Dual COX-2 and 15-LOX inhibition study of novel 4-arylidine-2-mercapto-1-phenyl-1H-imidazolidin-5(4H)-ones: Design, synthesis, docking and anti-inflammatory activity

Nermine A Osman; Mostafa K Soltan; Samar Rezq; Joseph Flaherty; Damian G Romero; Ahmed S Abdelkhalek

doi:10.1002/ardp.202300615

. Author manuscript; available in PMC: 2025 May 1.

Published in final edited form as: Arch Pharm (Weinheim). 2024 Feb 5;357(5):e2300615. doi: 10.1002/ardp.202300615

Dual COX-2 and 15-LOX inhibition study of novel 4-arylidine-2-mercapto-1-phenyl-1H-imidazolidin-5(4H)-ones: Design, synthesis, docking and anti-inflammatory activity

Nermine A Osman ^1,^*,^a, Mostafa K Soltan ^2,³, Samar Rezq ^4,^5,^6,^7,⁸, Joseph Flaherty ^5,^6,^7,⁸, Damian G Romero ^5,^6,^7,⁸, Ahmed S Abdelkhalek ^2,^*,^a

PMCID: PMC11073913 NIHMSID: NIHMS1959936 PMID: 38315093

Abstract

Novel arylidene-5(4H)-imidazolone derivatives 4a-r were designed and evaluated as multidrug-directed ligands (MTDLS), i.e., inflammatory, pro-inflammatory mediators and reactive oxygen species (ROS) inhibitors. All tested compounds showed cyclooxygenase (COX)-1 inhibitory effect more than celecoxib and less than indomethacin and also demonstrated an improved inhibitory activity against 15-lipoxygenase (15-LOX). Compounds 4f, 4l, and 4p exhibited COX-2 selectivity comparable to that of celecoxib, while 4k was the most selective COX-2 inhibitor. Interestingly, the screened results showed that compound 4k exhibited a superior inhibition effect against 15-LOX and was found to be the most selective COX-2 inhibitor over celecoxib, whereas compound 4f showed promising COX-2 and 15-LOX inhibitory activities besides its inhibitory effect against ROS production and its lowering effect of both TNF-α and IL-6 levels by ~80%. Moreover, compound 4f attenuated the LPS-mediated increase in NF-κB activation in RAW 264.7 macrophages. The preferred binding affinity of these molecules was confirmed by docking studies. We conclude that arylidene-5(4H)-imidazolone scaffolds provide promising hits for developing new synthons with anti-inflammatory and antioxidant activities.

Keywords: arylidene-5(4H)-imidazolone, synthesis, dual COX-2/15-LOX inhibitors, molecular docking, NF-κB

Graphical Abstract

Compound 4f, which had good 15-lipoxygenase (15-LOX) inhibitory activity (IC₅₀ = 3.99 μM) and cyclooxygenase-2 (COX-2) selectivity (SI = 131.59), showed significant anti-inflammatory activity against reactive oxygen species (ROS) production, outperforming celecoxib, while decreasing tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels by ~80%.

graphic file with name nihms-1959936-f0001.jpg

1. INTRODUCTION

Inflammation is a noxious event resulting from chemical or physical stimuli, or infections by microorganisms or parasites. Inflammation initial events start with irritation of the connective tissue and blood vessels at the site of the noxious event and result in redness, swelling, pain, and even loss of function. ^[1] In some diseases like rheumatoid arthritis, osteoarthritis, and psoriasis, the immune system fights against the body’s own cells, causing harmful inflammations that can last for years or even a lifetime with variable levels of severity. ^{[2, 3]} The most widely used treatment strategy for various inflammatory diseases is non-steroidal anti-inflammatory drugs (NSAIDs) owing to their efficacy in reducing pain and inflammation. ^[4]

Traditionally, NSAIDs were classified on the basis of their selectivity for inhibiting the cyclooxygenase enzyme (COX), which is responsible for the mechanism of NSAIDs’ analgesic, antipyretic, and anti-inflammatory properties. ^{[5, 6]} The cyclooxygenase (COX) enzyme or prostaglandin endoperoxide H synthase (PGHS) exists in two isoforms: PGHS-1 or COX-1 and PGHS-2 or COX-2. COX-1 modulates prostaglandin synthesis, which is responsible for gastrointestinal and renal protection, macrophage differentiation, platelet aggregation, and mucus production. ^{[7, 8]} Molecular studies have shown that COX-1 has a limited role in the inflammatory process; this can be demonstrated by the unchanged expression of COX-1 mRNA and protein during inflammation. ^[9] On the other hand, tissue injury triggers lipopolysaccharide (LPS), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), and tumour necrosis factor alpha (TNFα) to induce and activate COX-2 at the injury sites. Activation of COX-2 enhances the production of prostaglandins to mediate pain and support the inflammatory process. ^[10]

Most NSAIDs non-specifically inhibit both COX-1 and COX-2 at standard anti-inflammatory doses. However, inhibition of COX-2 isoform is responsible for anti-inflammatory and analgesic effects of these drugs; however gastric ulcers, perforations, and bleeding occur as a side effect resulting from concurrent inhibition of COX-1. ^[11] In the last century, selective anti-COX-2 drugs were discovered and marketed as the drug of choice for the treatment of inflammation with promising gastric safety, e.g, celecoxib (Celebrex^™, I) ^[12], valdecoxib (Bextra^™, II) ^[13], rofecoxib (Vioxx^™, III) ^[14] (Fig.1). However, some of these drugs (valdoeoxib and rofecoxib) were withdrawn from the market due to their serious cardiovascular thrombotic events, including myocardial infarction and stroke. ^{[15, 16]} The adverse effects were probably due to prolonged blocking of COX-2 inhibiting some desirable byproducts and activating the lipoxygenase (LOX) pathway. LOX catalyzes the oxidation of lipoproteins (LDL and HDL) to atherogenic forms. ^[17] Accordingly, simultaneously inhibiting LOX-mediated leukotriene synthesis and COX-mediated prostaglandin synthesishas gained much attention as dual anti-inflammatory targets. ^[18-20] Interestingly, NF-κB plays an important role in the regulation of inflammatory and immune responses. Cytokines and chemokines encoding genes are regulated by NF-κB promoting inflammation and inflammasome regulation. ^[21] Structurally, the basic requirements of selective COX-2 inhibitors involve two aryl groups attached to a heterocyclic core. Examples of those heterocyclic cores include, pyrazole in celecoxib I ^[12], furanone in rofecoxib II ^[14], and isoxazole in valdecoxib III ^[13] (Fig. 1). In addition, imidazole ring is a structural component of many anti-inflammatory molecules such as in flumizole IV ^[22], cimicoxib V,^[23] and azathioprine VI which are mainly used in the treatment of rheumatoid arthritis ^[24] (Fig. 1). Occasionally, the arylidene-5(4H)-imidazolone framework has been identified as a promising molecular target for the development of anti-inflammatory agents as in compounds VII and VIII ^{[25, 26]} (Fig. 2). Moreover, phenylsulphonyl substituents are essential for inducing COX-2 selectivity. ^{[27, 28]} On the other hand, some reports have shown that the replacement of a phenylsulphonyl substituent by a carboxamide linker resulted in a hydrophobic moiety such as compounds IX ^[29] and X ^[30] that showed remarkable selective inhibition against COX-2 (Fig. 2). Moreover, imidazole derivatives XI ^[31] and XII ^[32] were reported to have a promising LOX inhibitory activity (Fig. 2). Herein, we designed a series of compounds 4a-r, which exhibit a verified structural similarity to compounds (VII-XII) (i.e. arylidene-5(4H)-imidazolone as an anti-inflammatory pharmacophore, with carboxamide linkers bearing various lipophilic moieties) aiming to inhibit COX-2 and 15-LOX, and to act as multidrug-directed ligands (MTDLS) with antioxidant (ROS inhibition) and anti-inflammatory (TNF-α and IL-6 inflammatory cytokines inhibition) activities (Fig. 2).

Fig 1. — Commercially available V-shaped five membered ring molecules that selectively inhibit the COX-2 isosyme (**I-VI**).

Fig 2. — Design strategy and Structure-activity relationship (SAR) of the title compounds **4a-r** based on the structures of some reported anti-inflammatory active agents.

2. RESULTS AND DISCUSSION

2.1. Chemistry

N-Phenyl-2-thioxo-4-imidazolidinone (1) was prepared using the procedure reported by Ottanà et al ^[33] and Vanitha U. et al ^[34], while 2a-b derivatives were obtained using the procedure reported by Chazeau V. et al. ^[35] 2-Chloro-N-phenylacetamide derivatives (3a-r) were prepared according to the procedure reported by Tan A. et al. ^[36] The final target compounds (4a-r) were synthesized via S-alkylation reaction of 2a-b using 2-chloro-N-phenylacetamide derivatives (3 a-r) in CH₃CN using a catalytic agent; TEA at room temperature with good yields (scheme 1). Spectroscopic analysis techniques were used for confirmation of target chemical structures as ¹H-NMR, ¹³C-NMR, mass, and elemental analysis. ¹H-NMR showed characteristic peaks for amidic NH and methylene group (CH₂) at chemical shift range of 10.36 to 10.88 ppm and 4.05 to 4.30 ppm, respectively for all finals. Compounds 4i and 4r showed extra CH₂ group at 4.33 appeared as doublets related to PhCH₂ group because of coupling effect of adjacent NH group in addition to NH proton which showed as triplet signal at 8.86 ppm due to adjacent methylene group. According to ¹H-NMR spectrum the ‘Z’ configuration is the most promident one in which arylidene proton (7.64 – 7.59 ppm) appeared to be deshielded under the effect of adjacent C=O and these findings with in cope with a previously reported literature published by Rosaria Ottana et al. ^[37] In ¹³C-NMR spectra, compounds 4b, 4h (i.e containing 4-F), 4k and 4q (i.e containing CF₃) showed carbon-fluorine coupling interaction. ^[38] For instance, compound 4b spectrum revealed that C4 of N-phenylacetamide to which fluoride attached, resonated as a doublet at 158.09 (J = 240.0 Hz) whereas, the C1 resonated as doublets at 135.35 (J = 2.4 Hz). Furthermore, C2 and C6 coupled with fluorine and appeared as doublets at 120.82 (J = 7.8 Hz) ppm. Other C3 and C5 showed as doublets at 115.41 (J = 7.8 Hz) ppm due to fluorine interaction.Also, compound 4k spectra showed that C attached to F3 in CF₃ group appeared as quartet at 124.05 (J = 272 Hz) and C3 of N-phenylacetamide resonate as quartet at 129.54 (J = 31.3 Hz), while C2 and C4 appeared at 119.84 and 115.14 as quartet.

Scheme 1. — Synthesis of compounds **4a-r**. Reagent and conditions: (i) EtOH/H₂O, reflux, 24 h; (ii) arylaldehyde, NaOAC, EtOH, reflux 5 h; (iii) TEA, CH₃CN, rt, 6 h.

