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. 2024 Apr 19;16(10):963–981. doi: 10.4155/fmc-2023-0338

Exploring of novel oxazolones and imidazolones as anti-inflammatory and analgesic candidates with cyclooxygenase inhibitory action

Seham A Rayan 1, Riham F George 1, Nada M Mohamed 2, Mona F Said 1,*
PMCID: PMC11221545  PMID: 38639393

Abstract

Aim: Over the last few decades, therapeutic needs have led to a search for safer COX-2 inhibitors with potential anti-inflammatory and analgesic activity. Materials & methods: A new series of oxazolone and imidazolone derivatives 3a–c and 4a–r were synthesized and evaluated as anti-inflammatory and analgesic agents. COX-1/COX-2 isozyme selectivity testing and molecular docking were performed. Results: All compounds showed good activities comparable to those of the reference, celecoxib. The most active compounds 3a, 4a, 4c, 4e and 4f showed promising gastric tolerability with an ulcer index lower than that of celecoxib. The molecular docking of p-methoxyphenyl derivative 4c showed alkyl interaction with the side pocket His75 of COX-2 and achieved the best anti-inflammatory activity, with a COX-2 selectivity index better than that of celecoxib.

Keywords: : analgesic, anti-inflammatory, COX-2 inhibitors, imidazolone, oxazolone

GRAPHICAL ABSTRACT

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Plain language summary

Summary points.

Chemistry

  • Novel oxazolone and imidazolone derivatives were designed and synthesized.

Biological activity

  • The new compounds showed good analgesic and anti-inflammatory activities.

  • The most active compounds showed good COX-2 selectivity.

  • Compound 4c was the best anti-inflammatory agent.

  • The selected compounds showed gastric tolerability.

Molecular docking

  • Molecular docking found that the scaffold of the tested derivatives showed very similar binding pattern with the main hydrophobic channel residues of both isoenzymes.

  • The high activity of the derivative 4c was attributable to the additional alkyl interaction with the side pocket His75 of COX-2.

The inflammatory response is a complex process of the immune system associated with harmful stimuli and many diseases [1]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed drugs as anti-inflammatory and analgesic drugs [2]. Their anti-inflammatory activity is mediated by the inhibition of cyclooxygenase (COX) enzymes, which convert arachidonic acid into prostanoids such as thromboxane and prostaglandins, which play a key role in the generation of the inflammatory response [3–5]. COX enzymes exist in two isoforms: COX-1, which is expressed in the body and is responsible for various biological activities, specifically, cytoprotection of the gastric mucosa, renal hemodynamics and platelet thrombogenesis; and COX-2, which is an inducible isoform expressed mainly in inflamed tissues [6]. NSAIDs are classified into two main classes: nonselective NSAIDs, which inhibit both types of COX enzymes and consequently are associated with gastric irritation and ulcers; and selective COX-2 inhibitors, which inhibit COX-2 and have been developed to reduce the main side effects of nonselective NSAIDs [7,8]. However, some selective COX-2 inhibitors show cardiovascular adverse effects depending on their structures [9–12]. Thus there is always an effort to synthesize new anti-inflammatory drugs having enhanced safety profiles.

Nitrogen-containing heterocyclic compounds are key building blocks in pharmacologically active compounds [13,14]. Oxazole [15–19] and imidazole [20–25] compounds were reported to have anti-inflammatory and analgesic activities. Additionally, the di-arylheterocycle nucleus presents a privileged pharmacophore profile for the synthesis of selective COX-2 inhibitors such as coxibs, the most famous selective COX-2 inhibitors [26–28], in which two vicinal aryl rings are attached to a five-membered heterocyclic ring as pyrazole in celecoxib and one of the aryl rings is substituted at the para position with a sulfonyl moiety to increase COX-2 selectivity [29,30]. The di-phenyloxazolone derivative I bearing a sulfonyl moiety was found to possess high percentage inhibition (70.14% ± 1.71) with a selectivity index (SI) >50; additionally, the oxazolone carbonyl group forms a hydrogen bond with COX-2 [31]. Oxazolone derivative II (COX-2 IC50 = 0.019 μM) was the most potent derivative of the reported oxazolone series and was even more potent than celecoxib (COX-2 IC50 = 0.05 μM) [16].

The structures of the COX-1 and COX-2 isoforms are very similar; however, minor but key changes in the amino acid structures between the two enzymes result in differences in their active sites. Firstly, the binding site of the COX-2 enzyme possesses an extra space at the top, meaning that bulky inhibitors such as di- and tri-arylheterocycle compounds exhibit more selectivity toward COX-2 because they are too large to fit into the COX-1 active site [32–34]. Secondly, the COX-2 active site has a hydrophilic side pocket, so the inhibitors with a hydrophilic sulfonyl moiety exhibit more selectivity toward the COX-2 enzyme than the COX-1 enzyme [35–38]. For example, the tri-aryl imidazolone derivative III with a sulfonamide group (SO2NH2) attached to one of the aryl rings showed COX-2 selectivity (COX-2 IC50 = 0.74 μM; COX-1 IC50 = 3.9 μM) in comparison with the reference drug celecoxib (COX-2 IC50 = 0.87 μM; COX-1 IC50 = 7.7 μM), and it exhibited good gastrointestinal tolerance (ulcer index = 1.22) [39]. The introduction of an electron-donating methoxy group at the same position on the N-aryl imidazole in the derivative IVa showed high COX-2 potency and selectivity (IC50 = 0.060 μM; SI = 175) compared with the reference drug celecoxib (IC50 = 0.046 μM; SI = 315.22). Additionally, it was higher than the reference indomethacin and the para chloro-N-aryl derivative IVb (IC50 = 0.079 μM, SI = 12.53 and IC50 = 0.80 μM, SI = 7.5, respectively) [40]. The reported imidazolone V with an indole ring showed good anti-inflammatory activity with 80.20% inhibition and good analgesic activity with 80.74% protection; it also showed good gastrointestinal tolerance with a severity index of 0.71, which was better than that of the standard, indomethacin (severity index = 2.25) [41]. The introduction of a cyclic amino group in the form of pyrrolidine to the reported imidazothiazole derivative VI achieved the maximum COX-2 potency and good selectivity (COX-2 IC50 = 0.09; SI = 134.4) among the reported derivatives with different cyclic amino groups such as morpholine and piperidine against the reference celecoxib (COX-2 IC50 = 0.06 μM; SI = 405) [42].

According to the above findings and with the aim of developing safe and potent anti-inflammatory agents with a reasonable COX-2 selectivity, some oxazolone- and imidazolone-based derivatives have been designed and synthesized. This include the new oxazolone compound 3a and the reported compounds 3b and 3c [43,44] bearing different heterocyclic amines, and new imidazolone compounds in which N3-imidazolone was substituted by a phenyl, p-tolyl, p-methoxyphenyl, p-chlorophenyl, p-bromophenyl or p-sulfanilamide moiety to study the hydrophobic and electronic effects of different substituents on the biological activity (Figure 1).

Figure 1.

Figure 1.

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Reported compounds I–VI and the design strategy of the synthesized oxazolone and imidazolone scaffolds.

All the synthesized compounds were tested for their anti-inflammatory and analgesic activities. Derivatives that showed promising anti-inflammatory activity were further evaluated for their acute toxicity and gastric safety. Moreover, they were tested for their COX-1 and COX-2 inhibitory activities utilizing an in vitro COX enzyme inhibition assay.

Molecular docking simulations were also carried out for the most active compounds and the reference compound (celecoxib) into the active sites of both COX-1 and COX-2 enzymes to study their binding pattern and binding affinity and rationalize their significant experimental activity and their COX selectivity.

