Design and Synthesis of Novel 1,3,4-Oxadiazole and 1,2,4-Triazole Derivatives as Cyclooxygenase-2 Inhibitors with Anti-inflammatory and Antioxidant Activity in LPS-stimulated RAW264.7 macrophages

Mohamed MS Hamoud; Nermine A Osman; Samar Rezq; Hend A A Abd El-wahab; Abdalla E A Hassan; Hanan A Abdel-Fattah; Damian G Romero; Amany M Ghanim

doi:10.1016/j.bioorg.2022.105808

. Author manuscript; available in PMC: 2024 Mar 26.

Published in final edited form as: Bioorg Chem. 2022 Apr 13;124:105808. doi: 10.1016/j.bioorg.2022.105808

Design and Synthesis of Novel 1,3,4-Oxadiazole and 1,2,4-Triazole Derivatives as Cyclooxygenase-2 Inhibitors with Anti-inflammatory and Antioxidant Activity in LPS-stimulated RAW264.7 macrophages

Mohamed MS Hamoud ^a, Nermine A Osman ^a, Samar Rezq ^b,^c,^d,^e,^f, Hend A A Abd El-wahab ^g, Abdalla E A Hassan ^h, Hanan A Abdel-Fattah ^a, Damian G Romero ^c,^d,^e,^f, Amany M Ghanim ^a,^*

PMCID: PMC10965220 NIHMSID: NIHMS1968898 PMID: 35447409

Abstract

In an attempt to obtain new candidates with potential anti-inflammatory activity, two series of 1,3,4-oxadiazole based derivatives (8a-g) and 1,2,4-triazole based derivatives (10a,b and 11a-g) were synthesized and evaluated for their COX-1/COX-2 inhibitory activity. In vitro assays showed potent COX-2 inhibitory activity and selectivity of the novel designed compounds (IC₅₀ = 0.04 – 0.14 μM, SI = 60.71 – 337.5) compared to celecoxib (IC₅₀ = 0.045 μM, SI = 326.67). The anti-inflammatory and antioxidant activity of the synthesized compounds was investigated via testing their ability to inhibit pro-inflammatory [tumour necrosis factor (TNF-α) and interleukin-6 (IL-6)] and oxidative stress [nitric oxide (NO) and reactive oxygen species (ROS)] markers production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Most of the novel compounds exhibited potent anti-inflammatory and antioxidant activity. In particular, the novel compounds showed excellent IL-6 inhibitory activity (IC₅₀ = 0.96 – 11.14 μM) when compared to celecoxib (IC₅₀ = 13.04 μM) and diclofenac sodium (IC₅₀ = 22.97 μM). Moreover, the most potent and selective COX-2 inhibitor 11c (IC₅₀ = 0.04 μM, SI = 337.5) displayed significantly higher activity against NO and ROS production compared to celecoxib (IC₅₀ = 2.60 and 3.01 μM vs. 16.47 and 14.30 μM, respectively). Molecular modelling studies of the novel designed molecules into COX-2 active sites analysed their binding affinity. In-silico simulation studies indicated their acceptable physicochemical properties and pharmacokinetic profiles. This study suggests that the novel synthesized COX-2 inhibitors exert potent anti-inflammatory and antioxidant activity, highlighting their potential as promising therapeutic agents for the treatment of inflammation and oxidative stress-related diseases.

Keywords: COX-1/ COX-2 inhibitors; 1,3,4-oxadiazole; 1,2,4-triazole; anti-inflammatory; antioxidant

Graphical abstract

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1. Introduction

Inflammation is a defensive response to any cellular damage, resulting in the accumulation of immune cells in order to eliminate the stimulus. Despite that, prolonged inflammation is detrimental and can lead to different pathological disorders such as arthritis, atherosclerosis, neurodegenerative diseases and cancers [1–3]. The inflammatory response starts with the activation of phospholipase A₂, which induces the release of arachidonic acid (AA) from the lipid bilayer of the cell membrane. The rapid metabolism of the AA by the action of cyclooxygenase enzymes (COX-1 and COX-2) into prostaglandin H₂ (PGH₂), which in turn is converted to eicosanoids such as prostaglandin (PG), prostacyclin (PGI₂) and thromboxanes (TXA₂), all of which are responsible for the regulation of inflammatory cytokines generation and development of common inflammation symptoms such as fever, redness, swelling and pain [4,5].

The most frequently used traditional nonsteroidal anti-inflammatory drugs (NSAIDs) act by interfering with the COX pathway in order to block the formation of PGH₂. However, the advantageous therapeutic impact of NSAIDs has been usually limited by the severe gastrointestinal and renal side effects associated with their long-term use. This drawback of classical NSAIDs has been explained by the differential tissue distribution of the COX isoforms. COX-1 is expressed constitutively in gastrointestinal tract and kidneys and executes a significant role in the protection of gastric mucosa and the maintenance of renal perfusion [6], while COX-2 is usually induced by myriad of inflammatory stimuli for example bacterial lipopolysaccharide (LPS), growth factors, and cytokines such as tumour necrosis factor (TNF-α), interleukin-1 (IL-1), interleukin-2 (IL-2), and interleukin-6 (IL-6) [7].

The development of COX-2 selective inhibitors (coxibs) generated potent anti-inflammatory agents with diminished gastrointestinal toxicity (Fig. 1). However, a few years after their release to the market, rofecoxib and valdecoxib were withdrawn due to the increased incidence of myocardial infarction [8,9]. These findings encourage the search for preferential COX-2 inhibitors that can partially inhibit COX-1 to get around the overactivation of the prothrombotic eicosanoid TXA2 observed with selective COX-2 blockade, and hence avoid the potential cardiovascular risks [8,10].

Fig. 1. — Examples of coxibs as selective COX-2 inhibitors.

Many reports claimed that inflammation is frequently combined with oxidative stress, which occurs due to the accumulation of reactive oxygen species (ROS) and nitric oxide (NO). The metabolites of these species can induce inflammatory tissue injury by initiating lipid peroxidation that triggers many changes in permeability, fluidity and ion transport of cellular membranes [11–13]. These findings revealed that targeting the COX-2 inflammatory pathway along with oxidative stress would probably be a more effective strategy to attenuate and control inflammation [14,15].