2.2. Biological activity

2.2.1. COX1 and COX2 inhibitory activities

The efficacies of the tested compounds against COX-1 and COX-2 (IC₅₀) and the COX-2 selectivity indexes (SI values) are recorded in Table 1, using celecoxib and indomethacin as reference drugs. The in vitro assay revealed that all the screened compounds showed potent COX-2 inhibition with IC₅₀ value of 0.07-0.28 μM and COX-1 inhibition with IC₅₀ value of 3.11-13.04 μM in relation to celecoxib (COX-2 IC₅₀ = 0.08 μM, COX-1 IC₅₀ = 13.60 μM) and indomethacin (COX-2 IC₅₀ = 0.58 μM, COX-1 IC₅₀ = 0.11 μM). Compound 4k (IC₅₀ = 0.07μM) was the most potent COX-2 inhibitor among the tested molecules 4a-r. On the other hand, compounds 4f, 4i (Fig. 4), 4l, 4p and 4q were almost as potent as celecoxib (IC₅₀ = 0.08-0.09 μM). Regarding the SI, the highest selectivity was represented by compound 4k (SI = 186.28) which showed high selectivity even more than that of celecoxib (SI = 180.46). On the other hand, compounds 4f, 4l, and 4p had high selectivity index (SI = 131.59, 154.73, 152.23, respectively) compared with that of celecoxib, whereas compounds 4i, 4o, and 4q exhibited significant selectivity in relation to that of celecoxib. From the aforementioned data, it was clear that addition of the Cl group at the p-position of the benzylidine moiety enhanced the anti-COX-2 activity of 4k, 4l, 4p, and 4q by 1.85, 1.75, 1.75 and 3.1 folds compared with the unsabstituted compounds 4b, 4c, 4g, and 4h, respectively. Likewise, p-chlorobenzylidine influenced the selectivity of 4k, 4l, 4p, and 4q over that of 4b, 4c, 4g, and 4h; however, the introduction of Cl in the benzylidine reduced both COX-2 inhibition and selectivity of compound 4r relative to compound 4i with the same acetamide substitution (C₆H₅CH₂−) (Fig 3). Moreover, within the benzylidine series (4a-4i), compound 4i with methylene bridge in the acetamide part exhibited the highest potency and selectivity (0.09 μM, 121.60), indicating that methylene linker is required for optimal activity and selectivity. Additionally, compounds 4f and 4o having 4–methoxyphenyl substitutions in the acetamide showed high potency which indicates the importance of the electron-donating methoxy group.

Table 1.

In vitro COX-1, COX-2, and 15-LOX inhibition activities.

Compound	^aCOX1, IC₅₀ (μM)	^aCOX2, IC₅₀ (μM)	^bSI	15-LOX IC₅₀ (μM)
4a	7.83±1.19	0.12±0.060	62.97	5.71±2.56
4b	12.62±1.78	0.13±0.068	99.06	3.13±1.49
4c	9.12±1.40	0.14±0.062	64.62	3.24±1.46
4d	8.14±1.53	0.13±0.071	64.13	5.14±2.48
4e	9.12±1.40	0.10±0.048	90.85	5.14±2.48
4f	10.95±1.53	0.08±0.040	131.59	3.96±1.49
4g	9.83±1.65	0.14±0.073	68.07	2.82±1.27
4h	11.05±1.63	0.28±0.155	39.99	5.71±2.56
4i	10.59±1.62	0.09±0.041	121.60	2.82±1.27
4j	11.98±1.67	0.13±0.064	94.97	4.75±2.31
4k	13.04±1.82	0.07±0.034	186.28	2.17±1.04
4l	12.27±1.84	0.08±0.035	154.73	2.69±1.29
4m	8.00±1.31	0.18±0.082	44.12	4.21±1.53
4n	9.45±1.33	0.12±0.053	79.96	3.13±1.49
4o	10.95±1.53	0.10±0.044	111.27	3.24±1.46
4p	12.79±1.83	0.08±0.037	152.33	2.69±1.29
4q	10.14±1.61	0.09±0.040	111.90	3.99±1.90
4r	10.14±1.60	0.13±0.057	78.00	2.47±1.14
Celecoxib	13.60±2.25	0.08±0.037	180.46	-
Indomethacin	0.11±0.05	0.58±0.18	0.19	-
Quercetin	-	-	-	4.79±1.29

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IC_{50 in} (μM) concentration as expressed as mean ± SEM

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

Fig 4. — Effect of compounds 4f and 4l on COX-2 and 15-LOX activities and NF-κB activation. The inhibitory potential against COX2 (A, B) and 15-LOX (C, D) enzymatic activities was assessed for the tested compounds and expressed as % of control. (E) Phosphorylated NF-κB protein was quantified in cells treated with the vehicle or 25 μM of the tested compounds by Western blot and normalized to total NF-κB (N = 3/group).

Fig 3. — COX-2 structure activity relationship (SAR) of imidazole-2-thiol derivatives **4a-r**.

2.2.2. In vitro LOX inhibition activity

The inhibitory potential of the tested compounds (4a-r) for 15-LOX represented as the IC₅₀ concentrations for inhibiting 50% of enzymes compared to quercetin, the reference drug, is shown in Table 1. Fourteen out of 18 compounds showed strong inhibitory activity against 15-LOX with IC₅₀ values of 2.17-4.75 μM (Fig. 4), which is lower than quercetin (IC₅₀ = 4.79 μM). On the contrary, compounds 4a, 4d, 4e, and 4h had lower potency compared to quercetin. It was also observed that compounds 4a (IC₅₀ = 5.71 μM), 4d (IC₅₀ = 5.14 μM), 4e (IC₅₀ = 5.14 μM), and 4h (IC₅₀ = 5.71 μM) were less potent than similar compounds with the p-chlorobenzylidine substitution; 4j (IC₅₀ = 4.75 μM), 4m (IC₅₀ = 4.21 μM), 4n (IC₅₀ = 3.13 μM), and 4q (IC₅₀ = 3.99 μM), respectively. This observation is consistent with other results obtained with benzylidine-substituted compounds and their p-chlorobenzylidine-substituted compounds (Table 1). The aforementioned data might have attributed chloro substitution's role in improving the designed compounds' potency as 15-LOX inhibitors.

2.2.3. Effects on ROS production in LPS-activated RAW 264.7 macrophages

It was reported that the expression of COX-2 ^[39], as well as the production of ROS and other inflammatory mediators, are induced in RAW 264.7 cells exposed to the bacterial toxin LPS. ^[40] Moreover, several compounds with antioxidant potential have also been found to be effective against inflammatory diseases and cancer ^[40], highlighting oxidative stress as a possible target for developing new anti-inflammatory candidates. We examined the ability of 2-oxo-imidazole derivatives 4a-r to reduce reactive oxygen species (ROS) production in macrophages activated by LPS. The results recorded in Table 2 showed superior antioxidant potency with IC₅₀ of 21.78-41.00 μM for compounds; 4b, 4f (Fig. 4), 4i-j and 4m-p over celecoxib (IC₅₀ = 44.55 μM), while compounds 4a, 4h, and 4n revealed comparable potency to celecoxib with IC₅₀ of 44.55, 45,37, and 41.00 μM, repectively. Other 2-oxo-imidazole derivatives 4c-e, 4g, 4k, 4l, 4q, and 4r exhibited lower inhibitory activities (IC₅₀= 52.73 - 117.96 μM) than celecoxib (Table 2).

Table 2.

In vitro inhibitory activities of target compounds on LPS-induced ROS production in RAW 264.7 cells.

Compound	ROS (IC₅₀)
4a	44.55 ± 1.43
4b	23.82 ± 1.44
4c	61.60 ± 1.51
4d	62.09 ± 1.51
4e	117.96 ± 2.36
4f	22.77 ± 1.32
4g	65.89 ± 1.55
4h	45.37 ± 1.23
4i	21.78 ± 1.26
4j	33.35 ± 1.27
4k	71.02 ± 1.55
4l	52.73 ± 1.35
4m	38.40 ± 1.37
4n	41.00 ± 1.39
4o	24.24 ± 1.26
4p	25.36 ± 1.26
4q	56.16 ± 1.26
4r	72.35 ± 1.85
Celecoxib	44.55 ± 1.18
Indomethacin	54.96 ± 1.26

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Data is IC₅₀ in μM concentration as expressed as mean ± SEM. ROS: reactive oxygen species.

2.2.4. Effects on TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages

Soromou et al. ^[41] reported increased TNF-α and IL-6 cytokine production in RAW 264.7 macrophages upon LPS stimulation. Herein, we examined the ability of 2-oxo-imidazole derivatives 4a-r to inhibit the production of TNF-α and IL-6 in LPS-activated RAW 264.7 macrophages. The results are shown in Table 3. IL-6 findings revealed that 2-oxo-imidazole carrying p-chlorobenzylidine, 3-CF₃ groups; 4q (IC₅₀ = 4.54) or benzylidine, 3,4,5-OCH₃ groups; 4g (IC₅₀ =11.51) are more potent than celecoxib in lowering its levels (24.64 pg/mL). Moreover, 4d (26.35 pg/mL), 4f (Fig. 5, 22.21 pg/mL), 4h (23.42 pg/mL), 4i (21.00 pg/mL), 4l (25.57 pg/mL), and 4r (IC₅₀ = 20.29) showed a significant inhibitory activity comparable to that of celecoxib (24.64 pg/mL), while 4a-e, 4j-k, 4m-p (27.57-50.90 pg/mL) inhibited IL-6 to a lesser degree than celecoxib. Interestingly, the introduction of the Cl group to benzylidine series (4a-i) increased IL-6 inhibitory activity, especially for 4q (4.54 pg/mL; ~5 folds more than the unsubstituted benzylidine 4h) with the exception of compounds 4j (49.59 pg/mL), 4o (28.37 pg/mL), and 4p (30.70 pg/mL) (Table 3).

Table 3.

In vitro inhibitory activities of target compounds on LPS-induced TNF-α and IL-6 production in RAW 264.7 cells.

Compound	TNF-α (pg/mL)	IL-6 (pg/mL)
Control	7.38 ± 2.28	6.66 ± 2.28
Vehicle (LPS)	121.2 ± 25.98	120.5 ± 25.98
4a	57.28 ± 18.75	37.82 ± 1.06
4b	51.63 ± 10.35	50.90 ± 10.35
4c	49.81 ± 23.20	49.08 ± 23.20
4d	27.08 ± 4.22	26.35 ± 4.22
4e	58.19 ± 23.16	34.33 ± 1.82
4f	22.94 ± 6.32	22.21 ± 6.32
4g	12.23 ± 3.09	11.51 ± 3.09
4h	24.15 ± 1.05	23.42 ± 1.05
4i	46.98 ± 25.83	21.00 ± 9.39
4j	50.31 ± 10.22	49.59 ± 10.22
4k	28.29 ± 14.76	27.57 ± 14.76
4l	26.47 ± 5.33	25.75 ± 5.33
4m	27.89 ± 0.90	27.16 ± 0.9
4n	37.28 ± 14.95	36.56 ± 14.95
4o	29.10 ± 4.54	28.37 ± 4.54
4p	31.42 ± 3.70	30.70 ± 3.70
4q	5.26 ± 0.61	4.54 ± 0.61
4r	21.02 ± 5.03	20.29 ± 5.03
Celecoxib	25.36 ± 6.364	24.64 ± 6.36

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IL-6 and TNF-α levels were detected in cells treated with 25 μM of the tested compounds 4a-r.