Materials & methods

The apparatus used for the chemical syntheses and confirmation of the prepared compounds is listed in the Supplementary data (Supplementary S6.1). Compound 1 was synthesized as reported [45], while compounds 3b and 3c [43,44] were synthesized with some differences in the reported conditions.

Chemistry

General procedure for synthesis of compounds 3a–c

A mixture of hippuric acid (0.01 mol, 0.25 g), the appropriate aldehyde (0.01 mol) and anhydrous sodium acetate (0.03 mol, 0.34 g) was refluxed in acetic anhydride (10 ml) at 100°C for 6 h. Then the precipitate obtained was filtered, washed with water followed by aqueous ethanol and finally crystallized from ethanol.

2-Phenyl-4-(4-(pyrrolidin-1-yl)benzylidene)oxazol-5(4H)-one (3a)

Reddish-brown crystals; yield 40%; melting point (m.p.) 193–195°C; IR (KBr, cm-1): 3074, 3059 (CH aromatic), 2912, 2858 (CH aliphatic), 1766 (C=O), 1577, 1527 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 2.06–2.12 (m, 4H, 2CH2 pyrrolidine), 3.43 (t, J = 6.60 Hz, 4H, 2CH2N pyrrolidine), 6.64 (d, J = 8.96 Hz, 2H, ArH), 7.21 (s, 1H, olefinic H), 7.49–7.58 (m, 3H, ArH), 8.13–8.16 (m, 4H, ArH); 13C NMR (CDCl3, 100 MHz): 25.40 (2CH2 pyrrolidine), 47.79 (2CH2N pyrrolidine), 112.16, 121.57, 126.43, 127.68, 127.73, 128.77, 132.16, 133.67, 135.10, 149.80, 160.17 (aromatic C), 168.61 (C=O); electron impact mass spectroscopy (EIMS), m/z: 318.06 (M+); Anal. calcd. for C20H18N2O2 (318.38): C, 75.45; H, 5.70; N, 8.80. Found: C, 75.36; H, 5.84; N, 9.06.

2-Phenyl-4-(4-(piperidin-1-yl)benzylidene)oxazol-5(4H)-one (3b)

Orange crystals; yield 38.5%; m.p. 157–159°C; IR (KBr, cm-1): 3032 (CH aromatic), 2939, 2850 (CH aliphatic), 1762 (C=O), 1593, 1577 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 1.70 (s, 6H, 3CH2 of piperidine), 3.43 (s, 4H, 2CH2N of piperidine), 6.95 (d, J = 6.00 Hz, 2H, ArH), 7.20 (s, 1H, olefinic H), 7.50–7.59 (m, 3H, ArH), 8.12–8.17 (m, 4H, ArH); 13C NMR (CDCl3, 100 MHz): 24.36 (CH2, piperidine C-3), 25.34 (2CH2, piperidine C-2, C-4), 48.46 (2CH2N, piperidine), 114.08, 122.94, 126.26, 127.85, 128.81, 132.43, 132.83, 134.79, 152.87, 160.93 (aromatic C), 168.43 (C=O); Anal. calcd. for C21H20N2O2 (332.40): C, 75.88; H, 6.06; N, 8.43. Found: C, 75.69; H, 6.21; N, 8.70.

4-(4-Morpholinobenzylidene)-2-phenyloxazol-5(4H)-one (3c)

Yellowish-brown crystals; yield 48%; m.p. 56–58°C; IR (KBr, cm-1): 3066 (CH aromatic), 2951, 2897 (CH aliphatic), 1762 (C=O), 1597, 1581 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 3.38 (t, J = 4.96 Hz, 4H, 2CH2N of morpholine), 3.92 (t, J = 4.96 Hz, 4H, 2CH2O of morpholine), 7.01 (d, J = 8.76 Hz, 2H, ArH), 7.22 (s, 1H, olefinic H), 7.52–7.58 (m, 2H, ArH), 7.60–7.62 (m, 1H, ArH), 8.17–8.19 (m, 4H, ArH); 13C NMR (CDCl3, 100 MHz): 47.91 (2CH2N, morpholine), 66.37 (2CH2O, morpholine), 114.58, 125.15, 126.03, 128.02, 128.87, 130.30, 132.08, 132.77, 134.49, 152.16, 161.83 (aromatic C), 168.14 (C=O); Anal. calcd. for C20H18N2O3 (334.38): C, 71.84; H, 5.43; N, 8.38. Found: C, 71.98; H, 5.61; N, 8.54.

General procedure for the synthesis of compounds 4a–r

In a dry flask, a mixture of the appropriate oxazolone (3a–c) (0.01 mol), corresponding aniline (0.03 mol) and freshly fused sodium acetate (0.03 mol) was added in the presence of acetic acid (0.05 ml) as catalyst. Then the mixture was fused together at 90°C for 2 h. The obtained product was washed with water and crystallized from ethanol:dimethyl formamide (9:1).

2,3-Diphenyl-5-(4-(pyrrolidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4a)

Orange powder; yield 56%; m.p. 244–246°C; IR (KBr, cm-1): 3051 (CH aromatic), 2912, 2858 (CH aliphatic), 1705 (C=O), 1593, 1527 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 2.06–2.10 (m, 4H, 2CH2 of pyrrolidine), 3.43 (t, J = 6.44 Hz, 4H, 2CH2N of pyrrolidine), 6.64 (d, J = 8.80 Hz, 2H, ArH), 7.21 (d, J = 7.32 Hz, 2H, olefinic H and ArH), 7.29–7.34 (m, 3H, ArH), 7.38–7.45 (m, 4H, ArH), 7.57 (d, J = 7.40 Hz, 2H, ArH), 8.25 (d, J = 8.40 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 20.71 (2CH2, pyrrolidine), 42.91 (2CH2N, pyrrolidine), 107.26, 117.42, 122.67, 123.21, 123.44, 124.26, 124.52, 124.74, 125.77, 126.69, 129.17, 130.39, 130.51, 144.73, 151.88 (aromatic C), 165.60 (C=O); Anal. calcd. for C26H23N3O (393.49): C, 79.36; H, 5.89; N, 10.68. Found: C, 79.50; H, 6.08; N, 10.79.

2-Phenyl-5-(4-(pyrrolidin-1-yl)benzylidene)-3-(p-tolyl)-3,5-dihydro-4H-imidazol-4-one (4b)

Light-brown crystals; yield 60%; m.p. 241–243°C; IR (KBr, cm-1): 3070 (CH aromatic), 2947, 2846 (CH aliphatic), 1708 (C=O), 1589, 1512 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 2.05–2.08 (m, 4H, 2CH2 of pyrrolidine), 2.40 (s, 3H, CH3 of tolyl), 3.42 (t, J = 6.50 Hz, 4H, 2CH2N of pyrrolidine), 6.64 (d, J = 8.80 Hz, 2H, ArH), 7.09 (d, J = 8.20 Hz, 2H, ArH), 7.23 (d, J = 8.08 Hz, 2H, olefinic H and ArH), 7.30–7.33 (m, 3H, ArH), 7.38–7.42 (m, 1H, ArH), 7.58–7.60 (m, 2H, ArH), 8.25 (d, J = 8.44 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 21.23 (CH3, tolyl), 25.46 (2CH2, pyrrolidine), 47.63 (2CH2N, pyrrolidine), 111.97, 122.20, 127.21, 128.16, 129.01, 129.59, 129.96, 130.47, 131.25, 132.63, 134.05, 135.09, 137.94, 149.44, 156.76 (aromatic C), 170.60 (C=O); Anal. calcd. for C27H25N3O (407.52): C, 79.58; H, 6.18; N, 10.31. Found: C, 79.71; H, 6.24; N, 10.57.