Diarylheterocyclic scaffolds have been investigated as a common structural feature for many reported selective COX-2 inhibitors, among which 1,3,4-oxadiazole and 1,2,4-triazole functionalities were reported to exhibit potent anti-inflammatory activity. For example, the diaryl-substituted 1,3,4-oxadiazoles I and II [16], and 1,2,4-triazoles III and IV [17,18] showed significant and selective COX-2 inhibition over COX-1 (Fig.2). Alternatively, a number of reports examined the replacement of phenylsulphonyl substituent, which has been known to be essential for inducing COX-2 selectivity [19,20], by a carboxamide linker ended with a hydrophobic moiety such as compounds V [21] and VI [22] which exhibited remarkable selective inhibition against COX-2 (Fig. 2).

Fig. 2. — Reported 1,3,4-oxadiazoles and 1,2,4-triazoles with selective COX-2 inhibitory activity.

On the other hand, nicotinic acid derivatives represent some promising clinically available selective COX-2 inhibitor drugs like niflumic acid and talniflumate (Fig. 3). In addition, hybrids containing different derivatizations of nicotinic acid pulled in consideration within the field of anti-inflammatory drug design such as, compounds VII-IX (Fig. 3) [23–25]

Fig. 3. — Structures of clinically available selective COX-2 inhibitor drugs and some nicotinic acid derivatives with promising anti-inflammatory activity.

Keeping in view the therapeutic significance of combining two bioactive moieties in order to augment the anticipated anti-inflammatory activity, it was interesting to construct a molecular framework that not only provides the common structural feature of selective COX-2 inhibitors, a central di-substituted five-membered heterocycle, but is also hybridized with nicotinamide pharmacophore (Fig. 4). The designed structure allows different hydrophobic tail moieties to be connected to the core (1,3,4-oxadiazole and/or 1,2,4-triazole) by a carboxamide linker. Therefore, it provides the bulkiness necessary to fit into COX-2 rather than the smaller COX-1 active site, potentially enhancing the selectivity towards COX-2.

In this study, we aimed to synthesize novel 1,3,4-oxadiazole and 1,2,4-triazole derivatives and assess their COX-1/COX-2 inhibitory activity along with selectivity. Moreover, using LPS-activated RAW 264.7 macrophages to investigate their anti-inflammatory effect against the production of proinflammatory cytokines (TNF-α and IL-6) as well as their antioxidant activity against the generation of ROS and NO. Finally, molecular modelling was applied to rationalize the obtained biological results.

2. Results and Discussion

2.1. Chemistry

The methodology utilized to synthesize the target compounds 8a-g, 10a,b and 11a-g is outlined in scheme 1 and 2. Reaction of nicotinic acid (1) with benzotriazole in the presence of SOCl₂ in DCM at room temperature gave 1-nicotinoylbenzotriazole (2) which was converted to the ester 3 by the reaction with ethyl 4-aminobenzoate. The resulted ester was subjected to hydrazinolysis and gave the previously reported N-(4-(hydrazinecarbonyl) phenyl)nicotinamide (4) [26] Scheme 1.

Scheme 2. — Synthesis of oxadiazole derivatives 8a-g and triazole derivatives 10a, b and 11a-g. Reagents and conditions (i) CS₂, KOH, EtOH, rt, 12 hrs. (ii) reflux, 15 hrs. (iii) 2-Chloro-N-substituted phenylacetamide derivatives (7a-g), acetone, K₂CO₃, reflux. (iv) filter, dioxane, NH₂NH₂.H₂O, reflux, 20 hrs. (v) Ar’’-CHO, AcOH, reflux.

The 1,3,4-oxadiazole precursor 6 was obtained in a very good yield from the hydrazide 4 through potassium dithiocarbazate intermediate 5 in a single step synthesis. On the other hand, the reflux of the potassium dithiocarbazate 5 with hydrazine hydrate in dioxane gave the 1,2,4-triazole precursor 9 as depicted in Scheme 2. Alkylation of the oxadiazole 6 and the triazole 9 with the previously prepared 2-chloro-N-substituted phenylacetamide derivatives 7a-g in dry acetone under reflux delivered the target S-alkyl derivatives 8a-g and 11a-g respectively. Additionally, the free amino moiety in the triazole 9 was condesed with 4methylbenzaldehyde and 1H-indole-3-carboxaldehyde and provided the 4-arylideneamino-1,2,4-triazoles 10a,b respectively.

2.2. Biological activity

2.2.1. In vitro COX-1 and COX-2 inhibitory assay

Assessment of the target compounds 8a-g, 10a,b and 11a-g for their in vitro inhibition of human COX-1 and COX-2 enzymatic activity was carried out using celecoxib and diclofenac sodium as references. The efficacy for the tested compounds were determined by determining IC₅₀ (μM) (the inhibitor concentration needed to cause 50% enzymatic activity inhibition) and the COX-2 selectivity was calculated as COX-1 IC₅₀/COX-2 IC₅₀. The results are shown in Table 1.

Table 1:

In vitro COX-1 and COX-2 enzyme inhibitory activities and COX-2 selectivity indices of target compounds and references.

Compound	^a COX-1 IC₅₀(μM)	^a COX-2 IC₅₀(μM)	^b COX-2 SI
8a	11.5 ± 1.01^#	0.05 ± 0.01^#	230
8b	8.5 ± 0.60^*	0.14 ± 0.01^#	60.71
8c	11.5 ± 0.85^#	0.05 ± 0.01^#	230
8d	10.5 ± 2.98^#	0.16 ± 0.04^#	65.63
8e	7.5 ± 0.75^*	0.11 ± 0.01^#	68.18
8f	10.0 ± 3.17^*^#	0.08 ± 0.01^#	125
8g	8.5 ± 0.71^*	0.05 ± 0.01^#	170
10a	11.5 ± 0.98^#	0.11 ± 0.02^#	104.55
10b	8.0 ± 0.14^*	0.11 ± 0.01^#	72.73
11a	8.5 ± 0.62^*	0.11 ± 0.01^#	77.27
11b	11.5 ± 1.41^#	0.08 ± 0.01^#	143.75
11c	13.5 ± 1.63^#	0.04 ± 0.01^#	337.5
11d	10.5 ± 3.03^#	0.08 ± 0.01^#	131.25
11e	10.0 ± 2.34^*^#	0.06 ± 0.01^#	166.67
11f	9.5 ± 0.99^*^#	0.09 ± 0.01^#	105.56
11g	10.5 ± 2.25^#	0.08 ± 0.01^#	131.25
Celecoxib	14.7 ± 1.24	0.045 ± 0.02^#	326.67
Diclofenac sodium	3.8 ± 0.63^*	0.84 ± 0.01^*	4.52

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p < 0.05 vs celecoxib.

p < 0.05 vs diclofenac

IC_{50 in} (μM) concentration as expressed as mean ± SEM, for three replications.