Fig. 5. — Inhibitory effect of 4f on ROS (A), TNF-α (B) and IL-6 (C) production.

Regarding inhibition of TNF-α production, compounds 4q (5.26 pg/mL) and 4g (12.23 pg/mL) were found to be ~ 5- and 2-fold more potent than celecoxib (25.36 pg/mL), while compounds 4f (Fig. 5), 4h, and 4r (21.02-24.15 pg/mL) were as potent as celecoxib (24.64 pg/mL). Notably, the introduction of the Cl group to the benzylidine series (4a-i) enhanced TNF-α inhibitory activity, especially for 4q (concentration= 5.26 pg/mL; 4.6- fold more than the unsubstituted benzylidine; 4h). Furthermore, compound 4m (27.89 pg/mL) showed retained activity compared to unsubstituted 4d (27.08 pg/mL), while compounds 4o-p have 4-OCH₃ or 3,4,5-OCH₃ group/s had a reduced activity which might be explained on the basis of lipophilicity (i.e – π value) (Table 3).

2.2.5. Effect on NF-κB activation in LPS-activated RAW 264.7 macrophages

NF-κB plays an important role in the regulation of inflammatory and immune responses by inducing the gene expression of multiple proinflammatory cytokines and chemokines ^[21]. NF-κB activation is initiated in response to inflammation, cell proliferation and immune response ^[42]. LPS via binding to toll-like receptor4 (TLR4), induces the activation of NF-κB signalling, which is involved in LPS-mediated inflammatory responses ^[43]. Western blot analysis revealed that LPS-mediated increase in NF-κB activation was attenuated in RAW 264.7 macrophages pretreated with 4f and 4l (Fig. 4).

2.2.6. Cell viability assay

Cell viability testing for the most active compounds (4f, 4k, 4l) on RAW 264.7 cells demonstrated their non-toxic nature. These compounds exhibited a cell viability exceeding 90% of the control values at concentrations up to 25 μM. Furthermore, even at a concentration of 50 μM, the compounds maintained a cell viability percentage within the 83-105% range, indicating their safety and selectivity. (supplementary material Fig. S1).

2.3. Molecular modelling

2.3.1. COX-2 docking study

Validating docking procedures were initially performed by redocking the co-crystalized structure celecoxib into the 3LN1 active site (Fig. 6). The re-docked celecoxib studies showed 1.2959 Å for RMSD value. Compounds 4k (IC₅₀= 0.07 μM), 4f (IC₅₀= 0.08 μM), 4l (IC₅₀= 0.08 μM), and 4p (IC₅₀= 0.08 μM) showed COX-2 selectivity when docked at 3LN1 active site. The docking results of 4f revealed (1) HB with Leu338 as well as (3) H-π interactions with Ser339 and Ser516 (Fig. 7), whereas compound 4k showed (4) HBs with Arg106 ^[44], Phe504 and Leu517 besides (3) H-π interactions with Ser339, Tyr371 and Val509 (Fig. 8). Furthermore, 4l binding mode exhibited (2) HB with Phe504 and Ala513 in addition to (1) H-π interaction with Leu517 (Fig. 9). Finally, compound 4p formed (4) HBs with Leu338, Phe504 and Val509 besides (3) H-π interactions with Ala513 and Ser516 ^[45] (Fig. 10). Interestingly, superimposition of 4p with celecoxib (co-crystalized ligand) revealed similar binding interactions with amino acids Leu338, Phe504, Val509 and Ala513 (Fig. 11). The docking results of 4f, 4k, 4l, and 4p are summarized in Table 4.

Fig 7. — 4F (green sticks) binding interactions at COX-2 (pdb: 3LN1); (S = −7.9722, RMSD = 1.6837): A) 3D binding pose of 4f; B) 2D binding pose of 4f revealing different interactions with amino acids in binding pocket.

Fig 8. — 4k (gold sticks) binding interactions at COX-2 (pdb: 3LN1); (S= −6.9938, RMSD = 1.2340): (A) 3D binding pose of 4k; (B) 2D binding pose of 4k revealing different interactions with amino acids in binding pocket.

Fig 9. — 4l (cyan sticks) binding interactions at COX-2 (pdb: 3LN1); (S= −7.7415, RMSD = 1.1164): (A) 3D binding pose of 4l; (B) 2D binding pose of 4l revealing different interactions with amino acids in binding pocket.

Fig 10. — 4p (magenta sticks) binding interactions at COX-2 (pdb: 3LN1); (S= −6.2656, RMSD = 1.5103): (A) 3D binding pose of 4p; (B) 2D binding pose of 4p revealing different interactions with amino acids in binding pocket.

Fig 11. — 3D structure of superimposed 4f and celecoxib into 3LN1 active site showed similar interactions with Leu338, Phe504, Val509 and Ala513.

Table 4:

Results of docking studies for compounds 4f, 4k, 4l, and 4p at COX-2 (pdb: 3LN1) and 15-LOX (pdb: 4NRE), respectively, showing all possible binding interactions of selected compounds at the active sites of enzymes. Comparing to celecoxib CEL and C8E reference ligands.

Target (pdb)	Compound No.	Binding score (S)	Rmsd	HBs	H-π interactions
COX-2 (3LN1)	4f	−7.9722	1.6837	(1) Leu338	(2) Ser516, Ser339
	4k	− 6.9938	1.2340	(4) Arg106, Phe504, Leu517	(3) Tyr371, Ser339, Val509
	4l	−7.7415	1.1164	(2) Phe504, Ala513	(1) Leu517
	4p	− 6.2656	1.5103	(4) Leu338, Phe504, Val509	(3) Ala513, Ser516
	CEL	−9.3275	1.2959	(4) Leu338, Ser339, Arg499, Phe504	(4) Ser339, Val504, Ala513
LOX (4NRE)	4f	−6.7213	1.4433	(2) Phe184, Ala606	(3) Phe184, Leu420, Arg429
	4k	−7.3453	1.4531	(4) Ile412, Asp602, Ala606	(1) Leu420
	4l	− 6.8083	1.1369	(2) Leu419, Leu610	(4) Phe184, Leu420
	4p	− 7.3488	1.1975	(1) Leu609	(5) Phe184, Phe192, Leu415, Leu420
	C8E	−8.4285	1.1268	----	His378

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2.3.2. 15-LOX docking study

Docking validation was conducted by redocking of C8E (co-crystalized ligand) into the active site of 15-LOX (PDB: 4NRE). The results revealed that the re-docked ligand showed RMSD value of 1.5887 Å. All docking results are summarized in Table 4. Analysis of 4f binding mode (Fig. 12) exhibited (2) HBs with Phe184 and Ala606 in addition to (3) H-π interactions with Phe184, Leu420 and Arg429. Investigation of 4k binding interaction (Fig. 13) showed (4) HB interactions with Ile412, Asp602 and Ala606 and (1) H-π interaction with Leu420. Moreover, Compound 4l (Fig. 14) exhibited (2) HBs with Leu419 and Leu610 besides forming (4) H-π interactions with Phe184 and Leu420. In addition, compound 4p (Fig. 15) displayed (1) HB with Leu609 and (5) H-π interactions with Phe184, Phe192, Leu415, and Leu420. Overall, the docking and biological screening results suggest that compounds 4k, 4f, 4l, and 4p are potential anti-inflammatory candidates by inhibiting COX-2 and 15-LOX enzymes.

Fig 12. — 4f (green sticks) binding interactions at 15-LOX (pdb: 4NRE); (S= −6.7213, RMSD = 1.4531): (A) 3D binding pose of 4f; (B) 2D binding pose of 4f revealing different interactions with amino acids in binding pocket.

Fig 13. — 4k (green sticks) binding interactions at 15-LOX (pdb: 4NRE); (S= −7.3453, RMSD = 1.4531): (A) 3D binding pose of 4k; (B) 2D binding pose of 4k revealing different interactions with amino acids in binding pocket.

Fig 14. — 4l (cyan sticks) binding interactions at 15-LOX (pdb: 4NRE); (S= −6.8083, RMSD = 1.1369): (A) 3D binding pose of 4l; (B) 2D binding pose of 4l revealing different interactions with amino acids in binding pocket.

Fig 15. — 4p (magenta sticks) binding interactions at 15-LOX (pdb: 4NRE); (S= −7.3488, RMSD = 1.1975): (A) 3D binding pose of 4p; (B) 2D binding pose of 4p revealing different interactions with amino acids in binding pocket.

3. CONCLUSION

A series of N-aryl-thioacetamide linked imidazolidin-5(4H)-one hybrid derivatives 4a-r were successfully designed, synthesized and their chemical structures were confirmed using spectroscopic and analytical techniques. The target compounds were evaluated for their anti-inflammatory activity as dual COX-2 and 15-LOX inhibiting drug candidates. The in vitro assay revealed that all the tested compounds exhibited high potency and selectivity compared to Celexoxib as COX-2 inhibitor and quercetin as 15-LOX inhibitor reference drugs. Among the target compounds, 4k was observed to be the most potent COX-2 inhibitor with` high 15-LOX inhibition activity. On the other hand, the newly synthesized COX inhibitors were investigated for their in vitro inhibitory activities on the production of certain pro-inflammatory mediators: ROS, TNF-α and IL-6 in RAW 264.7 cells. Compounds 4b, 4f, 4i, 4o, and 4p exhibited two folds inhibitory activity against ROS production more potent than celecoxib indicating remarkable antioxidant activity. Regarding the TNF-α and IL-6 values, most of the target compounds i.e. 4f, 4g, 4h, 4q and 4r show potent inhibition on LPS-induced TNF-α and IL-6 production in RAW 264.7 cells. Dual COX-2 and 15-LOX anti-inflammatory activity was observed for compound 4f in addition to its ability to suppress other proinflammatory mediators such as ROS, TNF-α and IL-6 (80% inhibition). Docking studies were performed to deduce the most favoured binding poses. Based on our findings, we recommend to identify and develop novel imidazolidin-5(4H)-based hybrids as lead molecules for dual anti-inflammatory and antioxidant activities.

4. MATERIALS AND METHODS

4.1. Chemistry

4.1.1. General remarks

All reagents were commercially obtained with the highest percent of purity available, especially for synthesis, unless otherwise mentioned. Melting points were determined with a Gallenkamp (London, U.K.) melting point apparatus and are uncorrected. ¹H NMR and ¹³C NMR spectra (see the Supporting Information) were obtained using Bruker Ascend 400 MHz spectrometers. All spectra were recorded in deuterated solvents (CDCl₃ or DMSO-d₆), and the chemical shift values were expressed in parts per million (ppm) relative to the internal standard, tetramethylsilane (TMS). Elemental analyses were determined by the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Egypt, utilizing FLASH 2000 CHNS/O analyzer, Thermo Scientific. Mass spectra were conducted either on the Direct Inlet part to the mass analyzer in Thermo Scientific GCMS model ISQ, or 70 eV EI GC/MS-Qp2010 plus (Shimadzu) at the Regional Center for Mycology and Biotechnology, Al-Azhar University, and the Microanalytical Center, Faculty of Science, Cairo University, Giza, Egypt. Thin layer chromatography (TLC) was carried out on precoated silica plates (ALUGRAMVR SIL G/UV254) and visualised with UV light (254 nm) or iodine.