3-(4-Methoxyphenyl)-2-phenyl-5-(4-(pyrrolidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4c)

Reddish-brown crystals; yield 66%; m.p. 253–256°C; IR (KBr, cm-1): 3043 (CH aromatic), 2900, 2843 (CH aliphatic), 1701 (C=O), 1589, 1512 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 2.05–2.10 (m, 4H, 2CH2 of pyrrolidine), 3.43 (t, J = 6.50 Hz, 4H, 2CH2N of pyrrolidine), 3.85 (s, 3H, OCH3), 6.64 (d, J = 8.80 Hz, 2H, ArH), 6.95 (d, J = 8.80 Hz, 2H, ArH), 7.13 (d, J = 8.80 Hz, 2H, olefinic H and ArH), 7.30–7.34 (m, 3H, ArH), 7.38–7.42 (m, 1H, ArH), 7.60 (d, J = 7.30 Hz, 2H, ArH), 8.25 (d, J = 8.44 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 25.46 (2CH2 pyrrolidine), 47.62 (2CH2N pyrrolidine), 55.49 (OCH3), 111.97, 114.62, 122.19, 128.01, 128.19, 128.63, 129.01, 129.55, 130.48, 131.26, 134.00, 135.10, 149.45, 156.76, 159.15 (aromatic C), 170.78 (C=O); Anal. calcd. for C27H25N3O2 (423.52): C, 76.57; H, 5.95; N, 9.92. Found: C, 76.70; H, 5.87; N, 10.08.

3-(4-Chlorophenyl)-2-phenyl-5-(4-(pyrrolidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4d)

Reddish-brown crystals; yield 64%; m.p. 266–268°C; IR (KBr, cm-1): 3070, 3035 (CH aromatic), 2904, 2854 (CH aliphatic), 1701 (C=O), 1589, 1519 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 2.03–2.10 (m, 4H, 2CH2 of pyrrolidine), 3.43 (t, J = 6.50 Hz, 4H, 2CH2N of pyrrolidine), 6.64 (d, J = 8.80 Hz, 2H, ArH), 7.15 (d, J = 8.60 Hz, 2H, olefinic H and ArH), 7.33–7.43 (m, 6H, ArH), 7.55–7.57 (m, 2H, ArH), 8.25 (d, J = 8.44 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 25.45 (2CH2 pyrrolidine), 47.67 (2CH2N pyrrolidine), 112.05, 122.05, 128.35, 128.54, 128.96, 129.26, 129.46, 130.67, 131.91, 133.59, 133.67, 133.78, 135.26, 149.59, 156.02 (aromatic C), 170.07 (C=O); EIMS, m/z: 427.59 (M+); Anal. calcd. for C26H22ClN3O (427.93): C, 72.98; H, 5.18; N, 9.82. Found: C, 73.14; H, 5.35; N, 10.03.

3-(4-Bromophenyl)-2-phenyl-5-(4-(pyrrolidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4e)

Reddish-brown crystals; yield 60%; m.p. 263–265°C; IR (KBr, cm-1): 3051 (CH aromatic), 2954, 2858 (CH aliphatic), 1701 (C=O), 1589, 1527 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 2.06–2.10 (m, 4H, 2CH2 of pyrrolidine), 3.43 (t, J = 6.50 Hz, 4H, 2CH2N of pyrrolidine), 6.64 (d, J = 8.80 Hz, 2H, ArH), 7.08 (d, J = 8.60 Hz, 2H, olefinic H and ArH), 7.33–7.37 (m, 3H, ArH), 7.41–7.45 (m, 1H, ArH), 7.53–7.57 (m, 4H, ArH), 8.24 (d, J = 8.44 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 25.45 (2CH2 pyrrolidine), 47.68 (2CH2N pyrrolidine), 112.05, 121.68, 122.06, 128.36, 128.83, 128.96, 129.24, 130.68, 131.92, 132.43, 133.59, 134.29, 135.26, 149.59, 155.97 (aromatic C), 169.98 (C=O); Anal. calcd. for C26H22BrN3O (472.39): C, 66.11; H, 4.69; N, 8.90. Found: C, 66.35; H, 4.76; N, 9.14.

4-(5-Oxo-2-phenyl-4-(4-(pyrrolidin-1-yl)benzylidene)-4,5-dihydro-1H-imidazol-1-yl)benzene sulfonamide (4f)

Reddish-brown powder; yield 40%; m.p. 290–293°C; IR (KBr, cm-1): 3348, 3240 (NH2), 3089, 3059 (CH aromatic), 2916, 2858 (CH aliphatic), 1701 (C=O), 1593 (NH bending), 1527 (C=C aromatic); 1H NMR (DMSO, 400 MHz): 1.99 (t, J = 6.16 Hz, 4H, 2CH2 of pyrrolidine), 3.37 (t, J = 6.20 Hz, 4H, 2CH2N of pyrrolidine), 6.67 (d, J = 8.90 Hz, 2H, ArH), 7.21 (s, 1H, olefinic H), 7.39–7.50 (m, 9H, NH2SO2, D2O exch and ArH), 7.88 (d, J = 8.50 Hz, 2H, ArH), 8.24 (d, J = 8.28 Hz, 2H, ArH); 13C NMR (DMSO, 100 MHz): 25.38 (2CH2 pyrrolidine), 47.79 (2CH2N pyrrolidine), 112.50, 121.56, 127.07, 128.45, 128.94, 129.06, 129.45, 131.04, 131.29, 133.40, 135.38, 138.25, 143.71, 149.88, 156.22 (aromatic C), 169.38 (C=O); Anal. calcd. for C26H24N4O3S (472.56): C, 66.08; H, 5.12; N, 11.86. Found: C, 66.21; H, 5.31; N, 12.09.

2,3-Diphenyl-5-(4-(piperidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4g)

Orange powder; yield 62%; m.p. 183–185°C; IR (KBr, cm-1): 3051, 3061 (CH aromatic), 2939, 2831 (CH aliphatic), 1705 (C=O), 1593, 1516 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 1.61–1.76 (m, 6H, 3CH2 of piperidine), 3.42 (s, 4H, 2CH2N of piperidine), 7.02 (s, 2H, ArH), 7.20–7.22 (m, 2H, olefinic H and ArH), 7.31–7.34 (m, 3H, ArH), 7.39–7.45 (m, 4H, ArH), 7.56–7.58 (m, 2H, ArH), 8.25 (d, J = 8.00 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 24.22 (CH2, piperidine), 25.21 (2CH2, piperidine), 49.04 (2CH2N, piperidine), 114.68, 127.39, 128.09, 128.24, 129.08, 129.32, 130.80, 134.70, 135.09 (aromatic C), 170.45 (C=O); Anal. calcd. for C27H25N3O (407.52): C, 79.58; H, 6.18; N, 10.31. Found: C, 79.40; H, 6.23; N, 10.48.

2-Phenyl-5-(4-(piperidin-1-yl)benzylidene)-3-(p-tolyl)-3,5-dihydro-4H-imidazol-4-one (4h)

Brown crystals; yield 58%; m.p. 258–260°C; IR (KBr, cm-1): 3078 (CH aromatic), 2927 (CH aliphatic), 1708 (C=O), 1589 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 1.70 (s, 6H, 3CH2 of piperidine), 2.40 (s, 3H, CH3), 3.41 (s, 4H, 2CH2N of piperidine), 6.98 (s, 2H, ArH), 7.08 (d, J = 8.24 Hz, 2H, ArH), 7.22–7.34 (m, 5H, olefinic H and ArH), 7.40–7.43 (m, 1H, ArH), 7.58–7.61 (m, 2H, ArH), 8.24 (d, J = 8.40 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 21.23 (CH3 tolyl), 24.22 (CH2, piperidine), 25.24 (2CH2, piperidine), 48.95 (2CH2N, piperidine), 114.60, 127.18, 128.21, 129.08, 130.00, 130.74, 132.45, 134.65, 138.09 (aromatic C), 170.69 (C=O); EIMS, m/z: 421.99 (M+); Anal. calcd. for C28H27N3O (421.54): C, 79.78; H, 6.46; N, 9.97. Found: C, 79.85; H, 6.34; N, 10.15.

