹³C NMR were observed at expected positions in respective spectra (for details see experimental section).

Oral glucose tolerance test in normal rats (OGGT)

In the current study, oral hypoglycaemic effects of 30 newly synthesized compounds (I–XXX) were assessed in glucose-fed hyperglycaemic normal rats at the dose of 0.05 mM/kg b.w. The clinically used sulphonyl urea drug gliclazide at the dose 0.05 mM/kg b.w. was used as positive control.

Quantitative glucose tolerance of each animal was calculated by area under curve (AUC) method by using prism software. Comparing AUC of experimental and control groups determined the percentage antihyperglycaemic activity. Statistical comparison was made by Dunnett’s test. Samples showing significant inhibition (p < 0.05) on postprandial hyperglycaemia were considered as active samples.

The results are summarized in Table 1. Twenty-three compounds (III–XI, XIV–XVII, XIX–XXIV, XXVI and XXVIII–XXX) out of 30 compounds showed more or comparable area under the curve (AUC) reduction percentage (ranging from 21.9% to 35.5%) as compared to the standard drug gliclazide (22.0%). The compound VIII is the most active compound and exhibited 35.5% AUC reduction.

Table 1. In vivo OGTT results for the compounds (I–XXX).

Treatment 0.05 mmol/kg b.w.	AUC reduction %	Significance
Control	–	–
Gliclazide	22.0	p < 0.01
I	3.6	p > 0.05
II	10.5	p > 0.05
III	26.8	p < 0.01
IV	30.5	p < 0.01
V	25.8	p < 0.01
VI	30.1	p < 0.01
VII	25.5	p < 0.01
VIII	35.5	p < 0.01
IX	32.4	p < 0.01
X	34.9	p < 0.01
XI	27.3	p < 0.01
XII	20.2	p < 0.01
XIII	21.7	p < 0.01
XIV	33.4	p < 0.01
XV	25.7	p < 0.01
XVI	24.3	p < 0.01
XVII	27.8	p < 0.01
XVIII	15.0	p < 0.01
XIX	21.9	p < 0.01
XX	26.0	p < 0.01
XXI	22.0	p < 0.01
XXII	28.6	p < 0.01
XXIII	26.5	p < 0.01
XXIV	31.0	p < 0.01
XXV	17.8	p < 0.01
XXVI	24.9	p < 0.01
XXVII	19.8	p < 0.01
XXVIII	20.5	p < 0.01
XXIX	30.5	p < 0.01
XXX	30.0	p < 0.01

With regard to the SAR, it seems impossible to extract an obvious structure–activity relationship from the data shown in Table 1.

Docking

Docking studies of 30 synthesized compounds (I to XXX) were performed in order to get better comprehension of the aldose reductase inhibitory potency at molecular level and to shed light on the interactions in the active site of aldose reductase. Docking studies were performed using Glide module of the Schrodinger-9.4 software on the Aldose reductase (ALR) receptor (PDB id: 1EL3, 1USO, 2FZD, 2PDK). The most relevant poses were obtained with PDBID: 1EL3. However, the docking studies were performed with PDB ID: 1El3 because this structure is bound with IDD384, which is a sulphonamide derivative and is relevant with our study. The re-docking of reference ligand (IDD384) into active site of aldose reductase enzyme reveals that it occupies the same binding pocket with root mean square deviation (RMSD) of 0.61A which further validates the present docking protocol (Figure 3).

Figure 3. Binding interaction of cocrystallized ligand (IDD384) with ALR catalytic domain.

Docking results reveal that the compounds III and V bound tightly in the active site of aldose reductase (ALR2). These compounds (III and V) well occupied in the receptor cavity and forms hydrogen bonds and hydrophobic interactions (Figures 4 and 5). The sulfonamide group of III and V is anchored into the anion-binding site formed by Ala299, Leu300 and Trp111 and forms hydrogen-bonding with Trp111, which is key residue in binding and catalysis32,33. The 1,2,3,4-tetrahydronaphthalene ring core gets tightly trapped in the hydrophobic pocket formed by Phe122, Leu300, Leu301, Leu124 and Val130. Further docking analysis of compounds V and III with cocrystallized ligand (1DD384) by superimposition; it gives the same interaction pattern of binding with catalytic domain of ALR enzyme. The fact is also validated by Figure 6 where III and V were superimposed on cocrystal ligand. The binding pose of compound X (Figure 7A–C) reveals why it is inactive. It does not fit well in the receptor binding site and does not show hydrogen bonding like III and V. This may be explained on the basis that the cyclohexyl attached to the sulphonamide moiety renders the configuration of molecule. Moreover, all other groups attached to the pyridazinone ring show different poses when compared to the III and V (Figure 7B and C). Therefore, it misses all the important interactions required for ALR inhibition. The fact is also validated by (Figure 7B), where X was superimposed on V and it is clear that compound V has more relaxed conformation that compound X.

Figure 4. Compound III binding interaction with ALR catalytic domain (PDBID: 1El3).

Figure 5. Compound V binding interaction with ALR catalytic domain (PDBID: 1El3).

Figure 6. superimposition of compounds V and III with cocrystallized ligand (1dd384) against ALR enzyme (A) with, V with (B) III.

Figure 7. Comparison of binding interaction of inactive X with (A) cocrystallized ligand, (B) with V, (C) with III.

Aldose reductase inhibition

Eighteen synthesized compounds were evaluated in vitro for their ability to inhibit activity of partially purified rat lens aldose reductase (AR). It has been shown that there is an approximately 85% sequence similarity between rat lens and human aldose reductase (ALR2), while the proposed active sites of both enzymes are identical32. The performed assay was based on a spectrometric measurement, which is proven to be a reliable method33, with DL-glyceraldehyde as the substrate and NADPH as the cofactor. Quercetin, a known ARI was used as a positive control. Results are presented in Table 2. Out of the tested 18 compounds, only ten compounds showed aldose reductase activity with IC₅₀ ranging from 34 to 242 μM. Among these derivatives, compound IV (IC₅₀ = 45 μM) and V (IC₅₀= 34 μM) were found comparable with the known ARI quercetin (IC₅₀ = 41 μM). As regards to the structural activity relationship, no definite SAR could be extracted from Table 2.

Table 2. Aldose reductase (AR) inhibitory data.

Compounds	IC50 (μM)*
III	63
IV	45
V	34
VI	237
X	n.a.†
XII	77
XIII	n.a.
XV	n.a.
XVI	119
XVII	187
XVIII	75
XX	n.a.
XXI	384
XXII	n.a.
XXV	n.a.
XXVII	242
XXIX	n.a.
XXX	n.a.
Quercetin	41

*IC₅₀ values, represent the concentration required to produce 50% enzyme inhibition.

† n.a.: Not active.

Conclusion

In the present study, 30 new pyridazinone-substituted benzenesulphonylurea derivatives (I–XXX) were synthesized. The synthesized compounds are well supported by the spectroscopic data and elemental analysis. These compounds were screened for their antihyperglycaemic activity using in vivo OGTT assay (albino rats of Wistar strain). Eighteen compounds displaying good docking results were screened for their in vitro ability to inhibit rat lens aldose reductase. As a result, two compounds (IV and V) were identified possessing significant dual action (antihyperglycaemic and aldose reductase inhibition) and may be used as lead compounds for developing new drugs.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.