The InChI codes of the investigated compounds, together with some biological activity data, are provided as Supporting Information.

4.1.2. General scheme for the synthesis of compounds 4a-r

To a solution of 2 a-b (0.32 mmol) in CH₃CN, 2-chloro-N-substituted phenylacetamide derivatives 3 a-r (0.32 mmol) a catalytic amount of Et₃N (9 drops) were added and the reaction was stirred at room temperature for 6 h. The formed solid was collected by filtration, washed with CH₃CN and H₂O, and recrystallized from ethanol to afford target compounds 4a-r.

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-phenylacetamide (4a): Yield: % 82, white powder, m.p. 197-199 °C. ¹H NMR (400 MHz, DMSO) δ 10.51 (s, 1H, acetamide- NH), 8.20 (d, J = 7.5 Hz, 2H, phenylacetamide-C_2,6-H), 7.67 (s, 1H, 4-benzylidene =CH), 7.65-7.46 (m, 6H, phenylacetamide-C_3,5-H, 1-phenyl-C_2,6-H and 4-benzylidene-C_2,6-H), 7.24-7.20 (m, 5H, 1-phenyl-C_3,5-H and benzylidene-C_3,4,5-H), 7.06 (t, J = 7.3 Hz, 1H, 1-phenyl-C₄-H), 6.96 (s, 1H, phenylacetamide-C₄-H), 4.28 (s, 2H, acetamide-CH₂). ¹³C NMR (101 MHz, DMSO) δ 167.92 (s, imidazolinone-CO), 165.08 (s, cetamide-CONH), 164.27 (s, imidazolinone-NCN), 138.95 (s, phenylacetamide-C₁), 137.61 (s, benzylidene-C₁), 133.94 (s, 1-phenyl-C₁), 132.24 (s, imidazolinone-NCCO), 131.86 (s, 2C, 1-phenyl-C_3,5-H), 129.89 (s, 1-phenyl-C₄-H), 129.59 (s, 2C, phenylacetamide-C_3,5-H), 129.38 (s, phenylacetamide-C₄-H), 128.83 (s, 2C, benzylidene-C_3,5-H), 128.59 (s, 2C, benzylidene-C_2,6-H), 127.61 (s, 2C, 1-phenyl-C_2,6-H), 123.48 (s, benzylidene-C₄-H), 123.30 (s, 4-benzylidene =C-H), 119.04 (s, 2C, phenylacetamide-C_2,6-H), 35.51 (s, acetamide-CH₂). EIMS m/z (rel. int. %): 413.49 [M⁺]. Elemental analysis for C₂₄H₁₉N₃O₂S: Calculated/Found: 69.71/69.98 (%C); 4.63/4.80 (%H); 10.16/10.42 (%N).

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-(4-fluorophenyl) acetamide (4b): Yield: 78%, white powder, m.p. 225-227 °C. ¹H NMR (400 MHz, DMSO) δ 10.60 (s, 1H, acetamide- NH), 8.21 (d, J = 6.1 Hz, 2H, phenylacetamide-C_2,6-H), 7.69 (s, benylidene =C-H), 7.58-7.48 (m, 6H, ArH, 1-phenyl-C_3,5-H, 1-phenyl-C_2,6-H and benzylidene-C_2,6-H), 7.34 – 6.97 (m, 5H, benzylidene-C_3,4,5-H and phenylacetamide-C_3,5-H), 6.97 (s, 1H, 1-phenyl C₄-H), 4.28 (s, 2H, acetamide- CH₂).¹³C NMR (101 MHz, DMSO) δ 167.92 (s, imidazolinone-CO), 165.03 (s, acetamide-CONH), 164.24 (s, imidazolinone-NCN), 158.09 (d, ¹J_c,f = 240.1 Hz, phenylacetamide-C₄-F), 137.62 (s, benzylidene-C1), 135.35 (d, ⁴J_c,f = 2.4 Hz, phenylacetamide-C1), 133.96 (s, 1-phenyl-C1), 132.24 (s, imidazolinone-NCCO), 131.85 (s, 2C, 1-phenyl-C_3,5-H), 129.92 (s, 1-phenyl-C4), 129.58 (s, 2C, benzylidene-C_3,5-H), 129.37 (s, benzylidene-C4), 128.59 (s, 2C, benzylidene-C_2,6-H), 127.59 (s, 2C, 1-phenyl-C_2,6-H), 123.32 (s, benzylidene =C-H), 120.82 (d, ³J_c,f = 7.8 Hz, 2C, phenylacetamide-C_2,6-H), 115.41 (d, ²J_c,f = 22.2 Hz, 2C, phenylacetamide-C_3,5-H), 35.43 (s, acetamide-CH₂). EIMS m/z (rel. int. %): 341.64 [M⁺]. Elemental analysis for C₂₄H₁₈FN₃O₂S: Calculated/Found: 66.81/66.92 (%C); 4.20/4.37 (%H); 9.74/9.89 (%N).

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-(4-chlorophenyl) acetamide (4c): Yield: 90%, white powder, m.p. 228-229 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.98 (s, 1H, acetamide NH), 8.16 (dd, J = 7.8, 1.5 Hz, 2H, phenylacetamide-C_2,6-H), 7.59 – 7.44 (m, 6H, benylidene =C-H, benzylidene-C_2,6-H , phenylacetamide-C_3,5-H and benzylidene-C₄-H), 7.40 – 7.35 (m, 2H, benzylidene-C_3,5-H), 7.28 (s, 1H, 1-phenyl-C₄-H), 7.16 – 7.05 (m, 4H, 1-phenyl-C_2,3,5,6-H), 4.05 (s, 2H, acetamide-CH₂). ¹³C NMR (101 MHz, CDCl₃) δ 167.83 (s, imidazolinone-CO), 166.18 (s, acetamide-CONH), 162.44 (s, imidazolinone-NCN), 136.62 (s, phenylacetamide-C₁), 133.75 (s, benzylidene-C1), 131.76 (s, phenylacetamide-C4), 131.58 (s, 2C, phenylacetamide-C_3,5-H), 130.97 (s, 1-phenyl-C1), 130.00 (s, 2C, 1-phenyl-C_3,5-H), 129.97 (s, imidazolinone-NCCO), 129.39 (s, 2C, benzylidene-C_3,5-H), 129.27 (s, 1-phenyl-C₄-H), 128.98 (s, 2C, benzylidene-C_2,6-H), 127.28 (s, 2C, 1-phenyl-C_2,6-H), 126.40 (s, benzylidene-C₄-H), 120.76 (s, benzylidene =C-H), 115.16 (s, 2C, N-phenylacetamide-C_2,6-H), 35.10 (acetamide-CH₂). EIMS m/z (rel. int. %): 447.30 [M⁺]. Elemental analysis for C₂₄H₁₈ClN₃O₂S: Calculated/Found: 64.35/64.17 (%C); 4.05/4.29 (%H); 9.34/9.53 (%N).

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-(4-bromophenyl) acetamide (4d): Yield: 88%, white powder, m.p. 231-233 °C. ¹H NMR (400 MHz, DMSO) δ 10.66 (s, 1H, acetamide NH), 8.19 (d, J = 7.6 Hz, 2H, phenylacetamide-C_2,6-H), 7.64 – 7.45 (m, 9H, benzylidene=C-H, benzylidene-C_2,6-H, phenylacetamide-C_3,5-H and 1-phenyl-C_2,3,5,6-H), 7.34 (t, J = 7.2 Hz, 1H, benzylidene-C₄-H), 7.24 (t, J = 7.6 Hz, 2H, benzylidene-C_3,5-H), 6.95 (s, 1H, 1-phenyl-C₄-H), 4.27 (s, 2H, acetamide-CH₂). ¹³C NMR (101 MHz, DMSO) δ 167.87 (s, imidazolinone-CO), 165.29 (s, acetamide-CONH), 164.20 (s, imidazolinone-NCN), 138.28 (s, phenylacetamide-C₁), 137.58 (s, benzylidene-C₁), 133.93 (s, 1-phenyl-C₁), 132.21 (s, imidazolinone-NCCO), 131.82 (s, 2C, phenylacetamide-C_3,5-H), 131.65 (s, 2C, 1-phenyl-C_3,5-H), 129.91 (s, 1-phenyl-C₄-H), 129.58 (s, 2C, benzylidene-C_3,5-H), 129.37 (s, benzylidene-C₄-H), 128.58 (s, 2C, benzylidene-C_2,6-H), 127.59 (s, 2C, 1-phenyl-C_2,6-H), 123.28 (s, phenylacetamide-C₄), 120.96 (s, 2C, phenylacetamide-C_2,6-H), 115.01 (s, benzylidene =C-H), 35.46 (s, acetamide-CH₂). EIMS m/z (rel. int. %): 492.53 [M⁺]. Elemental analysis for C₂₄H₁₈BrN₃O₂S: Calculated/Found: 58.54/58.73 (%C); 3.68/3.85 (%H); 8.53/8.74 (%N).

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-(4-tolyl) acetamide (4e): Yield: 73%, white powder, m.p. 208-210 °C. ¹H NMR (400 MHz, DMSO) δ 10.42 (s, 1H, acetamide NH), 8.21 (d, J = 7.4 Hz, 2H, benzylidene-C_2,6-H), 7.61 – 7.52 (m, 5H, benzylidene-C=H, phenylacetamide-C_2,6-H and 1-phenyl-C_3,5-H), 7.47 – 7.42 (m, 3H, 1-phenyl-C_2,6-H and benzylidene-C₄-H), 7.26 (t, J = 7.5 Hz, 2H, benzylidene C_3,5-H), 7.12 (d, J = 8.3 Hz, 2H, phenylacetamide-C_3,5-H), 6.96 (s, 1H, 1-phenyl-C₄-H), 4.26 (s, 2H, acetamide-CH₂), 2.24 (s, 3H, CH₃). ¹³C NMR (101 MHz, DMSO) δ 167.98 (s, imidazolinone-CO), 164.78 (s, acetamide-CONH), 164.25 (s, imidazolinone-NCN), 137.61 (s, phenylacetamide-C₄), 136.43 (s, phenylacetamide-C₁), 133.95 (s, 2C, phenylacetamide-C_3,5-H), 132.38 (s, benzylidene-C₁), 132.23 (s, 1-phenyl-C₁), 131.85 (s, 1-phenyl-C₄-H), 129.89 (s, imidazolinone-NCCO), 129.56 (s, 2C, 1-phenyl-C_3,5-H), 129.35 (s, benzylidene-C₄-H), 129.16 (s, 2C, benzylidene C_3,5-H), 128.60 (s, 2C, benzylidene C_2,6-H), 127.59 (s, 2C, 1-phenyl-C_2,6-H), 123.24 (s, benzylidene-C=H), 119.03 (s, 2C, phenylacetamide-C_2,6-H), 35.48 (s, acetamide-CH₂), 20.42 (s, CH₃). EIMS m/z (rel. int. %): 423.26 [M⁺]. Elemental analysis for C₂₅H₂₁N₃O₂S: Calculated/Found: 70.24/70.47 (%C); 4.95/5.09 (%H); 9.83/10.08 (%N).