3-(4-methoxyphenyl)-2-phenyl-5-(4-(piperidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4i)

Orange powder; yield 59%; m.p. 248–250°C; IR (KBr, cm-1): 3043, 3001 (CH aromatic), 2927, 2904 (CH aliphatic), 1705 (C=O), 1593, 1512 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 1.70 (s, 6H, 3CH2 of piperidine), 3.41 (s, 4H, 2CH2N of piperidine), 3.85 (s, 3H, OCH3), 6.95 (d, J = 8.80 Hz, 4H, ArH), 7.13 (d, J = 8.80 Hz, 2H, olefinic H and ArH), 7.30–7.35 (m, 3H, ArH), 7.40–7.43 (m, 1H, ArH), 7.60 (d, J = 7.20 Hz, 2H, ArH), 8.24 (d, J = 8.52 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 24.24 (CH2, piperidine), 25.24 (2CH2, piperidine), 49.06 (2CH2N, piperidine), 55.49 (OCH3), 114.66, 127.82, 128.24, 128.61, 129.08, 129.36, 130.75, 134.66, 159.23 (aromatic C), 170.87 (C=O); Anal. calcd. for C28H27N3O2 (437.54): C, 76.86; H, 6.22; N, 9.60. Found: C, 77.05; H, 6.41; N, 9.87.

3-(4-Chlorophenyl)-2-phenyl-5-(4-(piperidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4j)

Brown crystals; yield 50%; m.p. 243–245°C; IR (KBr, cm-1): 3051 (CH aromatic), 2943, 2927 (CH aliphatic), 1705 (C=O), 1589 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 1.70 (s, 6H, 3CH2 of piperidine), 3.42 (s, 4H, 2CH2N of piperidine), 6.97 (s, 2H, ArH), 7.14 (d, J = 8.60 Hz, 2H, olefinic H and ArH), 7.31–7.46 (m, 6H, ArH), 7.56 (d, J = 7.20 Hz, 2H, ArH), 8.23 (d, J = 8.64 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 24.42 (CH2, piperidine), 25.42 (2CH2N, piperidine), 48.61 (2CH2N, piperidine), 114.20, 123.64, 128.37, 128.51, 129.00, 129.16, 129.50, 130.84, 131.09, 133.66, 133.77, 134.72, 134.90, 152.77, 156.87 (aromatic C), 170.19 (C=O); Anal. calcd. for C27H24ClN3O (441.96): C, 73.38; H, 5.47; N, 9.51. Found: C, 73.66; H, 5.60; N, 9.62.

3-(4-Bromophenyl)-2-phenyl-5-(4-(piperidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one (4k)

Brown crystals; yield 56%; m.p. 254–256°C; IR (KBr, cm-1): 3051 (CH aromatic), 2927, 2846 (CH aliphatic), 1705 (C=O), 1581 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 1.70–1.74 (m, 6H, 3CH2 of piperidine), 3.42 (s, 4H, 2CH2N of piperidine), 7.00 (s, 2H, ArH), 7.08 (d, J = 8.60 Hz, 2H, olefinic H and ArH), 7.31–7.38 (m, 3H, ArH), 7.43–7.46 (m, 1H, ArH), 7.54–7.57 (m, 4H, ArH), 8.23 (d, J = 8.48 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 24.29 (CH2, piperidine), 25.28 (2CH2, piperidine), 48.87 (2CH2N, piperidine), 114.48, 121.81, 128.40, 128.81, 129.02, 130.92, 132.47, 134.13, 134.86 (aromatic C), 170.09 (C=O); Anal. calcd. for C27H24BrN3O (486.41): C, 66.67; H, 4.97; N, 8.64. Found: C, 66.89; H, 5.15; N, 8.79.

4-(5-Oxo-2-phenyl-4-(4-(piperidin-1-yl)benzylidene)-4,5-dihydro-1H-imidazol-1-yl)benzene sulfonamide (4l)

Reddish-brown powder; yield 40%; m.p. 268–270°C; IR (KBr, cm-1): 3356, 3236 (NH2), 3093, 3062 (CH aromatic), 2939, 2835 (CH aliphatic), 1708 (C=O), 1635 (NH bending), 1593, 1516 (C=C aromatic); 1H NMR (DMSO, 400 MHz): 1.61 (s, 6H, 3CH2 of piperidine), 3.42 (s, 4H, 2CH2N of piperidine), 7.03 (d, J = 9.04 Hz, 2H, ArH), 7.20 (s, 1H, olefinic H), 7.40–7.45 (m, 4H, ArH), 7.47–7.50 (m, 5H, NH2SO2, D2O exch and ArH), 7.87 (d, J = 8.50 Hz, 2H, ArH), 8.22 (d, J = 8.80 Hz, 2H, ArH); 13C NMR (DMSO, 100 MHz): 24.45 (CH2, piperidine), 25.42 (2CH2, piperidine), 48.15 (2CH2N, piperidine), 114.23, 122.98, 127.08, 128.49, 128.96, 129.13, 129.35, 130.24, 131.45, 134.42, 135.16, 138.12, 143.78, 152.72, 157.10 (aromatic C), 169.49 (C=O); Anal. calcd. for C27H26N4O3S (486.59): C, 66.65; H, 5.39; N, 11.51. Found: C, 66.80; H, 5.18; N, 11.43.

5-(4-Morpholinobenzylidene)-2,3-diphenyl-3,5-dihydro-4H-imidazol-4-one (4m)

Orange powder; yield 50%; m.p. 218–220°C; IR (KBr, cm-1): 3055, 3035 (CH aromatic), 2958, 2889 (CH aliphatic), 1708 (C=O), 1597, 1512 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 3.35 (t, J = 4.80 Hz, 4H, 2CH2N of morpholine), 3.89 (t, J = 4.96 Hz, 4H, 2CH2O of morpholine), 6.96 (d, J = 8.92 Hz, 2H, ArH), 7.19–7.22 (m, 2H, olefinic H and ArH), 7.30–7.34 (m, 3H, ArH), 7.39–7.46 (m, 4H, ArH), 7.56–7.58 (m, 2H, ArH), 8.26 (d, J = 8.80 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 47.64 (2CH2N morpholine), 66.62 (2CH2O morpholine), 114.26, 125.40, 127.37, 128.14, 128.26, 129.10, 129.24, 129.34, 130.00, 130.90, 134.54, 135.00, 135.93, 152.40, 158.30 (aromatic C), 170.49 (C=O); Anal. calcd. for C26H23N3O2 (409.49): C, 76.26; H, 5.66; N, 10.26. Found: C, 75.98; H, 5.74; N, 10.48.