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

The results revealed the potent inhibitory activity of the synthesized oxadiazoles (8a-g) and triazoles (10a,b and 11a-g) against COX-2 (IC₅₀ = 0.04 – 0.14 μM ) and COX-1 (IC₅₀ = 7.5 – 13.5 μM ) compared to celecoxib (COX-2 IC₅₀ = 0.045 μM, COX-1 IC₅₀ = 14.7 μM) and diclofenac sodium (COX-2 IC₅₀ = 0.84 μM, COX-1 IC₅₀ = 3.8 μM). With respect to the selectivity towards COX-2, all tested compounds showed significantly improved selectivity (SI = 60.71 – 337.5) compared to the reference NSAID diclofenac (SI = 4.52). The most potent and selective COX-2 inhibitor among the tested compounds was the triazole 11c (IC₅₀ = 0.04 μM, SI = 337.5) which showed a comparable potency and selectivity to the reference celecoxib (IC₅₀ = 0.045 μM, SI = 326.67) and much greater potency and 74-fold higher selectivity than diclofenac sodium (IC₅₀ = 0.84 μM, SI = 4.52).

Based on the obtained in vitro COX-1/COX-2 inhibition assay results, a number of SAR (structure activity relationship) are noticeable. Within the triazole series, it was clear that S-alkyl derivatives (11a-g) have better activity and selectivity towards COX-2 (IC₅₀ = 0.04 – 0.11 μM, SI = 77.27 – 337.5) compared to aryldiene derivatives (10a,b) (IC₅₀ = 0.11, 0.11 μM, SI = 104.55, 72.73 respectively). Comparing SI of the oxadiazole series (8a-g) with their triazole analogues (11a-g) indicated that presence of one methyl group to the hydrophobic tail in para position seems to be more suitable for developing the best selectivity among each of the two series (8c SI = 230, and 11c SI = 337.5). On the other hand, including two methyl groups decreases the selectivity dramatically compared to the mono-methylated analogues as exhibited in pairs 8d/8c (SI = 65.63 vs 230) and 11d/11c (SI = 131.25 vs 337.5). With the exception of the p-acetyl substituent (11a), substitution on the hydrophobic tail at para position improved both COX-2 inhibition and selectivity of the triazole COX-2 inhibitors when compared to their oxadiazole analogues as in pairs 11b/8b (IC₅₀ = 0.08 vs 0.14 μM, SI = 143.75 vs 60.71), 11c/8c (IC₅₀ = 0.04 vs 0.05 μM, SI = 337.5 vs 230) and 11e/8e (IC₅₀ = 0.06 vs 0.11 μM, SI = 166.67 vs 68.18). While substitution at meta- or ortho- positions gave the preference to the oxadiazole derivatives as in compounds 8f/11f (IC₅₀ = 0.08 vs 0.09 μM, SI = 125 vs 105.56) and 8g/11g (IC₅₀ = 0.05 vs 0.08 μM, SI = 170 vs 131.25, respectively). Among the halogenated oxadiazole series, the chlorinated derivative 8g exhibited enhanced activity and selectivity towards COX-2 (IC₅₀ = 0.05 μM, SI = 170) compared to bromo- and fluoro- derivatives 8f,e (IC₅₀ = 0.08, 0.11 μM, SI = 125, 68.18 respectively). While the fluorinated triazole 11e displayed better COX-2 activity and selectivity (IC₅₀ = 0.06 μM, SI = 166.67) related to chlorinated and brominated derivatives 11g,f (IC₅₀ = 0.08, 0.09 μM, SI = 131.25, 105.56 respectively).

2.2.2. Inhibitory activity against NO and ROS production in LPS-activated RAW 264.7 macrophages.

Endogenous nitric oxide (NO) is one of the proinflammatory mediators that is produced from activated macrophages during the inflammatory response. NO plays an important role in maintaining chronic inflammatory reactions either by switching cell death from apoptosis into necrosis [27] and/or upregulating COX-2 gene expression [28]. Additionally, NO can react with superoxide to produce peroxynitrite and increase oxidative stress, a major contributor to the pathogenesis of inflammatory diseases [29]. Treatment of RAW 264.7 macrophages with Gram-negative bacterial lipopolysaccharide (LPS) stimulates the formation of NO via inducing the expression of inducible nitric oxide synthase (iNOS). Also, LPS-stimulated RAW264.7 macrophages generate reactive oxygen species (ROS), including hydrogen peroxide, superoxide radicals and hydroxyl radicals. The activation of polymorphonuclear neutrophils (PMNs) during the elevated inflammatory response increases ROS production leading to molecular oxidative damage and progression of inflammatory disorders [30]. Therefore, inhibition of NO and ROS production has a great influence on developing novel anti-inflammatory agents. Interestingly, multiple studies reported the antioxidant potential of both 1,3,4-oxadiazole and 1,2,4-triazole derivatives [31–35]. We investigated the influence of the newly synthesized COX inhibitors on the production of NO and ROS in LPS-induced RAW 264.7 macrophages (Table 2).

Table 2:

In vitro inhibitory activities of target compounds on LPS-induced NO and ROS production in RAW 264.7 cells.