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-(4-methoxyphenyl)acetamide (4f): Yield: 69%, white powder, m.p. 191-193 °C. ¹H NMR (400 MHz, DMSO) δ 10.37 (s, 1H, acetamide NH), 8.22 (d, J = 7.3 Hz, 2H, benzylidene-C_2,6-H), 7.59 – 7.53 (m, 5H, benzylidene =C-H, phenylacetamide-C_2,6-H and 1-phenyl-C_3,5-H), 7.47 – 7.45 (m, 2H, 1-phenyl-C_2,6-H), 7.35 (t, J = 7.3 Hz, 1H, benzylidene-C₄-H), 7.27 (t, J = 7.5 Hz, 2H, benzylidene C_3,5-H), 6.96 (s, 1H, 1-phenyl-C₄-H), 6.90 – 6.88 (m, 2H, phenylacetamide-C_3,5-H), 4.25 (s, 2H, acetamide-CH₂), 3.71 (s, 3H, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 167.92 (s, imidazolinone-CO), 164.52 (s, acetamide-CONH), 164.28 (s, imidazolinone-NCN), 155.34 (s, phenylacetamide-C₄), 137.63 (s, benzylidene-C₁), 133.97 (s, 1-phenyl-C₁), 132.24 (s, phenylacetamide-C₁), 132.08 (s, 1-phenyl-C₄-H), 131.87 (s, 2C, 1-phenyl-C_3,5-H), 129.91 (s, imidazolinone-NCCO), 129.57 (s, benzylidene-C₄-H), 129.36 (s, 2C, benzylidene C_3,5-H), 128.62 (s, 2C, benzylidene C_2,6-H), 127.61 (s, 2C, 1-phenyl-C_2,6-H), 123.24 (s, benzylidene =C-H), 120.57 (s, 2C, phenylacetamide-C_2,6-H), 113.93 (s, 2C, phenylacetamide-C_3,5-H), 55.17 (s, OCH₃), 35.42 (s, actamide-CH₂). EIMS m/z (rel. int. %): 443.60 [M⁺]. Elemental analysis for C₂₅H₂₁N₃O₃S: Calculated/Found: 67.70/67.51 (%C); 4.77/4.85 (%H); 9.47/9.62 (%N).

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-(3,4,5-trimethoxyphenyl) acetamide (4g): Yield: 76%, white powder, m.p. 195-196 °C. ¹H NMR (400 MHz, DMSO) δ 10.44 (s, 1H, acetamide NH), 8.23 (d, J = 7.6 Hz, 2H, benzylidene-C_2,6-H), 7.60 – 7.52 (m, 3H, benzylidene =C-H and benzylidene-C_2,6-H), 7.46 (d, J = 7.6 Hz, 2H, 1-phenyl-C_3,5-H), 7.37-7.27 (m, 3H, 1-phenyl-C_2,6-H and benzylidene-C₄-H), 7.00 (s, 2H, benzylidene-C_3,5-H), 6.96 (s, 1H, 1-phenyl-C₄-H), 4.25 (s, 2H, actamide CH₂), 3.70 (s, 6H, 3,5-OCH₃), 3.60 (s, 3H, 4-OCH₃).¹³C NMR (101 MHz, DMSO) δ 167.88 (s, imidazolinone-CO), 164.95 (s, acetamide-CONH), 164.25 (s, imidazolinone-NCN), 152.75 (s, 2C, phenylacetamide C_3,5), 137.61 (s, benzylidene-C₁), 135.03 (s, phenylacetamide C₄), 133.98 (s, phenylacetamide C₁), 133.58 (s, 1-phenyl C₁), 132.22 (s, imidazolinone-NCCO), 131.91 (s, 2C, 1-phenyl-C_3,5-H), 129.89 (s, 1-phenyl-C₄-H), 129.57 (s, benzylidene-C₄-H), 129.37 (s, 2C, benzylidene-C_3,5-H), 128.64 (s, 2C, benzylidene-C_2,6-H), 127.58 (s, 2C, 1-phenyl-C_2,6-H), 123.27 (s, benzylidene =C-H), 96.89 (s, 2C, phenylacetamide-C_2,6-H), 60.12 (s, 4-OCH₃), 55.69 (s, 3,5-OCH₃), 35.46 (s, acetamide-CH₂). EIMS m/z (rel. int. %): 503.53 [M^+v], 502.29 [M⁺−1]. Elemental analysis for C₂₇H₂₅N₃O₅S: Calculated/Found: 64.40/64.62 (%C); 5.00/5.17 (%H); 8.34/8.52 (%N).

(Z)-2-[(4-Benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]-N-[3-(trifluoromethyl)phenyl]acetamide (4h): Yield: 81%, white powder, m.p. 194-195 °C. ¹H NMR (400 MHz, DMSO) δ 10.88 (s, 1H, actamide NH), 8.18 (d, J = 7.6 Hz, 2H, benzylidene-C_2,6-H), 8.12 (s, 1H, N-phenylacetamide C₂-H), 7.87 (d, J = 8.0 Hz, 1H, N-phenylacetamide C₆-H), 7.57 (m, 4H, 1-phenyl-C_2,3,5,6-H), 7.47 (d, J = 7.2 Hz, 2H, benzylidene-C_3,5-H), 7.42 (d, J = 7.8 Hz, 1H, N-phenylacetamide C₄-H), 7.30 (t, J = 7.3 Hz, 1H, N-phenylacetamide C₅-H), 7.20 (t, J = 7.6 Hz, 2H, benzylidene-C₄-H and 1-phenyl-C₄-H), 6.96 (s, 1H, benzylidene =C-H), 4.30 (s, 2H, acetamide CH₂). ¹³C NMR (101 MHz, DMSO) δ 167.87 (s, imidazolinone CO), 165.80 (s, acetamide CONH), 164.23 (s, imidazolinone-NCN), 139.65 (s, acetamide C₁), 137.59 (s, benzylidene-C₁), 133.94 (s, 1-phenyl-C₁), 132.22 (s, imidazolinone-NCCO), 131.79 (s, 2C, 1-phenyl-C_3,5-H), 130.17 (s, phenylacetamide C₅-H), 130.01 (s, 1-phenyl C₄-H), 129.83 (s, benzylidene-C₄-H), 129.61 (s, 2C, benzylidene-C_3,5-H), 129.54 (q, ²J_c,f = 31.3 Hz, phenylacetamide C₃), 128.51 (s, 2C, benzylidene-C_2,6-H), 127.60 (s, 2C, 1-phenyl-C_2,6-H), 124.05 (q, ¹J_c,f = 272 Hz, 3-CF₃), 123.34 (s, phenylacetamide C₆-H), 122.61 (s, benzylidene =C-H), 119.84, 115.14 (2q, ³J_c,f = 3.9, 4.1 Hz, 2C, phenylacetamide C_2,4-H), 35.40 (s, acetamide CH₂). EIMS m/z (rel. int. %): 481.25 [M⁺]. Elemental analysis for C₂₅H₁₈F₃N₃O₂S: Calculated/Found: 62.36/62.49 (%C); 3.77/3.80 (%H); 8.73/8.94 (%N).

(Z)-N-Benzyl-2-[(4-benzylidene-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]acetamide (4i): Yield: 74%, white powder, m.p. 250-252 °C. ¹H NMR (400 MHz, DMSO) δ 8.86 (t, J = 5.8 Hz, 1H, actamide NH), 8.28 (dd, J = 6.6, 2.8 Hz, 2H, benzylidene-C_2,6-H), 7.59-7.52 (m, 3H, benzylidene =C-H and 1-phenyl C_2,6-H), 7.46– 7.42 (m, 5H, 1-phenyl C_3,5-H, benzylidene-C_3,5-H and benzylidene-C₄-H), 7.26 – 7.18 (m, 5H, benzylacetamide C_2,3,4,5,6-H), 6.99 (s, 1H, 1-phenyl C₄-H), 4.33 (d, J = 5.9 Hz, 2H, benzyl-CH₂), 4.18 (s, 2H, acetamide CH₂). ¹³C NMR (101 MHz, DMSO) δ 168.00 (s, imidazolinone-CO), 166.22 (s, acetamide-CONH), 164.19 (s, imidazolinone-NCN), 138.89 (s, phenylacetamide C₁), 137.67 (s, benzylidene C1), 134.05 (s, 1-phenyl C1), 132.24 (s, imidazolinone-NCCO), 131.89 (s, 2C, 1-phenyl C_3,5-H), 130.00 (s, 1-phenyl C₄-H), 129.54 (s, 2C, benzylidene C_3,5-H), 129.33 (s, benzylidene C₄-H), 128.73 (s, 2C, benzylidene C_2,6-H), 128.20 (s, 2C, phenylacetamide C_3,5-H), 127.60 (s, 2C, 1-phenyl C_2,6-H), 127.15 (s, 2C, phenylacetamide C_2,6-H), 126.78 (s, phenylacetamide C₄-H), 123.22 (s, benzylidene =C-H), 42.65 (s, benzyl CH₂), 34.43 (s, acetamide CH₂). EIMS m/z (rel. int. %): 427.91 [M⁺]. Elemental analysis for C₂₅H₂₁N₃O₂S: Calculated/Found: 70.24/70.43 (%C); 4.95/5.07 (%H); 9.83/10.07 (%N).

(Z)-2-{[4-(4-Chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl]thio}-N-phenylacetamide (4j): Yield: 87%, white powder, m.p. 258-260 °C. ¹H NMR (400 MHz, DMSO) δ 10.50 (s, 1H, acetamide NH), 8.20 (d, J = 8.5 Hz, 2H, benzylidene C_2,6-H), 7.67 (d, J = 7.9 Hz, 2H, phenylacetamide C_2,6-H), 7.60-7.52 (m, 3H, benzylidene =C-H + benzylidene C_3,5-H), 7.47 (d, J = 7.1 Hz, 2H, 1-phenyl C_3,5-H), 7.33 (t, J = 7.8 Hz, 2H, phenylacetamide C_3,5-H), 7.15 (d, J = 8.5 Hz, 2H, 1-phenyl C_2,6-H), 7.08 (t, J = 7.3 Hz, 1H, 1-phenyl C₄-_H), 6.96 (s, 1H, phenylacetamide C₄-H), 4.25 (s, 2H, acetamide CH₂). ¹³C NMR (101 MHz, DMSO) δ 167.74 (s, imidazolinone CO), 165.03 (s, acetamide CONH), 164.93 (s, imidazolinone NCN), 138.98 (s, phenylacetamide C₁), 138.04 (s, benzylidene C₄), 134.29 (s, benzylidene C₁), 133.29 (s, 2C, benzylidene C_2,6-H), 132.90 (s, 1-phenyl C₁), 132.16 (s, imidazolinone NCCO), 129.60 (s, 2C, 1-phenyl C_3,5-H), 129.42 (s, 1-phenyl C₄-H), 128.87 (s, 2C, phenylacetamide C_3,5-H), 128.56, (s, 2C, benzylidene C_3,5-H) 127.60 (s, 2C, 1-phenyl C_2,6-H), 123.49 (s, phenylacetamide C₄-H), 121.59 (s, benzylidene =C-H), 118.95 (s, 2C, phenylacetamide C_2,6-H), 35.51 (s, acetamide CH₂). EIMS m/z (rel. int. %): 447.36 [M⁺]. Elemental analysis for C₂₄H₁₈ClN₃O₂S: Calculated/Found: 64.35/64.47 (%C); 4.05/4.21 (%H); 9.83/10.08 (%N).