5-(4-Morpholinobenzylidene)-2-phenyl-3-(p-tolyl)-3,5-dihydro-4H-imidazol-4-one (4n)

Brown crystals; yield 70%; m.p. 238–240°C; IR (KBr, cm-1): 3082 (CH aromatic), 2920, 2889 (CH aliphatic), 1708 (C=O), 1589, 1512 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 2.40 (s, 3H, CH3), 3.34 (t, J = 4.80 Hz, 4H, 2CH2N of morpholine), 3.89 (t, J = 4.90 Hz, 4H, 2CH2O of morpholine), 6.97 (d, J = 8.88 Hz, 2H, ArH), 7.08 (d, J = 8.16 Hz, 2H, ArH), 7.23 (d, J = 8.08 Hz, 2H, olefinic H and ArH), 7.31–7.35 (m, 3H, ArH), 7.41–7.44 (m, 1H, ArH), 7.60 (d, J = 7.32 Hz, 2H, ArH), 8.26 (d, J = 8.80 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 16.50 (CH3), 42.97 (2CH2N morpholine), 61.84 (2CH2O morpholine), 109.59, 120.80, 122.42, 123.50, 124.37, 124.56, 124.99, 125.28, 126.12, 127.61, 129.75, 131.30, 133.40, 147.51, 153.71 (aromatic C), 165.93 (C=O); Anal. calcd. for C27H25N3O2 (423.52): C, 76.57; H, 5.95; N, 9.92. Found: C, 76.71; H, 6.12; N, 10.15.

3-(4-Methoxyphenyl)-5-(4-morpholinobenzylidene)-2-phenyl-3,5-dihydro-4H-imidazol-4-one (4o)

Dark-orange powder; yield 73%; m.p. 240–242°C; IR (KBr, cm-1): 3047 (CH aromatic), 2900, 2866 (CH aliphatic), 1708 (C=O), 1589, 1512 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 3.34 (t, J = 4.80 Hz, 4H, 2CH2N of morpholine), 3.85 (s, 3H, OCH3), 3.90 (t, J = 4.90 Hz, 4H, 2CH2O of morpholine), 6.96 (t, J = 8.96 Hz, 4H, ArH), 7.12 (d, J = 8.84 Hz, 2H, olefinic H and ArH), 7.30–7.35 (m, 3H, ArH), 7.41–7.44 (m, 1H, ArH), 7.61 (d, J = 7.32 Hz, 2H, ArH), 8.26 (d, J = 8.84 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 43.04 (2CH2N morpholine), 50.75 (OCH3), 61.81 (2CH2O morpholine), 109.66, 109.93, 120.91, 122.94, 123.52, 123.84, 124.37, 124.51, 124.93, 126.14, 129.75, 131.28, 147.41, 153.76, 154.52 (aromatic C), 166.10 (C=O); Anal. calcd. for C27H25N3O3 (439.52): C, 73.79; H, 5.73; N, 9.56. Found: C, 74.02; H, 5.89; N, 9.72.

3-(4-Chlorophenyl)-5-(4-morpholinobenzylidene)-2-phenyl-3,5-dihydro-4H-imidazol-4-one (4p)

Brown crystals; yield 50%; m.p. 256–258°C; IR (KBr, cm-1): 3078 (CH aromatic), 2962, 2889 (CH aliphatic), 1708 (C=O), 1589, 1516 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 3.36 (t, J = 4.72 Hz, 4H, 2CH2N of morpholine), 3.91 (t, J = 4.92 Hz, 4H, 2CH2O of morpholine), 6.99 (d, J = 8.76 Hz, 2H, ArH), 7.14 (d, J = 8.60 Hz, 2H, ArH), 7.31 (s, 1H, olefinic H), 7.34–7.47 (m, 5H, ArH), 7.57 (d, J = 7.36 Hz, 2H, ArH), 8.26 (d, J = 8.76 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 47.74 (2CH2N morpholine), 66.53 (2CH2O morpholine), 114.39, 125.46, 128.43, 128.50, 128.97, 129.06, 129.54, 130.32, 131.08, 133.50, 133.87, 134.64, 135.67, 152.25, 157.81 (aromatic C), 170.14 (C=O); Anal. calcd. for C26H22ClN3O2 (443.93): C, 70.35; H, 5.00; N, 9.47. Found: C, 70.61; H, 5.12; N, 9.39.

3-(4-Bromophenyl)-5-(4-morpholinobenzylidene)-2-phenyl-3,5-dihydro-4H-imidazol-4-one (4q)

Brown crystals; yield 60%; m.p. 253–255°C; IR (KBr, cm-1): 3097, 3078 (CH aromatic), 2889, 2846 (CH aliphatic), 1708 (C=O), 1585, 1516 (C=C aromatic); 1H NMR (CDCl3, 400 MHz): 3.35 (t, J = 4.80 Hz, 4H, 2CH2N of morpholine), 3.89 (t, J = 4.90 Hz, 4H, 2CH2O of morpholine), 6.95 (d, J = 8.92 Hz, 2H, ArH), 7.07 (d, J = 8.60 Hz, 2H, ArH), 7.31 (s, 1H, olefinic H), 7.34–7.47 (m, 2H, ArH), 7.34–7.38 (m, 1H, ArH), 7.54–7.57 (m, 4H, ArH), 8.25 (d, J = 8.84 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 42.85 (2CH2N morpholine), 61.85 (2CH2O morpholine), 109.49, 117.13, 120.49, 123.69, 124.04, 124.23, 124.31, 125.71, 126.31, 127.75, 129.27, 129.91, 130.83, 147.71, 152.92 (aromatic C), 165.34 (C=O); Anal. calcd. for C26H22BrN3O2 (488.39): C, 63.94; H, 4.54; N, 8.60. Found: C, 63.85; H, 4.70; N, 8.84.

4-(4-(4-Morpholinobenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl)benzene sulfonamide (4r)

Orange crystals; yield 38%; m.p. 248–250°C; IR (KBr, cm-1): 3309, 3213 (NH2), 3074, 3047 (CH aromatic), 2889, 2854 (CH aliphatic), 1693 (C=O), 1639 (NH bending), 1593, 1516 (C=C aromatic); 1H NMR (DMSO, 400 MHz): 3.34 (t, J = 4.80 Hz, 4H, 2CH2N of morpholine), 3.75 (t, J = 5.08 Hz, 4H, 2CH2O of morpholine), 7.07 (d, J = 9.04 Hz, 2H ArH), 7.23 (s, 1H, olefinic H), 7.40–7.49 (m, 5H, NH2SO2, D2O exch and ArH), 7.50–7.51 (m, 4H, ArH), 7.88 (d, J = 8.50 Hz, 2H, ArH), 8.24 (d, J = 8.80 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz): 47.17 (2CH2N morpholine), 66.32 (2CH2O morpholine), 114.28, 124.32, 127.10, 128.51, 128.98, 129.18, 129.25, 129.92, 131.59, 134.86, 135.14, 138.03, 143.84, 152.84, 157.80 (aromatic C), 169.56 (C=O); Anal. calcd. for C26H24N4O4S (488.56): C, 63.92; H, 4.95; N, 11.47. Found: C, 64.14; H, 5.13; N, 11.69.

Biological activity

In vivo screening

All pharmacological in vivo procedures were carried out depending on the standard guidelines. Handling and experimentation were carried out according to the international ethical guidelines concerning the care and use of laboratory animals and approved by the Research Ethics Committee, Faculty of Pharmacy, Cairo University, Egypt (PC: 3337).

Anti-inflammatory activity

The in vivo anti-inflammatory activity of all the targeted compounds 3a–c and 4a–r and celecoxib as a reference standard was determined using the carrageenan-induced rat paw edema method reported by Winter et al. [46–48] (Supplementary S6.2.1.1).

Analgesic activity

The analgesic activity of all the targeted compounds 3a–c and 4a–r and celecoxib as a reference standard was determined using the standardized method of acetic acid-induced abdominal writhing in mice [47–49] (Supplementary S6.2.1.2).