Compound	NO IC₅₀ (μM)	ROS IC₅₀ (μM)
8a	11.37±0.02^*^#	25.55 ± 0.09^*
8b	0.40 ± 0.80^*^#	0.03 ± 0.69^*^#
8c	18.90 ± 0.25^*^#	12.09 ± 0.03^*^#
8d	60.23±0.06^*^#	14.09 ± 0.03^#
8e	>100 μM	67.22 ± 0.06^*^#
8f	>100 μM	8.06 ± 0.09^*^#
8g	47.33±0.03^*^#	9.00±0.11^*^#
10a	47.17±0.03^*^#	0.49 ± 0.74^*^#
10b	2.19± 1.32^*^#	1.78 ± 0.51^*^#
11a	1.22±1.25^*^#	9.69 ± 0.93^*^#
11b	1.92±1.27^*^#	3.31± 0.37^*^#
11c	2.60±1.24^*^#	3.01± 1.06^*^#
11d	9.58±0.26^*^#	44.34± 0.09^*^#
11e	10.11±0.07^*^#	27.91 ± 0.08^*^#
11f	92.52±0.13^*^#	9.77± 0.08^*^#
11g	7.83±0.09^*^#	0.07±0.78^*^#
Celecoxib	16.47±0.07	14.30±0.18
Diclofenac sodium	29±0.01^*	25.35±0.05^*

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IC_{50 in} (μM) concentration as expressed as mean ± SEM, for three replicates.

p < 0.05 vs celecoxib.

p < 0.05 vs diclofenac. NO (Nitric Oxide); ROS (Reactive Oxygen Species).

Among the displayed results in Table 2, the oxadiazole 8b exhibited the best inhibitory effect against both NO and ROS production (IC₅₀ = 0.40 and 0.03 μM, respectively) showing more than 41-fold better NO IC₅₀ and about 476-fold lower ROS IC₅₀ values to the reference celecoxib (IC₅₀ = 16.47 and 14.30 μM, respectively) (Fig. 5). This potent antioxidant activity of 8b could be explained by the strong free radical scavenging properties exerted by p-nitro substitution at the phenyl ring [36,37]. On the other hand, it was notable that oxadiazole derivatives – except 8b – showed lower NO inhibition than their triazole analogues as shown in pairs 8a/11a (IC₅₀ = 11.37 vs 1.22 μM), 8c/11c (IC₅₀ = 18.90 vs 2.60 μM), 8d/11d (IC₅₀ = 60.23 vs 9.58 μM), 8e/11e (IC₅₀ = >100 vs 10.11 μM), 8f/11f (IC₅₀ = >100 vs 92.52 μM) and 8g/11g (IC₅₀ = 47.33 vs 7.83 μM). Halogenated derivatives in oxadiazole series (8e-g) and triazole series (11e-g) inhibited NO production to lower extent in comparison to other monosubstituted derivatives within each series. All tested compounds - except 8a,e and 11d,e - revealed improved inhibition for ROS production (IC₅₀ = 0.03 – 14.09 μM ) compared to the references celecoxib (IC₅₀ = 14.30 μM) and diclofenac sodium (IC₅₀ = 25.35 μM).

Fig. 5. — Effect of compound 8b on the production of ROS and NO in LPS-stimulated RAW264.7 macrophages. Measurements of ROS (A, C) and NO (B, D) were performed in RAW264.7 macrophages that were pretreated with different concentrations (12.5, 25, 50 or 100 μM) of 8b (A, B) or celecoxib (C, D) for 2 h followed by LPS challenge (1 μg/mL) for an additional 20 h. The results shown are representative of three independent experiments. *p < 0.05 vs untreated controls (vehicle). #p < 0.05 vs LPS-treated cells. NO (Nitric Oxide); ROS (Reactive Oxygen Species).

Interestingly, the most potent and selective COX-2 inhibitor 11c displayed significantly higher activity against ROS and NO production (IC₅₀ = 3.01 and 2.60 μM respectively) when compared to celecoxib (IC₅₀ = 14.30 and 16.47 μM respectively) consequently, providing better antioxidant activity (Fig. 6A and B).

Fig. 6. — Effect of compound **11c** on the production of ROS, NO, TNF-α, IL-6 in LPS-stimulated RAW264.7 macrophages. Measurements of ROS (A), NO (B), TNF-α (C), and IL-6 (D) were performed in RAW264.7 macrophages that were pretreated with different concentrations (12.5, 25, 50 or 100 μM) of **11c** or celecoxib for 2 h followed by LPS challenge (1 μg/mL) for an additional 20 h. The results shown are representative of three independent experiments. *p < 0.05 vs celecoxib. IL-6 (interleukin-6); NO (Nitric Oxide); ROS (Reactive Oxygen Species); TNF-α (Tumour necrosis factor-α).

2.2.3. Inhibitory activity against TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages.

Activated macrophages are the main source of proinflammatory cytokines such as tumour necrosis factor (TNF) and interleukins (ILs) [38]. Among these cytokines, IL-6 has a great importance in activating the immune system and boosting the inflammatory response [39]. Moreover, TNF-α is highly involved in the pathophysiological processes occurring in many inflammatory diseases like osteoarthritis [40]. We utilized LPS-activated RAW 264.7 macrophages to investigate the anti-inflammatory effect of the synthesized compounds via testing their inhibitory potential against LPS-induced proinflammatory cytokines (TNF-α and IL-6) production, and results are summarized in 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 IC₅₀ (μM)	IL-6 IC₅₀ (μM)
8a	17.88±1.07^*^#	1.40±0.66^*^#
8b	2.76±1.23	5.63±0.33^*^#
8c	3.51±1.17	2.43±1.15^*^#
8d	1.87±1.89^*^#	1.04±0.50^*^#
8e	3.37±1.23	1.95±0.39^*^#
8f	8.29±1.04	1.93±0.67^*^#
8g	10.31±1.14^*	7.76±0.04^*^#
10a	7.73±1.07	0.96±0.65^*^#
10b	34.62 ±1.10^*^#	49.74±0.23^*^#
11a	19.70±1.06^*^#	16.98±0.01^*^#
11b	7.05±1.08	9.68±0.01^*^#
11c	11.11±1.07^*	11.14±0.01^#
11d	4.30±1.06^*	9.64±0.11^*^#
11e	7.08±1.11	1.66±0.24^*^#
11f	3.01±1.06	5.63±0.03^*^#
11g	35.36±1.00^*^#	5.61±0.03^*^#
Celecoxib	5.55±1.08	13.04±0.01
Diclofenac sodium	7.46±1.27	22.97±0.07^*

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IC₅₀ (μM) is expressed as mean ± SEM, for three replicates.

p < 0.05 vs celecoxib.

p < 0.05 vs diclofenac. TNF-α (Tumour necrosis factor-α); IL-6 (interleukin-6).