(Z)-2-{[4-(4-Chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl]thio}-N-(4-fluorophenyl)acetamide (4k): Yield: 68%, white powder, m.p. 242-244 °C. ¹H NMR (400 MHz, DMSO) δ 10.57 (s, 1H, acetamide NH), 8.20 (d, J = 8.4 Hz, 2H, benzylidene C_2,6-H), 7.69-7.65 (m, 2H, phenylacetamide C_2,6-H), 7.60-7.52 (m, 3H, benzylidene =C-H + benzylidene C_3,5-H), 7.46 (d, J = 7.2 Hz, 2H, 1-phenyl C_3,5-H), 7.17-7.15 (m, 4H, 1-phenyl C_2,6-H and phenylacetamide C_3,5-H), 6.96 (s, 1H, 1-phenyl C₄-H), 4.25 (s, 2H, acetamide CH₂). ¹³C NMR (101 MHz, DMSO) δ 167.73 (s, imidazolinone CO), 164.97 (s, acetamide CONH), 164.90 (s, imidazolinone NCN), 158.12 (d, ¹J _C,F = 240 Hz, phenylacetamide C₄), 138.05 (s, benzylidene C₄), 135.37 (d, ⁴J _C,F = 2.4 Hz, phenylacetamide C₁), 134.31 (s, benzylidene C₁), 133.28 (s, 2C, benzylidene C_2,6-H), 132.92 (s, 1-phenyl C₁), 132.15 (s, imidazolinone NCCO), 129.59 (s, 2C, 1-phenyl C_3,5-H), 129.41 (s, 1-phenyl C₄-H), 128.54 (s, 2C, benzylidene C_3,5-H), 127.58 (s, 2C, 1-phenyl C_2,6-H), 121.60 (s, benzylidene =C-H), 120.73 (d, ³J _C,F = 7.8 Hz, 2C, phenylacetamide C_2,6-H), 115.43 (d, ²J_C,F = 22.3 Hz, 2C, phenylacetamide C_3,5-H), 35.43 (s, acetamide CH₂). EIMS m/z (rel. int. %): 465.53 [M⁺]. Elemental analysis for C₂₄H₁₇ClFN₃O₂S: Calculated/Found: 61.87/62.04 (%C); 3.68/3.89 (%H); 9.02/9.28 (%N).

(Z)-2-{[4-(4-Chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl]thio}-N-(4-chlorophenyl)acetamide (4l): Yield: 88%, white powder, m.p. 248-250 °C. ¹H NMR (400 MHz, DMSO) δ 10.64 (s, 1H, acetamide NH), 8.19 (d, J = 8.6 Hz, 2H, phenylacetamide C_2,6-H), 7.68 (d, J = 8.8 Hz, 2H, benzylidene C_2,6-H), 7.59 – 7.57 (m, 3H, benzylidene =C-H and phenylacetamide C_3,5-H), 7.47 – 7.45 (m, 2H, benzylidene C_3,5-H), 7.39 (d, J = 8.8 Hz, 2H, 1-phenyl C_2,6-H), 7.17 (d, J = 8.6 Hz, 2H, 1-phenyl C_3,5-H), 6.96 (s, 1H, 1-phenyl C₄-H), 4.26 (s, 2H, acetamide CH₂). ¹³C NMR (101 MHz, DMSO) δ 167.72 (s, imidazolinone CO), 165.24 (s, acetamide CONH), 164.88 (s, imidazolinone NCN), 138.03 (s, phenylacetamide C₁), 137.90 (s, benzylidene C₄), 134.32 (s, phenylacetamide C₄), 133.26 (s, 2C, phenylacetamide C_3,5-H), 132.90 (s, benzylidene C₁), 132.14 (s, 1-phenyl C₁), 129.60 (s, 2C, benzylidene C_2,6-H), 129.43 (s, imidazolinone NCCO), 128.79 (s, 2C, 1-phenyl C_3,5-H), 128.55 (s, 2C, benzylidene C_3,5-H), 127.59 (s, 2C, 1-phenyl C_2,6-H), 127.11 (s, 1-phenyl C₄-H), 121.62 (s, benzylidene =C-H), 120.53 (s, 2C, phenylacetamide C_2,6-H), 35.46 (s, acetamide CH₂). EIMS m/z (rel. int. %): 482.72 [M⁺]. Elemental analysis for C₂₄H₁₇Cl₂N₃O₂S: Calculated/Found: 59.76/59.89 (%C); 3.55/3.62 (%H); 8.71/8.84 (%N).

(Z)-N-(4-Bromophenyl)-2-[(4-(4-chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]acetamide (4m): Yield: 85%, white powder, m.p. 252-253 °C. ¹H NMR (400 MHz, DMSO) δ 10.64 (s, 1H, acetamide NH), 8.19 (d, J = 8.6 Hz, 2H, phenylacetamide C_2,6-H), 7.64 – 7.47 (m, benzylidene =C-H, benzylidene C_2,6-H, phenylacetamide C_3,5-H and benzylidene C_3,5-H), 7.47 – 7.45 (m, 2H, 1-phenyl C_2,6-H), 7.17 (d, J = 8.6 Hz, 2H, 1-phenyl C_3,5-H), 6.96 (s, 1H, 1-phenyl C₄-H), 4.25 (s, 2H, acetamide CH₂).¹³C NMR (101 MHz, DMSO) δ 167.70 (s, imidazolinone CO), 165.25 (s, acetamide CONH), 164.87 (s, imidazolinone NCN), 138.30 (s, phenylacetamide C₁), 138.02 (s, benzylidene C₄), 134.31 (s, benzylidene C₁), 133.24 (s, 2C, phenylacetamide C_3,5-H), 132.89 (s, 1-phenyl C₁), 132.13 (s, imidazolinone NCCO), 131.69 (s, 2C, benzylidene C_3,5-H), 129.59 (s, 2C, 1-phenyl C_3,5-H), 129.42 (s, 1-phenyl C₄-H), 128.54 (s, 2C, benzylidene C_3,5-H), 127.58 (s, 2C, 1-phenyl C_2,6-H), 121.61 (s, benzylidene =C-H), 120.89 (s, 2C, phenylacetamide C_2,6-H), 115.08 (s, phenylacetamide C₄), 35.47 (s, acetamide CH₂). EIMS m/z (rel. int. %): 526.20 [M⁺]. Elemental analysis for C₂₄H₁₇BrClN₃O₂S: Calculated/Found: 54.72/54.68 (%C); 3.25/3.34 (%H); 7.98/8.15 (%N).

(Z)-2-{[4-(4-Chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl]thio}-N-(4-tolyl)acetamide (4n): Yield: 65%, white powder, m.p. 240-242 °C. ¹H NMR (400 MHz, DMSO) δ 10.41 (s, 1H, acetamide NH), 8.20 (d, J = 8.3 Hz, 2H, benzylidene C_2,6-H), 7.60-7.54 (m, 5H, benzylidene =C-H, phenylacetamide C_2,6-H and benzylidene C_3,5-H), 7.46 (d, J = 7.2 Hz, 2H, phenylacetamide C_3,5-H), 7.15 – 7.12 (m, 4H, 1-phenyl C_2,3,5,6-H), 6.96 (s, 1H, 1-phenyl C₄-H), 4.23 (s, 2H, acetamide CH₂), 2.25 (s, 3H, CH₃). ¹³C NMR (101 MHz, DMSO) δ 167.98 (s, imidazolinone CO), 165.08 (s, acetamide CONH), 165.04 (s, imidazolinone NCN), 138.20 (s, phenylacetamide C₄), 136.63 (s, phenylacetamide C₁), 134.54 (s, benzylidene C₄), 133.48 (s, 2C, phenylacetamide C_3,5-H), 133.00 (s, benzylidene C₁), 132.81 (s, 1-phenyl C₁), 132.29 (s, 1-phenyl C₄-H), 129.83 (s, 2C, benzylidene C_2,6-H), 129.66 (s, imidazolinone NCCO), 129.42 (s, 2C, 1-phenyl C_3,5-H), 128.79 (s, 2C, benzylidene C_3,5-H), 127.73 (s, 2C, 1-phenyl C_2,6-H), 121.87 (s, benzylidene =C-H), 119.18 (s, 2C, phenylacetamide C_2,6-H), 35.63 (s, acetamide CH₂), 20.59 (s, CH₃). EIMS m/z (rel. int. %): 461.92 [M⁺]. Elemental analysis for C₂₅H₂₀ClN₃O₂S: Calculated/Found: 65.00/64.89 (%C); 4.36/4.52 (%H); 9.10/9.27 (%N).

(Z)-2-{[4-(4-Chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl]thio}-N-(4-methoxyphenyl)acetamide (4o): Yield: 62%, white powder, m.p. 244-245 °C. ¹H NMR (400 MHz, DMSO) δ 10.36 (s, 1H, acetamide NH), 8.21 (d, J = 8.6 Hz, 2H, benzylidene C_2,6-H), 7.61 – 7.52 (m, 5H, benzylidene =C-H, phenylacetamide C_2,6-H and benzylidene C_3,5-H), 7.51 – 7.45 (m, 2H, 1-phenyl C_2,6-H), 7.19 (d, J = 8.6 Hz, 2H, 1-phenyl C_3,5-H), 6.96 (s, 1H, 1-phenyl C₄-H), 6.92 – 6.88 (m, 2H, phenylacetamide C_3,5-H), 4.23 (s, 2H, acetamide CH₂), 3.72 (s, 3H, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 167.75 (s, imidazolinone CO), 164.94 (s, acetamide CONH), 164.47 (s, phenylacetamide C₄), 155.42 (s, imidazolinone NCN), 138.06 (s, benzylidene C₄), 134.30 (s, benzylidene C₁), 133.31 (s, 2C, benzylidene C_2,6-H), 132.92 (s, 1-phenyl C₄-H), 132.16 (s, 2C, phenylacetamide C₂ and 1-phenyl C₁), 129.59 (s, 2C, 1-phenyl C_3,5-H), 129.41 (s, imidazolinone NCCO), 128.59 (s, 2C, benzylidene C_3,5-H), 127.60 (s, 2C, 1-phenyl C_2,6-H), 121.56 (s, benzylidene =C-H), 120.45 (s, 2C, phenylacetamide C_2,6-H), 113.99 (s, 2C, phenylacetamide C_3,5-H), 55.21 (s, OCH₃), 35.43 (s, acetamide CH₂). EIMS m/z (rel. int. %): 477.10 [M⁺]. Elemental analysis for C₂₅H₂₀ClN₃O₃S: Calculated/Found: 62.82/62.68 (%C); 4.22/4.31 (%H); 8.79/8.95 (%N).