Toxicological studies

Toxicological studies were performed for the most active compounds 3a, 4a, 4c, 4e and 4f using Finney’s method [50] (Supplementary S6.2.1.3).

Ulcerogenic effect

The ulcerogenic effects of the most active anti-inflammatory pyrrolidine derivatives 3a, 4a, 4c, 4e and 4f were investigated in comparison with the reference standard, celecoxib, by the standard method [51], and the ulcer index was calculated according to the standard method [52] (Supplementary S6.2.1.4).

Histopathological examination

Microscopical examination of rat gastric mucosa from the fundic region was carried out to evaluate the severity of ulcers induced by the most active compounds (3a, 4a, 4b, 4e and 4f) in comparison with the severity produced by the reference standard, celecoxib [53,54] (Supplementary S6.2.1.5).

In vitro COX enzyme inhibition assay

The compounds 4c, 4e and 4f and the reference standard celecoxib were examined for their ability to inhibit COX-1 and COX-2 (Supplementary S6.2.2).

Molecular docking simulation

The molecular docking simulation was performed using the open-source PyRx (https://pyrx.sourceforge.io/) embedded Vina tool. Both x-ray protein crystals of COX-1 and COX-2 were obtained from the Protein Data Bank (PDB) [55] as PDB IDs 3KK6 [56] and 3LN1 [57], respectively. The best-quality chains of the corresponding protein structures were prepared using the embedded DockPrep tool of Chimera v.17.1 then saved in .pdb format. This preparation tool includes removal of unnecessary water, ions and the cocrystallized ligand followed by the addition of hydrogen atoms and assigning charges to protein atoms. The tested derivatives were drawn and energy minimized using OSIRIS DataWarrior, then saved in .sdf format (https://openmolecules.org/datawarrior/). The docking simulation utilized a grid box that contained the cocrystallized celecoxib binding site using the default MMFF94x force field. For COX-1, the grid box dimensions were x = 10.942 Å, y = 18.415 Å and z = 19.387 Å, while for COX-2 the dimensions were x = 14.046 Å, y = 11.020 Å and z = 12.122 Å. The resulting binding energy was recorded as docking score function. The output binding modes of the tested derivatives were visualized by BIOVIA Discovery Studio Visualizer 2021 (https://discover.3ds.com/discovery-studio-visualizer-download).

Results & discussion

Chemistry

The synthetic pathway of the target compounds is illustrated in Figure 2. The hippuric acid (1) was prepared according to the reported procedure [45]. Different aldehydes (2a–c) were prepared through the reflux of 4-fluorobenzaldehyde and appropriate secondary amine (piperidine, pyrrolidine or morpholine) in dimethyl formamide in the presence of potassium carbonate [58]. Refluxing benzoyl glycine with different substituted aldehydes in acetic anhydride in the presence of sodium acetate provided oxazolone derivatives 3a–c (Figure 2A) [39,45 59,60].

Figure 2.

Figure 2.

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Synthetic pathways for the targeted compounds.

(A) Synthesis of target compounds 3a–c. Reagents and reaction conditions: (i) acetic anhydride, sodium acetate, reflux 6 h. (B) Synthesis of target compounds 4a–r. Reagents and reaction conditions: (i) acetic acid, sodium acetate, fusion 2 h.

Fusion of the prepared oxazolones (3a–c) with the appropriate anilines in the presence of acetic acid and sodium acetate afforded imidazolone derivatives 4a–r. (Figure 2B).

The new synthesized derivatives were confirmed by spectral and elemental analysis as described in detail above.

Biological activity

In vivo screening

Anti-inflammatory activity

The in vivo anti-inflammatory activity of all new compounds (3a–c and 4a–r) and celecoxib as reference standard was screened using a carrageenan-induced rat paw edema assay according to the reported procedure [46]. This technique is used to evaluate the ability of the tested compounds to reduce the edema produced in the rat paw after being injected with carrageenan. The results (Table 1) showed that all the tested compounds exhibited promising anti-inflammatory activity, with up to 90.08% inhibition.

Table 1.

In vivo anti-inflammatory effect of celecoxib and the newly synthesized compounds on carrageenan-induced edema of the hind paw in rats (n = 5) at 0.028 mmol/kg.

Compound No. Edema (mm) ± SEM (% inhibition) % potency‡
1 h 2 h 3 h 4 h
Control 1.71 ± 0.02 1.81 ± 0.07 1.98 ± 0.28 1.86 ± 0.16 0
Celecoxib 1.28 ± 0.15† (25.15%) 0.83 ± 0.15† (54.14%) 0.77 ± 0.16† (61.11%) 1.06 ± 0.09† (43.01%) 100
3a 0.83 ± 0.11† (51.46%) 0.27 ± 0.10† (85.08%) 0.39 ± 0.19† (80.30%) 0.36 ± 0.03† (80.64%) 131
3b 0.73 ± 0.08† (57.31%) 0.82 ± 0.81† (54.69%) 1.04 ± 0.39† (47.47%) 0.65 ± 0.39† (65.05%) 77
3c 0.85 ± 0.19† (50.29%) 0.42 ± 0.09† (76.79%) 0.51 ± 0.10† (74.24%) 0.69 ± 0.05† (62.90%) 121
4a 1.02 ± 0.12† (40.35%) 0.73 ± 0.22† (59.67%) 0.24 ± 0.08† (87.88%) 0.34 ± 0.09† (81.72%) 143
4b 0.67 ± 0.08† (60.82%) 0.98 ± 0.04† (45.86%) 0.54 ± 0.08† (72.73%) 0.55 ± 0.06† (70.43%) 119
4c 0.13 ± 0.03† (92.39%) 0.27 ± 0.09† (85.08%) 0.19 ± 0.03† (90.40%) 0.21 ± 0.07† (88.71%) 147
4d 0.89 ± 0.04† (47.95%) 0.86 ± 0.25† (52.49%) 0.56 ± 0.21† (71.72%) 0.50 ± 0.02† (73.12%) 117
4e 0.58 ± 0.09† (66.08%) 0.80 ± 0.09† (55.80%) 0.26 ± 0.07† (86.87%) 0.63 ± 0.07† (66.13%) 142
4f 0.30 ± 0.08† (82.46%) 0.50 ± 0.13† (72.37%) 0.20 ± 0.06† (89.89%) 0.35 ± 0.04† (81.18%) 147
4g 0.36 ± 0.18† (78.95%) 0.88 ± 0.21† (51.38%) 1.04 ± 0.20† (47.47%) 0.97 ± 0.17† (47.85%) 77
4h 0.47 ± 0.17† (72.51%) 0.79 ± 0.14† (56.35%) 0.72 ± 0.21† (63.64%) 0.66 ± 0.07† (64.52%) 104
4i 1.13 ± 0.06† (33.92%) 1.72 ± 0.06† (4.97%) 0.81 ± 0.30† (59.09%) 0.61 ± 0.29† (67.20%) 96
4j 0.53 ± 0.08† (69.00%) 1.08 ± 0.14† (40.33%) 0.86 ± 0.09† (56.56%) 1.55 ± 0.09† (16.66%) 92
4k 1.17 ± 0.11† (31.58%) 1.16 ± 0.17† (35.91%) 0.82 ± 0.23† (58.58%) 1.15 ± 0.30† (38.17%) 95
4l 0.21 ± 0.06† (87.72%) 0.30 ± 0.08† (83.42%) 0.56 ± 0.07† (71.72%) 0.52 ± 0.03† (72.04%) 117
4m 0.29 ± 0.17† (83.04%) 1.23 ± 0.13† (32.04%) 0.92 ± 0.18† (53.53%) 0.79 ± 0.11† (57.53%) 87
4n 0.51 ± 0.19† (70.17%) 1.26 ± 0.20† (30.39%) 1.35 ± 0.20† (31.82%) 1.03 ± 0.17† (44.62%) 52
4o 1.09 ± 0.13† (36.26%) 1.28 ± 0.28† (29.28%) 1.44 ± 0.31† (27.27%) 1.36 ± 0.15† (26.88%) 44
4p 0.73 ± 0.20† (57.31%) 1.01 ± 0.07† (44.19%) 1.38 ± 0.03† (30.30%) 1.03 ± 0.09† (44.62%) 49
4q 0.75 ± 0.14† (56.14%) 0.85 ± 0.08† (53.04%) 0.95 ± 0.04† (52.02%) 0.59 ± 0.09† (68.28%) 85
4r 0.40 ± 0.14† (76.61%) 0.52 ± 0.17† (71.27%) 0.60 ± 0.30† (69.69%) 1.11 ± 0.17† (40.32%) 114
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Statistical analysis was carried out by one-way analysis of variance.