The results clearly demonstrated that all tested compounds – except 10b and 11a – exhibited excellent IL-6 inhibitory activity (IC₅₀ = 0.96 – 11.14 μM) when compared to celecoxib (IC₅₀ = 13.04 μM) and diclofenac sodium (IC₅₀ = 22.97 μM) as references. Compound 11c revealed a considerable TNF-α IC₅₀ value (11.11 μM) with a remarkable activity against IL-6 (IC₅₀ = 11.14 μM) (Fig. 6C and D).

It was remarkable that the oxadiazole series (8a-g) had enhanced activity against the production of TNF-α and IL-6 compared to their analogues in the triazole series (11a-g) with only a few exceptions (8f/11f: TNF-α IC₅₀ = 8.29 vs. 3.01 μM, 8g/11g: IL-6 IC₅₀ = 7.76 vs. 5.61 μM, and 8e/11e: IL-6 IC₅₀ = 1.95 vs. 1.66 μM).

2.3. In-silico study

2.3.1. Molecular Modelling

To study and simulate the behaviour of the designed molecules toward COX-2 enzyme, the orientation of the designed compounds (8a-g), (10a,b) and (11a-g) within the active binding site of the cyclooxygenase-2 enzyme (COX-2) co-crystallized with SC-558 (PDB entry 1CX2) [41] were investigated using Molecular Operating Environment (MOE) version MOE 2020.09 (Chemical Computing Group, Montreal, CA) Docking setup was first validated by re-docking of the co-crystallized ligand SC-558. The redocking RMSD value is 0.647 Å with energy score (S) = −7.639 kcal/mol. All the key interactions accomplished by the co-crystallized ligand SC-558 with the key amino acids in the binding site are reproducible using the followed docking setup. The validated setup was then used to investigate the ligand-receptor interactions at the binding site for the compounds 8a-g, 10a,b and 11a-g.

Most of the protein-ligand interactions in COX-2 active site are stabilized by van der Waals forces at the hydrophobic pocket formed by Tyr348, Phe381, Leu384, Tyr385, Trp387, Gly526, Ala527 and Ser530 amino acids. COX-2 binding site contains another pocket, which is mainly responsible for inducing selectivity for COX-2, composed of amino acids His90, Gln192, Leu352, Ser353, Arg513, Ala516, Ile517, Phe518 and Val523. This “selectivity pocket” is occupied by the phenylsulphonamide moiety of SC-558, where it is easily accessible due to the presence of smaller residue Val523, which provide enough space for binding with the COX-2 inhibitor. On the contrary, COX-1 active site has a reduced volume side pocket, due to substitution of valine to isoleucine at the same position-523 [42–45].

The docking scores for the compounds 8a-g, 10a,b and 11a-g within COX-2 active site ranged from −6.748 to −4.613 kcal/mol. All the compounds fitted at the COX-2 active site, where the hydrophobic tail placed in the hydrophobic pocket. The 2-(thio)acetamide linker placed in the vicinity of the selectivity pocket and formed hydrogen bonds with one or two key amino acids as in compounds 8a-c and 11b-e (supporting material Table 1). This could explain why triazole derivatives 10a and 10b, which lack the 2-(thio)acetamide linker, have lower COX-2 activity and selectivity than the S-alkyl triazole series (11a-g).

Based on COX-2 enzyme assay experimental results, the most active and selective compound was 11c (IC₅₀= 0.04 μM, SI=337.5). These results agree with the virtual screening results where compound 11c was well placed in the active site and showed hydrogen bond interaction with His 90 through the oxygen of the 2-(thio)acetamide group and Pi-H interactions with Asp 515 and His 90 via phenyl and triazole rings respectively. Moreover, the aminotriazole group enhanced the selectivity through the hydrogen bonding of the amino group with Gln192 and Ser353 with a score of −5.522 Kcal/mol (Fig. 7).

Fig. 7. — 2-D and 3-D binding interaction pattern of **11c** in the binding site of COX-2 (PBD:1CX2).

2.3.2. Molecular dynamic simulation

To validate docking poses of 11c, it was subjected to 50 ns molecular dynamic simulation (MDS). The simulation of 11c-protien complex showed that compound 11c achieved good stability inside the COX-2 binding site over 29.6 ns of the simulation time with an average RMSD of 2.5 Å from the starting docking pose. Thereafter, it began to deviate significantly with elevated fluctuations to reach its maximum deviation at 30.7 ns (RMSD = 5.3 Å). At 42.7 ns its deviations returned back to be around RMSD = 1.6 Å until the end of simulation. Compound 11c showed good binding stability inside the COX-2 binding site, however, it was less than that of the co-crystalized ligand SC-558 (PDB entry 1CX2) which maintained stable orientation over the whole simulation with lower deviations (average RMSD = 0.88 Å) (Fig. 8). Accordingly, its estimated absolute binding free energy (ΔG_binding) with COX-2 was higher than that of the co-crystalized ligand SC-558 (PDB entry 1CX2) (ΔG_binding = −7.6 and –9.9 kcal/mol).

Fig. 8. — RMSDs of both compound **11c** and the co-crystalized ligand SC-558 (PDB entry 1CX2) inside the active site of Cox-2 over 50 ns of MDS (A). RMSF of the protein bound to **11c** and the co-crystalized ligand SC-558 (PDB entry 1CX2) (B).

Analyzing the simulation results and comparing with docking pose with lowest binding free energy of the selected compound 11c, the hydrogen bond interaction with Ser353 and Gln 192 either directly or through water bridge was maintained by interaction percentage 50%, 40% respectively through the 50 ns simulation. Intrestingly, an additional hydrogen bond with Arg 513, Asp 515 with interaction percentage 100% either directly or through water bridge and hydrophobic bond interaction with val 523 and Phe 513 with interaction percentage 80, 60 % respectively was observed. These additional interactions with the above mentioned amino acids confirms the binding of compound 11c in the selectivity pocket of COX-2 enzyme and explains the selectivity of compound 11c. (Fig. 9).