(Z)-2-{[4-(4-Chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl]thio}-N-(3,4,5-trimethoxyphenyl)acetamide (4p): Yield: 59%, white powder, m.p. 246-247 °C. ¹H NMR (400 MHz, DMSO) δ 10.44 (s, 1H, acetamide NH), 8.24 (d, J = 8.6 Hz, 2H, benzylidene C_2,6-H), 7.61 – 7.55 (m, 3H, benzylidene =C-H, and benzylidene C_3,5-H), 7.54 – 7.55 (m, 2H, 1-phenyl C_2,6-H), 7.24 (d, J = 8.6 Hz, 2H, 1-phenyl C_3,5-H), 7.04 (s, 2H, phenylacetamide C_2,6-H), 6.97 (s, 1H, 1-phenyl C₄-H), 4.22 (s, 2H, acetamide CH₂), 3.72 (s, 6H, 3,5-OCH₃), 3.62 (s, 3H, 4-OCH₃).¹³C NMR (101 MHz, DMSO) δ 167.70 (s, imidazolinone CON), 164.93 (s, acetamide CONH), 152.82 (s, imidazolinone NCN and phenylacetamide C_3,5), 138.06 (s, phenylacetamide C₄), 135.13 (s, benzylidene C₄), 134.26 (s, benzylidene C₁), 133.61 (s, phenylacetamide C₁), 133.39 (s, 2C, benzylidene C_2,6-H), 132.97 (s, 1-phenyl C₁), 132.13 (s, imidazolinone NCCO), 129.59 (s, 2C, 1-phenyl C_3,5-H), 129.42 (s, 1-phenyl C₄-H), 128.61 (s, 2C, benzylidene C_3,5-H), 127.55 (s, 2C, 1-phenyl C_2,6-H), 121.55 (s, benzylidene =C-H), 96.71 (s, 2C, phenylacetamide C_2,6-H), 60.16 (s, 4-OCH₃), 55.68 (s, 3,5-OCH₃), 35.48 (s, acetamide CH₂). EIMS m/z (rel. int. %): 538.34 [M⁺]. Elemental analysis for C₂₇H₂₄ClN₃O₅S: Calculated/Found: 60.28/60.19 (%C); 4.50/4.62 (%H); 7.81/8.04 (%N).

(Z)-2-{[4-(4-Chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl]thio}-N-[3-(trifluoromethyl) phenyl]acetamide (4q): Yield: 86%, white powder, m.p. 187-188 °C. ¹H NMR (400 MHz, DMSO) δ 10.86 (s, 1H, actamide NH), 8.19 (d, J = 8.4 Hz, 2H, benzylidene C_2,6-H), 8.06 (s, 1H, phenylacetamide C₂-H), 7.94 (s, 1H, phenylacetamide C₆-H), 7.61 – 7.42 (m, 7H, benzylidene =C-H, benzylidene C_3,5-H and 1-phenyl C_2,3,5,6-H), 7.15 (d, J = 8.3 Hz, 2H, phenylacetamide C_4,5-H), 6.96 (s, 1H, 1-phenyl C₄-H), 4.27 (s, 2H, acetamide CH₂).¹³C NMR (101 MHz, DMSO) δ 167.73 (s, imidazolinone CO), 165.77 (s, acetamide CONH), 164.91 (s, imidazolinone NCN), 139.68 (s, phenylacetamide C₁), 138.02 (s, benzylidene C₄), 134.36 (s, benzylidene C₁), 133.44 (s, 1-phenyl C₁), 133.24 (s, 2C, 1-phenyl C_3,5-H), 132.88 (s, imidazolinone NCCO), 132.14 (s, phenylacetamide C₅-H), 130.25 (s, 1-phenyl C₄-H), 129.64 (q, ²J c,f= 32 Hz, phenylacetamide C₃), 129.64 (s, 1-phenyl C₂-H), 129.17 (s, 1-phenyl C₆-H), 128.50 (s, , 2C, benzylidene C_3,5-H), 127.59 (s, 2C, benzylidene C_2,6-H), 124.03 (q, ¹J c,f= 272.3 Hz, 3-CF₃), 122.55 (s, phenylacetamide C₆-H), 121.67 (s, benzylidene =C-H), 119.87, 115.08 (2q, ³J c,f= 3.9, 3.5 Hz, 2C, phenylacetamide C_2,4-H), 35.41 (s, acetamide CH₂). EIMS m/z (rel. int. %): 515.18 [M⁺]. Elemental analysis for C₂₅H₁₇ClF₃N₃O₂S: Calculated/Found: 58.20/58.47 (%C); 3.32/3.46 (%H); 8.14/8.23 (%N).

(Z)-N-Benzyl-2-[(4-(4-chlorobenzylidene)-5-oxo-1-phenyl-4,5-dihydro-1H-imidazol-2-yl)thio]acetamide (4r): Yield: 74%, white powder, m.p. 254-256 °C. ¹H NMR (400 MHz, DMSO) δ 8.86 (t, J = 5.7 Hz, 1H, acetamide NH), 8.29 (d, J = 8.5 Hz, 2H, benzylidene C_2,6-H), 7.59 – 7.42 (m, 7H, benzylidene =C-H, benzylidene C_3,5-H ,1-phenyl C_2,6-H and 1-phenyl C_2,6-H), 7.25-7.00 (m, 5H, benzyl C_2.3.4,5,6-H), 7.00 (s, 1H, 1-phenyl C₄-H), 4.32 (d, J = 5.8 Hz, 2H, benzyl CH₂), 4.17 (s, 2H, acetamide CH₂). ¹³C NMR (101 MHz, DMSO) δ 167.89 (s, imidazolinone CO), 166.30 (s, CONH), 164.84 (s, imidazolinone NCN), 138.87 (s, benzyl C1), 138.10 (s, benzylidene C₄), 134.47 (s, benzylidene C₁), 133.44 (s, 2C, benzylidene C_2,6-H), 133.01 (s, 1-phenyl C₁), 132.17 (s, imidazolinone NCCO), 129.60 (s, 2C, 1-phenyl C_3,4-H), 129.41 (s, 2C, benzylidene C_3,5-H),128.77 (s, 2C, benzyl C_3,5-H), 128.19 (s, 1-phenyl C_2,6-H), 127.60 (s, 2C, benzyl C_2,6-H), 127.27 (s, 1-phenyl C₄-H), 126.84 (s, benzyl C₄-H), 121.62 (s, benzylidene =C-H), 42.76 (s, benzyl CH₂), 34.45 (s, acetamide CH₂). EIMS m/z (rel. int. %): 461.23 [M⁺]. Elemental analysis for C₂₅H₂₀ClN₃O₂S: Calculated/Found: 65.00/65.07 (%C); 4.36/4.53 (%H); 9.10/9.34 (%N).

4.2. Biological activity

The potential of the compounds to inhibit COX1, COX2, and 15-LOX enzymes was tested in vitro using the corresponding inhibitory assay. RAW 264.7 macrophages (ATCC, Manassas, VA) were used to perform cell culture studies. RAW 264.7 cells were cultured in Dulbecco's minimal essential media (DMEM, Invitrogen) containing 10% HI-FBS, sodium pyruvate (1 mM), penicillin (100 I.U./mL), and streptomycin (100 μg/mL). ^[46] The antioxidant (ROS generation) and anti-anti-inflammatory (TNF-α, IL-6, and NF-κB activation) properties of the synthesized compounds were evaluated in macrophages subjected to an 18-hour challenge with LPS (1 μg/mL), as outlined below.

4.2.1. In vitro COX-1/COX-2 inhibitory assay

All novel compounds were screened for their ability to inhibit ovine COX-1 and human recombinant COX-2 isozymes using a COX inhibitor screening assay kit (Item No. 560131, Cayman Chemical, Ann Arbor, MI, USA). Enzyme Immunoassay (EIA) was conducted in triplicates following manufacturer's instructions against the tested compounds, celecoxib, and indomethacin.

4.2.2. In vitro 15-LOX inhibitory assay

A lipoxygenase inhibitor screening assay kit from Cayman Chemical (item no. 760700) was used to test every synthesized compound for its ability to inhibit soybean 15-LOX isozyme. Colorimetric assays were performed in triplicates using quercetin as reference compound.

4.2.3. Assessment of ROS and cytokines production in LPS-activated RAW 264.7 macrophages

RAW 264.7 cells were cultured in black 96-well plates (200,000 cells/mL, 100 μl/well) for 24 hours. Subsequently, the cells were treated with individual test compounds or reference drugs at varying concentrations (12.5, 25, and 50 μM) for 2 hours at 37°C before introducing LPS (1 μg/mL) for 18 hours. The supernatant from the cell culture was collected for TNF-α and IL-6 levels measurement (1:10 dilution) using DuoSet ELISA kits from R&D Systems (Minneapolis, MN, USA) following the manufacturer's instructions. Additionally, ROS levels were measured using the fluorescence probe of oxidative species 2,7-dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes) as detailed in our previous studies ^[47].

4.2.4. Assessment of NF-κB activation in LPS-activated RAW 264.7 macrophages

RAW 264.7 cells treated with the test compounds (25 μM) for 2 hours followed by LPS (1 μg/mL) for 18 hours. Then, were lysed with RIPA buffer containing Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). NF-κB activation (by phosphorylation) was assessed using 10 micrograms of total protein and phospho and total NF-κBb p65 antibodies (#3033 and #8242; 1:1,000; Cell Signaling Technology).

4.2.5. Cell viability assay

To evaluate the possible non-selective cytotoxic effect of the most active compounds compounds (4f, 4k, and 4l), their effect on the cell viability of RAW 264.7 macrophages cells line was assessed at different concentrations (12.5, 25, 50, and 100 μM). Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, Wisconsin) according to the manufactrurer’s instructions.

4.3. Molecular modelling

The X-ray crystal structures of COX-2 (PDB code: 3LN1), 15-LOX (PDB code: 4NRE), were downloaded from the Protein Data Bank (http://www.rcsb.org). MOE 2019.0102 (Chemical Computing Group, Montreal, CA, https://www.chemcomp.com/) was used to implement the molecular docking studies. Each crystal structure was prepared by adding hydrogen, partial charges and protonated 3D. The selected compounds were generated with Chemdraw program, transferred to MOE followed by the addition of partial charges, energy minimization and protonated 3D structure. The docking protocol was validated by redocking the co-crystallized compounds; celecoxib and C8E into 3LN1 and 4NRE, respectively, by measuring the RMSD value. The default docking protocol parameters were used and the docking poses were selected based on both energy scores and 2D and 3D interactions visual inspection.