†

Significant difference from the control value at p < 0.05.

‡

% Potency = % edema thickness inhibition relative to that of the standard reference (celecoxib) at 3 h.

SEM: Standard error of the mean.

Analgesic activity

All the new target compounds 3a–c and 4a–r were screened for their analgesic activity using the standard method of acetic acid-induced abdominal writhing in mice [49]. As shown in Supplementary Table 2, all the newly synthesized compounds not only had promising anti-inflammatory activity but also exhibited promising analgesic activity, higher than that of celecoxib. Their analgesic activity ranged between 26.12 and 100% protection. Compounds containing a pyrrolidine and piperidine ring showed high protection, especially the pyrrolidine phenyl bromo derivative 4d, which exhibited 100% protection. The morpholine-containing derivatives showed low, moderate or high analgesic activity, while compound 4r carrying a benzene sulfonamide moiety exhibited 100% protection.

Toxicological studies

Toxicological study of the most active anti-inflammatory pyrrolidine derivatives 3a, 4a, 4c, 4e and 4f was performed using a standardized method [50]. All tested compounds revealed a high safety margin with no animal death after intraperitoneal injection of doses up to 0.28 mmol/kg body weight (i.e., up to tenfold of the used anti-inflammatory dose) within 24 h of observation.

Ulcerogenic effect

The ulcerogenic effects of the most active anti-inflammatory pyrrolidine derivatives 3a, 4a, 4c, 4e and 4f were investigated in comparison with the reference standard, celecoxib, by the standard method [51], and the ulcer index was calculated according to the standard method [52]. As presented in Supplementary Table 3, it was noted that all the tested compounds exhibited a remarkable gastrointestinal tolerance compared with celecoxib. The pyrrolidine derivatives bearing methoxy phenyl 4c and benzene sulfonamide 4f were the best, with an ulcer index of 8.20.

Histopathological examination

Microscopic examination of rat gastric mucosa from the fundic region was carried out to evaluate the severity of ulcers induced by the most active compounds (3a, 4a, 4b, 4e and 4f) in comparison with the severity produced by the reference standard, celecoxib.

Stomach tissue sections from control animals showed normal architecture of the fundic region lined by pale mucosal cells with basal oval nuclei. The fundic glands were lined with mucous neck cells, oxyntic cells and peptic cells and they opened to the surface in gastric pits. The mucous neck cells were simple columnar epithelium with pale staining. The oxyntic cells were large and rounded with extensive acidophilic cytoplasm and central rounded nuclei. The peptic cells had basophilic cytoplasm; their nuclei were basal and rounded (score 0) (Figure 3A).

Figure 3.

Figure 3.

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Effect of treatment with different compounds on histological alterations of the gastric mucosal tissue.

Photomicrographs of hematoxylin and eosin-stained sections of stomach from control group (A), celecoxib-treated group (B and C), 3a-treated group (D), 4a-treated group (E), 4c-treated group (F), 4e-treated group (G), and 4f-treated group (H); inside box 200×. (A) Intact mucosal epithelial lining and gastric glands (black arrow). (B) Necrosis of surface epithelium and swelling of glandular epithelial cells (red arrow). (C) Submucosal edema, congestion and leukocytic infiltration (dark red arrow). (D) Desquamation of surface epithelial lining with intact subepithelial glands (brown arrow). (E) Marked submucosal edema (purple arrow). (F & G) Mild sloughing of surface epithelium with slight dilatation of the gastric glands (yellow arrows for G) and (green arrows for F). (H) Mild dilatation of the gastric glands and few mononuclear inflammatory cells infiltrating the submucosal layer (blue arrow). Hematoxylin and eosin, ×200.

Stomach tissue sections of rats treated with the reference drug celecoxib showed necrosis of the surface epithelium and swelling of glandular epithelial cells, especially mucous neck cells, oxyntic cells and peptic cells (score 3). Mononuclear inflammatory cell infiltration was seen in the lamina propria and submucosa. Submucosal edema and congestion were also seen (Figure 3B and C).

Similarly, gastric mucosa of animals treated with 3a or 4a showed desquamation of the surface epithelial lining with intact subepithelial glands score. Marked submucosal edema was prominent in animals treated with 4a. Low numbers of infiltrating leukocytes were seen in the submucosal layer (score 2) (Figure 3D and E).

Conversely, in animals treated with 4e or 4c, the gastric mucosa revealed mild sloughing of surface epithelium with slight dilatation of the gastric glands. The submucosa showed mild edema and few infiltrating mononuclear inflammatory cells (score 1) (Figure 3F and G). Animals treated with 4f revealed mild improvement of the gastric mucosa in comparison with the reference drug. Mild exfoliation of epithelial surface gastric mucosa was seen. Infiltration of leukocytes (mainly lymphocytes and macrophages) in the mucosal and submucosal layers was seen (score 2). Dilatation of the gastric glands and mild submucosal edema were also noticed (Figure 3H). The scores of these histological changes are recorded in Supplementary Table 4.

Structure–activity relationships

A brief study of the structure–activity relationships of the newly synthesized compounds mainly at 3 h (the time at which the reference compound, celecoxib, exhibited its maximum anti-inflammatory activity) revealed the following. The presence of a pyrrolidine ring is preferable for activity. The oxazolone 3a and the imidazolones (4a–f) that carried a pyrrolidine ring showed the best anti-inflammatory activity, ranging between 71.72 and 90.40%. Regarding the imidazolones, it was observed that the high anti-inflammatory activity of the pyrrolidine derivatives 4f, 4l and 4r was related to the presence of a benzene sulfonamide moiety, with inhibition of 89.89, 71.72 and 69.69%, respectively. Also, the presence of an electron-donating methoxy group in the pyrrolidine derivative 4c revealed the most promising anti-inflammatory activity (90.40% inhibition) compared with other substituted and unsubstituted derivatives, while the presence of the electron-donating groups like methyl and methoxy groups in the piperidine derivatives 4h and 4i was more favorable for activity (63.64 and 59.09% inhibition, respectively) than that of electron-withdrawing groups like chloro (4j) and bromo (4k) (56.56 and 58.58% inhibition, respectively). On the other hand, the unsubstituted morpholine derivative 4m exhibited higher anti-inflammatory activity (53.53% inhibition) than morpholine derivatives with electron-donating (4n and 4o) and electron-withdrawing substituents (4p and 4q), with anti-inflammatory activity of 31.82, 27.27, 30.30 and 52.02% inhibition, respectively.