Fig. 9. — **Protein-11c** contacts inside the COX-2 binding site over 50 ns of MDS

2.3.3. In-silico prediction of physicochemical properties and pharmacokinetic profile.

Molinspiration Chemoinformatics server [46] was used to predict the oral bioavailability of the newly synthesized compounds in the light of Lipinski’s rule of five, topological polar surface area (TPSA), and the number of rotatable bonds (NROTB) (supporting material Table 2). The examined compounds – except 8b, 8f, 11b, and 11f - did not violate Lipiniski’s rule, hence revealing appropriate oral bioavailability.

Pre-ADMET software [47] estimates the rate and extent of human intestinal absorption (HIA) via utilizing in vitro assays such as Caco2 (derived from human colon adenocarcinoma) or MDCK (Madin-Darby Canine Kidney) cell-based studies. Besides, it can predict in vivo data on blood brain barrier (BBB) penetration and percent of drug bound to plasma protein (PPB) (supporting material Table 3). The calculated parameters indicate that all studied compounds showed excellent human intestinal absorption (82.23 % – 98.34 %) as well as celecoxib (96.69%). The tested compounds also showed low potential to cross the BBB (permeability values 0.03 – 0.15) and strong plasma protein binding capacity (93.82 % - 100 %). Finally, all compounds were predicted to be non-substrate/non-inhibitors of CYP2D6 enzyme, which is involved in the metabolism of numerous drugs, indicating the minimal possibility of drug-drug interaction. Collectively, the in-silico simulation studies showed acceptable physicochemical properties and pharmacokinetic profiles for the novel synthesized compounds.

3. Conclusion

To summarize, two series of 1,3,4-oxadiazole derivatives (8a-g) and 1,2,4-triazole derivatives (10a,b and 11a-g) were synthesized and evaluated in vitro as COX-1/COX-2 inhibitors. All compounds exhibited potent and selective COX-2 inhibitory profiles. The triazole 11c was the most potent and selective COX-2 inhibitor among the tested compounds (IC₅₀ = 0.04, SI = 337.5), showing a comparable potency and selectivity to the reference celecoxib. LPS-activated RAW 264.7 macrophages were utilized to investigate the anti-inflammatory effect of the synthesized compounds via testing their inhibitory potential against production of NO, ROS, TNF-α and IL-6. The oxadiazole 8b exhibited remarkable inhibitory activity against both NO and ROS production (IC₅₀ = 0.40 and 0.03 μM, respectively), showing more than 41-fold better NO IC₅₀ and about 476-fold lower ROS IC₅₀ values to the reference celecoxib. Compound 11c displayed significantly higher activity against NO and ROS production (IC₅₀ = 2.60 and 3.01 μM, respectively) when compared to celecoxib (IC₅₀ = 16.47 and 14.30 μM, respectively) hence, offering improved antioxidant activity. Most of the tested compounds showed excellent IL-6 inhibition (IC₅₀ = 0.96 – 11.14 μM) when compared to celecoxib (IC₅₀ = 13.04 μM) and diclofenac sodium (IC₅₀ = 22.97 μM) as references. Molecular modelling studies explored the binding affinity of the designed molecules into COX-2 active sites and MD simulation was performed for validation of the docking results and further explation of the COX-2 selectivity of compound 11c. Moreover, the designed compounds showed promising physicochemical properties and pharmacokinetic profiles in accordance with the results obtained from in-silico simulation studies. Based on the findings in this study, the novel synthesized COX-2 inhibitors reported are potent COX-2 inhibitors and moderate COX-1 inhibitors with a strong antioxidant and anti-inflammatory activity. These novel COX-2 inhibitors represent promising novel therapeutic agents to overcome the limitations of current COX-2 inhibitors.

4. Experimental

4.1. Chemistry

All chemical reagents were purchased from commercial sources with the highest purity available. The melting points (°C) of the synthesized compounds were determined in open capillaries using Stuart Melting Point apparatus and are uncorrected. NMR spectra, IR spectra and elemental analyses (C, H, N) were carried out at Applied Nucleic Acid Research Center, Faculty of Sciences, Zagazig University, Zagazig, Egypt. Mass spectra were carried out at the Regional Center of Mycology and Biotechnology, Al-Azhar University, Nasr City, Egypt. The IR-spectra (KBr, cm⁻¹) of the compounds were recorded on Bruker Alpha FT-IR spectrometer. ¹H NMR and ¹³C APT NMR spectra were recorded on Bruker high performance Digital FT-NMR spectrometer advance III 400 MHz using dimethyl sulfoxide (DMSO)-d₆ as solvent. Chemical shifts are reported in δ (ppm) relative to the internal tetramethylsilane (TMS) standard. Mass spectra were obtained using a GC/MS Mat 112 S mass spectrometer under EI⁺ ionization technique/mode. Elemental analyses were determined using the Vario MICRO cube (Elementar) CHNS analyzer. The HRMS and LC-HRMS were recorded on LC/Q-TOF, 6530 (Agilent Technologies, Santa Clara, CA, USA) at Faculty of pharmacy, Fayoum University. All reactions were monitored by thin layer chromatography (TLC) (Rf) on silica gel 60 GF245 (E-Merck, Germany) using an UV lamp for visualization at a wavelength (λ) of 254 nm. Compounds 7a-g were prepared according to reported methods [48].

N-(4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl) phenyl)nicotinamide (6).