Supplementary Material

Supinfo1

NIHMS1959936-supplement-Supinfo1.doc^{(83KB, doc)}

Supinfo2

NIHMS1959936-supplement-Supinfo2.docx^{(12.3MB, docx)}

Acknowledgements:

The in vitro anti-inflammatory and antioxidant assays were supported by the National Institute of General Medical Sciences of the National Institutes of Health grant P20GM121334 (D.G.R. and S.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Declaration of competing interest:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supinfo1

NIHMS1959936-supplement-Supinfo1.doc^{(83KB, doc)}

Supinfo2

NIHMS1959936-supplement-Supinfo2.docx^{(12.3MB, docx)}

[R1] [1].Abdellatif KRA; Abdelall EKA; Elshemy HAH; Lamie PF; Elnahaas E; Amin DME, Design, synthesis of new anti-inflammatory agents with a pyrazole core: COX-1/COX-2 inhibition assays, anti-inflammatory, ulcerogenic, histopathological, molecular Modeling, and ADME studies. J. Mol. Struct, 2021, 1240, 130554. [Google Scholar]

[R2] [2].Duan L; Rao X; Sigdel KR; Hindawi, 2019; Vol. 2019. [Google Scholar]

[R3] [3].Shen H-H; Yang Y-X; Meng X; Luo X-Y; Li X-M; Shuai Z-W; Ye D-Q; Pan H-F, NLRP3: a promising therapeutic target for autoimmune diseases. Autoimmun. Rev, 2018, 17, (7), 694–702. [DOI] [PubMed] [Google Scholar]

[R4] [4].Gunaydin C; Bilge SS, Effects of Nonsteroidal Anti-Inflammatory Drugs at the Molecular Level. Eurasian J Med, 2018, 50, (2), 116–121. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] [5].Chandrasekharan NV; Dai H; Roos KL; Evanson NK; Tomsik J; Elton TS; Simmons DL, COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc. Natl. Acad. Sci. U. S. A, 2002, 99, (21), 13926–13931. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] [6].Minghetti L., Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J. Neuropathol. Exp. Neurol, 2004, 63, (9), 901–910. [DOI] [PubMed] [Google Scholar]

[R7] [7].Bozimowski G., A review of nonsteroidal anti-inflammatory drugs. AANA J., 2015, 83, (6). [PubMed] [Google Scholar]

[R8] [8].Osafo N; Agyare C; Obiri DD; Antwi AO, Mechanism of action of nonsteroidal anti-inflammatory drugs. Nonsteroidal anti-inflammatory drugs, 2017, 1, 15. [Google Scholar]

[R9] [9].Smith CJ; Zhang Y; Koboldt CM; Muhammad J; Zweifel BS; Shaffer A; Talley JJ; Masferrer JL; Seibert K; Isakson PC, Pharmacological analysis of cyclooxygenase-1 in inflammation. Proceedings of the National Academy of Sciences, 1998, 95, (22), 13313–13318. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] [10].Gandhi J; Khera L; Gaur N; Paul C; Kaul R, Role of modulator of inflammation cyclooxygenase-2 in gammaherpesvirus mediated tumorigenesis. Frontiers in microbiology, 2017, 8, 538. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] [11].Simon LS, Role and regulation of cyclooxygenase-2 during inflammation. The American journal of medicine, 1999, 106, (5), 37S–42S. [DOI] [PubMed] [Google Scholar]

[R12] [12].Penning TD; Talley JJ; Bertenshaw SR; Carter JS; Collins PW; Docter S; Graneto MJ; Lee LF; Malecha JW; Miyashiro JM, Synthesis and biological evaluation of the 1, 5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1-yl] benzenesulfonamide (SC-58635, celecoxib). J. Med. Chem, 1997, 40, (9), 1347–1365. [DOI] [PubMed] [Google Scholar]

[R13] [13].Talley JJ; Brown DL; Carter JS; Graneto MJ; Koboldt CM; Masferrer JL; Perkins WE; Rogers RS; Shaffer AF; Zhang YY, 4-[5-Methyl-3-phenylisoxazol-4-yl]-benzenesulfonamide, valdecoxib: a potent and selective inhibitor of COX-2. J. Med. Chem, 2000, 43, (5), 775–777. [DOI] [PubMed] [Google Scholar]

[R14] [14].Prasit P; Wang Z; Brideau C; Chan C-C; Charleson S; Cromlish W; Ethier D; Evans J; Ford-Hutchinson A; Gauthier J, The discovery of rofecoxib,[MK 966, VIOXX^®, 4-(4′-methylsulfonylphenyl)-3-phenyl-2 (5H)-furanone], an orally active cyclooxygenase-2 inhibitor. Bioorg. Med. Chem. Lett, 1999, 9, (13), 1773–1778. [DOI] [PubMed] [Google Scholar]

[R15] [15].McGettigan P; Henry D, Cardiovascular risk and inhibition of cyclooxygenase: a systematic review of the observational studies of selective and nonselective inhibitors of cyclooxygenase 2. JAMA, 2006, 296, (13), 1633–1644. [DOI] [PubMed] [Google Scholar]

[R16] [16].Lamie PF; Philoppes JN; Rárová L, Design, synthesis, and biological evaluation of novel 1, 2-diaryl-4-substituted-benzylidene-5 (4H)-imidazolone derivatives as cytotoxic agents and COX-2/LOX inhibitors. Arch. Pharm, 2018, 351, (3-4), 1700311. [DOI] [PubMed] [Google Scholar]

[R17] [17].Abdelall EK; Kamel GM, Synthesis of new thiazolo-celecoxib analogues as dual cyclooxygenase-2/15-lipoxygenase inhibitors: Determination of regio-specific different pyrazole cyclization by 2D NMR. Eur. J. Med. Chem, 2016, 118, 250–258. [DOI] [PubMed] [Google Scholar]

[R18] [18].Arora M; Choudhary S; Singh PK; Sapra B; Silakari O, Structural investigation on the selective COX-2 inhibitors mediated cardiotoxicity: A review. Life Sci., 2020, 251, 117631. [DOI] [PubMed] [Google Scholar]

[R19] [19].Catella-Lawson F; Crofford LJ, Cyclooxygenase inhibition and thrombogenicity. The American journal of medicine, 2001, 110, (3), 28–32. [DOI] [PubMed] [Google Scholar]

[R20] [20].Abdelkhalek AS; Kothayer H; Rezq S; Orabi KY; Romero DG; El-Sabbagh OI, Synthesis of new multitarget-directed ligands containing thienopyrimidine nucleus for inhibition of 15-lipoxygenase, cyclooxygenases, and pro-inflammatory cytokines. Eur. J. Med. Chem, 2023, 115443. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] [21].Liu T; Zhang L; Joo D; Sun S-C, NF-κB signaling in inflammation. Signal Transduction and Targeted Therapy, 2017, 2, (1), 17023. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] [22].Husain A; AlAsmari AF; Azmi SNH; Ali N; Sarker MMR; Alharbi M; Ishtikhar M; Khan SA, Rational drug design, synthesis, and in vivo biological activity of new indolyl-imidazolone hybrids as potential and safer non-steroidal anti-inflammatory agents. Journal of King Saud University-Science, 2022, 34, (4), 102023. [Google Scholar]

[R23] [23].Giorgi M; Kim T-W; Saba A; Rouini M-R; Yun H; Ryschanova R; Owen H, Detection and quantification of cimicoxib, a novel COX-2 inhibitor, in canine plasma by HPLC with spectrofluorimetric detection: Development and validation of a new methodology. J. Pharm. Biomed. Anal, 2013, 83, 28–33. [DOI] [PubMed] [Google Scholar]

[R24] [24].Siwach A; Verma PK, Synthesis and therapeutic potential of imidazole containing compounds. BMC chemistry, 2021, 15, 1–69. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] [25].El-Araby M; Omar A; Hassanein HH; El-Helby A-GH; Abdel-Rahman AA, Design, Synthesis and in Vivo Anti-inflammatory Activities of 2, 4-Diaryl-5-4 H-imidazolone Derivatives. Molecules, 2012, 17, (10), 12262–12275. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] [26].Kumar S; Aghara JC; Manoj A; Alex AT; Mathew AJ; Joesph A, Novel quinolone substituted imidazol-5 (4H)-ones as anti-inflammatory, anticancer agents: Synthesis, biological screening and molecular docking studies. Indian Journal of Pharmaceutical Education and Research, 2020, 54, (3), 771–780. [Google Scholar]

[R27] [27].Elshemy HA; Abdelall EK; Azouz AA; Moawad A; Ali WA; Safwat NM, Synthesis, anti-inflammatory, cyclooxygenases inhibitions assays and histopathological study of poly-substituted 1, 3, 5-triazines: Confirmation of regiospecific pyrazole cyclization by HMBC. Eur. J. Med. Chem, 2017, 127, 10–21. [DOI] [PubMed] [Google Scholar]

[R28] [28].Abdelall EK; Lamie PF; Ahmed AK; EL-Shaymaa E-N, COX-1/COX-2 inhibition assays and histopathological study of the new designed anti-inflammatory agent with a pyrazolopyrimidine core. Bioorg. Chem, 2019, 86, 235–253. [DOI] [PubMed] [Google Scholar]

[R29] [29].Abd-Ellah HS; Abdel-Aziz M; Shoman ME; Beshr EA; Kaoud TS; Ahmed A-SF, Novel 1, 3, 4-oxadiazole/oxime hybrids: Synthesis, docking studies and investigation of anti-inflammatory, ulcerogenic liability and analgesic activities. Bioorg. Chem, 2016, 69, 48–63. [DOI] [PubMed] [Google Scholar]

[R30] [30].Mohassab AM; Hassan HA; Abdelhamid D; Gouda AM; Gomaa HA; Youssif BG; Radwan MO; Fujita M; Otsuka M; Abdel-Aziz M, New quinoline/1, 2, 4-triazole hybrids as dual inhibitors of COX-2/5-LOX and inflammatory cytokines: Design, synthesis, and docking study. J. Mol. Struct, 2021, 1244, 130948. [Google Scholar]

[R31] [31].Moutevelis-Minakakis P; Papavassilopoulou E; Michas G; Georgikopoulou K; Ragoussi M-E; Neophytou N; Zoumpoulakis P; Mavromoustakos T; Hadjipavlou-Litina D, Synthesis, in silico docking experiments of new 2-pyrrolidinone derivatives and study of their anti-inflammatory activity. Biorg. Med. Chem, 2011, 19, (9), 2888–2902. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Dual COX-2 and 15-LOX inhibition study of novel 4-arylidine-2-mercapto-1-phenyl-1H-imidazolidin-5(4H)-ones: Design, synthesis, docking and anti-inflammatory activity

Nermine A Osman

Mostafa K Soltan

Samar Rezq

Joseph Flaherty

Damian G Romero

Ahmed S Abdelkhalek

Abstract

Graphical Abstract

1. INTRODUCTION

Fig 1.

Fig 2.

2. RESULTS AND DISCUSSION

2.1. Chemistry

Scheme 1.

2.2. Biological activity

2.2.1. COX1 and COX2 inhibitory activities

Table 1.

Fig 4.

Fig 3.

2.2.2. In vitro LOX inhibition activity

2.2.3. Effects on ROS production in LPS-activated RAW 264.7 macrophages

Table 2.

2.2.4. Effects on TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages

Table 3.

Fig. 5.

2.2.5. Effect on NF-κB activation in LPS-activated RAW 264.7 macrophages

2.2.6. Cell viability assay

2.3. Molecular modelling

2.3.1. COX-2 docking study

Fig. 6:

Fig 7.

Fig 8.

Fig 9.

Fig 10.

Fig 11.

Table 4:

2.3.2. 15-LOX docking study

Fig 12.

Fig 13.

Fig 14.

Fig 15.

3. CONCLUSION

4. MATERIALS AND METHODS

4.1. Chemistry

4.1.1. General remarks

4.1.2. General scheme for the synthesis of compounds 4a-r

4.2. Biological activity

4.2.1. In vitro COX-1/COX-2 inhibitory assay

4.2.2. In vitro 15-LOX inhibitory assay

4.2.3. Assessment of ROS and cytokines production in LPS-activated RAW 264.7 macrophages

4.2.4. Assessment of NF-κB activation in LPS-activated RAW 264.7 macrophages

4.2.5. Cell viability assay

4.3. Molecular modelling

Supplementary Material

Acknowledgements:

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

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