In vitro COX enzyme inhibition assay

The compounds 4c, 4e and 4f were chosen from the most active anti-inflammatory compounds to examine their ability to inhibit COX-1 and COX-2, using celecoxib as a reference standard. The compounds’ IC50 values for COX-1 and COX-2, and their IC50 ratio and SI, were determined and are listed in Table 2. The results revealed that the tested compounds showed promising potency as COX inhibitors compared with celecoxib. Compound 4c exhibited potent COX-2 inhibitory activity with an IC50 of 0.05 μM, higher than that of the reference celecoxib and relative to other tested compounds. All the tested compounds showed a good COX-2 selectivity (SI range of 1.64–2.08) in comparison with the reference celecoxib (SI = 1.98).

Table 2.

IC50 values for COX-1 and COX-2 and selectivity indices of compounds 4c, 4e, 4f and celecoxib.

Compound IC50 (μM)† Selectivity index‡ Ref.
COX-1 COX-2  
Celecoxib 0.188 0.095 1.98 [31]
4c 0.10 0.05 2.00  
4e 0.52 0.25 2.08  
4f 0.79 0.48 1.64  
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†

IC50 is the concentration needed to cause 50% inhibition of COX-1 and COX-2 enzymatic activity.

‡

Selectivity index = COX-1 IC50/COX-2 IC50.

Molecular docking simulation of COX-1 & COX-2

To articulate the achieved IC50 values of both COX-1 and COX-2, molecular docking simulation was performed for the most potent derivatives 4c, 4e and 4f. Before proceeding to the actual simulation, the docking protocol was validated through redocking the cocrystallized celecoxib to both isoenzyme active sites, demonstrating root mean square deviations of 0.77 and 0.44 Å for COX-1 and COX-2, respectively, as calculate by DockRMSD webserver [61] (Supplementary Figure 1A & B). The achieved docking results are summarized in Supplementary Table 1 and the predicted binding modes of the active derivatives are presented in Figures 4–6.

Figure 4.

Figure 4.

Open in a new tab

The predicted 2D- and 3D-binding mode of 4c with COX-1 and COX-2.

(A–D) The predicted 2D and 3D binding mode of 4c, illustrated as a green stick model, to (A and B) COX-1 and (C and D) COX-2 using PDB IDs 3KK6 and 3LN1, respectively. (E) Superimposed conformation of 4c and the yellow stick model of celecoxib.

PDB: Protein Data Bank.

Figure 5.

Figure 5.

Open in a new tab

The predicted 2D and 3D binding mode of 4e, illustrated as a green stick model.

(A & B) COX-1 and (C & D) COX-2 using PDB IDs 3KK6 and 3LN1, respectively.

PDB: Protein Data Bank.

Figure 6.

Figure 6.

Open in a new tab

The predicted 2D binding mode of 4f to COX-1 and COX-2.

(A) COX-1 and (B) COX-2 using PDB IDs 3KK6 and 3LN1, respectively.

PDB: Protein Data Bank.

It was observed from the docked conformations that the main 2-phenyl-5-(4-(pyrrolidin-1-yl)benzylidene)-3,5-dihydro-4H-imidazol-4-one scaffold of the tested derivatives showed a very similar binding pattern with the main hydrophobic channel residues of both isoenzymes. For instance, several alkyl and π–alkyl interactions were observed between the common scaffold and COX-1 Tyr355, Gly526 and Leu384 as well as COX-2 Leu338, Ala502 and Val509 (Supplementary Table 1) [62]. Nevertheless, the difference in the side chain phenyl substitution of 4c, 4e and 4f greatly affected their COX inhibitory potency. The p-methoxyphenyl analog 4c showed the best inhibitory activity of COX-1 and COX-2 as justified from its binding conformations (Figure 4). The p-methoxy moiety managed to form five hydrophobic interactions with COX-1 active site residues Met113, Ile345, Leu117, Leu535 and Leu531 (Figure 4A). Similarly, the p-methoxy moiety of 4c exhibited several hydrophobic interactions with COX-2 residues Leu345, Val102, Leu517 and Gln178 (Figure 4C and D) [63]. Moreover, the main scaffold pyrrolidine of 4c demonstrated additional alkyl interaction with the side pocket His75 of COX-2 (Figure 4C) [62]. Upon examining the binding conformation of 4c relative to celecoxib, three extended π–alkyl interactions were observed between the phenyl–pyrrolidine moiety of 4c and COX-2 Arg499 and Ala502 (Figure 5C and D). These extended interactions could enhance the fitting of 4c inside the COX-2 active site compared with celecoxib, albeit with less favorable binding energy. The overall binding energy of 4c was calculated to be -8.50 kcal/mol relative to -11.90 kcal/mol of celecoxib.

In a similar fashion to 4c, the p-bromophenyl analog 4e established four hydrophobic interactions with COX-1 residues Leu117, Met113, Leu531 and Ile345 (Figure 5A & B). Nonetheless, 4e showed higher COX-1 binding energy than 4c (-6.80 kcal/mol compared with -7.60 kcal/mol; Supplementary Table 1), which explained the fivefold drop in its inhibitory activity. Regarding COX-2, the p-bromo group formed only one π–alkyl interaction, with Tyr341 (Figure 5C & D), maintaining the previously mentioned common scaffold interactions with the hydrophobic channel residue. As with COX-1, the calculated binding energy of 4e to COX-2 was less favorable than that of 4c (-6.20 and -8.50 kcal/mol, respectively) and resulted in a fourfold drop in its activity.

On the other hand, substituting the side chain phenyl moiety with a para-sulfonamide group, as in 4f, caused an eightfold decrease in its potency compared with its congener 4c regarding both isoenzymes. This could be explained by the major increment in its overall binding energy to both isoenzymes relative to its congeners by 1–2 integral score unit in addition to a lower number of interactions with the active sites’ residues than 4c and 4e (Supplementary Table 1). As reported, the sulfonamide group was found to be deprotonated at physiological pH, exposing a negative charge [64]. This charged part of 4f revealed a possible site of hydrogen bond formation with the catalytic site Arg120 in COX-1 and electrostatic interaction with Arg106 in COX-2 [65]. Moreover, the common interaction pattern observed earlier with 4c and 4e by their main phenyl–imidazolidine–phenyl–pyrrolidine scaffold lacked many interactions with the isoenzyme’s hydrophobic pocket in the case of 4f, further explaining its lower potency (Figure 6A & B).

Conclusion

The current study showed that the oxazolone- and imidazolone-based derivatives are promising candidates for designing safe and effective anti-inflammatory and analgesic agents with an enhanced gastric tolerability compared with the reference drug celecoxib and previously reported active compounds I–VI. All the new target compounds showed promising anti-inflammatory activity, especially derivatives carrying a pyrrolidine ring (3a, 4a, 4c, 4e and 4f), which revealed the best anti-inflammatory activity with good gastric tolerability. All the new target compounds also showed potent analgesic activity. The pyrrolidinyl benzylidene imidazolone derivatives 4c, 4e and 4f showed the most potent anti-inflammatory and analgesic activities with remarkable gastric tolerability compared with the other derivatives. The p-methoxyphenyl derivative 4c is considered the most promising analog, showing 90.40% inflammation inhibition, 95.49% writhing protection in analgesia testing, COX-2 IC50 of 0.05 μM and SI of 2.00; it also showed an ulcer index of 8.20. Molecular docking studies predicted the binding pattern of the most potent compounds in COX-1 and COX-2 enzymes, confirming their ability to satisfy the structural features required for binding and rationalizing their selectivity based on their docking binding patterns and scores.

Supplementary Material

Supplementary Material
IFMC_A_2340180_SM0001.pdf (5.6MB, pdf)

Supplemental data

To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.4155/fmc-2023-0338

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

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