A solution of the hydrazide 4 (0.512 g, 2 mmol) in 10 ml of absolute ethanol was treated with a solution of KOH (0.112 g, 2 mmol) in 2 ml H₂O and CS₂ (0.296 ml, 6 mmol) and stirred at room temprature for 12 hrs. Then, the mixture was heated under reflux for 15 hrs. The solvent was evaporated. The residue was dissolved in H₂O, filtered, and acidified with glacial acetic acid. The precipitate was filtered off, washed with H₂O, and recrystallized from absolute ethanol. Yellow crystals (76 % yield). M.p. 280–282 °C. IR: ν_max/cm⁻¹; 3276, 3065, 1661, 1603, 1501. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 14.66 (s, H, NH, exchangeable with D₂O), 10.76 (s, 1H, NH, exchangeable with D₂O), 9.12 (d, J = 1.8 Hz, 1H, C₂-H of pyridine), 8.78 (dd, J =4.7, 1.8 Hz, 1H, C₆-H of pyridine), 8.31 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 8.00 (d, J = 8.8 Hz, 2H, Ar-H), 7.90 (d, J = 8.8 Hz, 2H, Ar-H), 7.59 (dd, J =8.0, 4.7 Hz, 1H, C₅-H of pyridine). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 177.31 (CS), 164.5 (CO), 160.4 (C2-oxadiazole), 152.4 (CH, pyridine), 148.7 (CH, pyridine), 142.2 (Ar-C), 135.6 (CH, pyridine), 130.3 (C3 pyridine), 126.9 (Ar-CH), 123.5 (CH, pyridine), 120.4 (Ar-CH), 117.6 (Ar-C). Analysis calcd. for C₁₄H₁₀N₄O₂S: C, 56.37; H, 3.38; N, 18.78. Found: C, 56.46; H, 3.42; N, 18.82.

General procedure for the synthesis of N-(4-(5-((2-((substituted phenyl)amino)-2-oxoethyl) thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8a-g)

To a solution of the oxadiazole 6 (0.60 g, 2 mmol) and K₂CO₃ (0.55 g, 4 mmol) in 10 ml dry acetone, the appropriate 2-chloro-N-substituted phenyl acetamide derivatives 7a-g (2 mmol) was added and heated under reflux for 4 hrs. The progress of the reaction was monitored by TLC, and after the completion of the reaction, the reaction mixture was concentrated under reduced pressure. The precipitate was washed with water then filtered off and purified by crystalization in methanol to obtain compounds 8a-g.

N-(4-(5-((2-((4-Acetylphenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8a).

White crystals (86 % yield). M.p. 195–197°C. IR: ν_max/cm⁻¹; 3325, 3065, 2959, 1669, 1591, 1520. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.77 (s, 1H, NH, exchangeable with D₂O), 10.76 (s, 1H, NH, exchangeable with D₂O), 9.12 (s, 1H, C₂-H of pyridine), 8.78 (app.s, 1H, C₆-H of pyridine), 8.31 (d, J = 7.0 Hz, 1H, C₄-H of pyridine), 8.04 – 7.91 (m, 6H, Ar-H), 7.73 (d, J = 7.8 Hz, 2H, Ar-H), 7.59 (app.s, 1H, C₅-H of pyridine), 4.38 (s, 2H, CH₂), 2.53 (s, 3H, CH₃). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 196.5 (CO), 165.6 (CO), 165.0 (CO), 164.5 (C5-oxadiazole), 162.8 (C2-oxadiazole), 152.4 (CH, pyridine), 148.7 (CH, pyridine), 142.9 (Ar-C), 142.2 (Ar-C), 135.6 (CH, pyridine), 132.1 (Ar-C), 130.3 (C3 pyridine), 129.6 (Ar-CH), 127.2 (Ar-CH), 123.6 (CH, pyridine), 120.4 (Ar-CH), 118.4 (Ar-CH), 118.0 (Ar-C), 36.9 (CH₂), 26.4 (CH₃). Analysis calcd. for C₂₄H₁₉N₅O₄S: C, 60.88; H, 4.04; N, 14.79. Found: C, 60.97; H, 4.18; N, 14.93. HRMS (ESI): m/z cald C₂₄H₁₉N₅O₄S (M+H)⁺= 474.12305, found 474.12337.

N-(4-(5-((2-((4-Nitrophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8b)

White crystals (92 % yield). M.p. 168–170°C. IR: ν_max/cm⁻¹; 3297, 3060, 2954, 1650, 1600, 1531. 1350. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.05 (s, 1H, NH, exchangeable with D₂O), 10.76 (s, 1H, NH, exchangeable with D₂O), 9.12 (d, J= 1.8 Hz, 1H, C₂-H of pyridine), 8.78 (dd, J= 4.8, 1.8 Hz, 1H, C₆-H of pyridine), 8.31 (d, J= 8.0 Hz, 1H, C₄-H of pyridine), 8.25 (d, J= 9.2 Hz, 2H, Ar-H), 8.04–7.92 (m, 4H, Ar-H), 7.85 (d, J= 9.2 Hz, 2H, Ar-H), 7.59 ((dd, J= 8.0, 4.8 Hz, 1H, C₅-H of pyridine), 4.41 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 166.1 (CO), 165.0 (CO), 164.5 (C5-oxadiazole), 162.7 (C2-oxadiazole), 152.4 (CH, pyridine), 148.7 (CH, pyridine),144.7 (Ar-C), 142.5 (Ar-C), 142.2 (Ar-C), 135.6 (CH, pyridine), 130.3 (C3 pyridine), 127.2 (Ar-CH), 125.1 (Ar-CH), 123.5 (CH, pyridine), 120.4 (Ar-CH),118.9 (Ar-CH), 118.0 (Ar-C), 36.9 (CH₂). Analysis calcd. for C₂₂H₁₆N₆O₅S: C, 55.46; H, 3.38; N, 17.64. Found: C, 55.61; H, 3.52; N, 17.56. HRMS (ESI): m/z cald C₂₂H₁₆N₆O₅S ([M+H] ⁺) 477.09756, found 477.09816.

N-(4-(5-((2-((4-Methylphenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8c).

Yellow crystals (84% yield). M.p. 215–217 °C. IR: ν_max/cm⁻¹;3322, 3034, 2970, 2911, 1725, 1667, 1319. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.76 (s, 1H, NH, exchangeable with D₂O), 10.34 (s, 1H, NH, exchangeable with D₂O), 9.12 (d, J= 1.6 Hz, 1H, C₂-H of pyridine), 8.79 (dd, J= 4.8, 1.6 Hz, 1H, C₆-H of pyridine), 8.31 (d, J= 8.1 Hz, 1H, C₄-H of pyridine), 8.09–7.95 (m, 4H, Ar-H), 7.59 (dd, J = 8.1, 4.8 Hz, 1H, C₅-H of pyridine ), 7.47 (d, J = 8.4 Hz, 2H, Ar-H), 7.13 (d, J = 8.4 Hz, 2H, Ar-H), 4.32 (s, 2H, CH₂), 2.26 (s, 3H, CH₃). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 164.8 (CO), 164.6 (CO), 164.4 (C5-oxadiazole), 163.0 (C2-oxadiazole), 152.4 (CH, pyridine), 148.8 (CH, pyridine),142.2 (Ar-C), 138.3 (Ar-C), 135.6 (CH, pyridine), 132.8 (Ar-C), 130.3 (C3 pyridine), 129.6 (Ar-CH), 127.3 (Ar-CH), 123.6 (CH, pyridine), 120.5 (Ar-CH),119.2 (Ar-CH), 118.1 (Ar-C), 36.8 (CH₂), 20.8 (CH₃). Analysis calcd. for C₂₃H₁₉N₅O₃S: C, 62.01; H, 4.30; N, 15.72. Found: C, 62.16; H, 4.51; N, 15.93. HRMS (ESI): m/z cald C₂₃H₁₉N₅O₃S ([M+H] ⁺) 446.12813, found 446.12832.

N-(4-(5-((2-((2,5-Dimethylphenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl) nicotinamide (8d).

White crystals (79 % yield). M.p. 255–257 °C. IR: ν_max/cm⁻¹; 3364, 3232, 2993, 2923, 1725, 1646, 1537. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.68 (s, 2H, 2 x NH, exchangeable with D₂O), 9.11 (d, J = 1.6 Hz, 1H, C₂-H of pyridine), 8.77 (dd, J = 4.8, 1.6 Hz, 1H, C₆-H of pyridine), 8.31 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 7.88–7.80 (m, 4H, Ar-H), 7.58 (dd, J = 8.0, 4.8 Hz, 1H, C₅-H of pyridine ), 8.25 (d, J = 7.7 Hz, 1H, Ar-H), 7.18 (d, J = 7.7 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 4.28–4.16 (m, 2H, CH₂), 2.31 (s, 3H, CH₃), 2.15 (s, 3H, CH₃). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 171.3 (CO), 164.4 (CO), 163.2 (C5-oxadiazole), 162.6 (C2-oxadiazole), 152.3 (CH, pyridine), 148.8 (CH, pyridine), 141.7 (Ar-C), 136.1 (Ar-C), 135.6 (Ar-CH), 134.0 (Ar-C), 132.7 (Ar-C), 130.6 (CH, pyridine), 130.3 (C3 pyridine), 129.8 (Ar-CH), 128.9 (Ar-CH), 128.5 (Ar-C), 128.1 (Ar-CH), 123.5 (Ar-CH), 119.5 (CH, pyridine), 32.6 (CH₂), 20.3 (CH₃), 16.7 (CH₃). Analysis calcd. for C₂₄H₂₁N₅O₃S: C, 62.73; H, 4.61; N, 15.24. Found: C, 62.79; H, 4.72; N, 15.35. HRMS (ESI): m/z cald C₂₄H₂₁N₅O₃S ([M+H] ⁺) 460.14379, found 460.14469.

N-(4-(5-((2-((4-Fluorophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8e).

White crystals (75 % yield). M.p. 174–176 °C. IR: ν_max/cm⁻¹; 3330, 3063, 2912, 1667, 1646, 1512. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.76 (s, 1H, NH, exchangeable with D₂O), 10.49 (s, 1H, NH, exchangeable with D₂O), 9.12 (d, J = 1.7 Hz, 1H, C₂-H of pyridine), 8.78 (dd, J = 4.8, 1.7 Hz, 1H, C₆-H of pyridine), 8.31 (app. d, J = 8.1 Hz, 1H, C₄-H of pyridine), 8.00–7.95 (m, 4H, Ar-H), 7.62 −7.58 (m, 3H, 2Ar-H and C₅-H of pyridine ), 7.19 −7.15 (m, 2H, Ar-H), 4.32 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 165.1 (CO), 165.0 (CO), 164.6 (C5-oxadiazole), 163.0 (C2-oxadiazole), 158.3 (d, J = 240.2 Hz, C-F), 152.5 (CH, pyridine), 148.9 (CH, pyridine), 142.3 (Ar-C), 135.7 (CH, pyridine), 135.1 (Ar-C), 130.4(C3 pyridine), 127.3 (Ar-CH), 123.7 (CH, pyridine), 121.1 (d, J = 7.9 Hz, Ar-CH), 120.5 (Ar-CH), 118.2 (Ar-C), 115.6 (d, J = 22.3 Hz, Ar-CH), 36.8 (CH₂). Analysis calcd. for C₂₂H₁₆FN₅O₃S: C, 58.79; H, 3.59; N, 15.58. Found: C, 58.94; H, 3.63; N, 15.62.MS, m/z: 449.75 (M⁺).

N-(4-(5-((2-((3-Bromophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8f).

White crystals (89 % yield). M.p. 235–237 °C. IR: ν_max/cm⁻¹; 3287, 3019, 2920, 1647, 1587, 1321. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.76 (s, 1H, NH, exchangeable with D₂O), 10.61 (s, 1H, NH, exchangeable with D₂O), 9.12 (d, J = 1.7 Hz, 1H, C₂-H of pyridine), 8.78 (dd, J = 4.8, 1.7 Hz, 1H, C₆-H of pyridine), 8.31 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 8.01–7.94 (m, 5H, Ar-H), 7.59 (dd, J = 8.0, 4.8 Hz, 1H, C₅-H of pyridine ), 7.50 – 7.47 (m, 1H, Ar-H), 7.33 – 7.25 (m, 2H, Ar-H), 4.34 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 165.6 (CO), 165.2 (CO), 164.7 (C5-oxadiazole), 163.0 (C2-oxadiazole), 152.5 (CH, pyridine), 148.8 (CH, pyridine), 142.3 (Ar-C), 140.2 (Ar-C), 135.8 (CH, pyridine),131.0 (Ar-CH), 130.4 (C3 pyridine), 127.4 (Ar-CH), 126.5 (Ar-CH), 123.7 (CH, pyridine), 121.8 (Ar-C),121.6 (Ar-CH), 120.6 (Ar-CH), 118.2 (Ar-C), 118.1 (Ar-CH), 36.8 (CH₂). Analysis calcd. for C₂₂H₁₆BrN₅O₃S: C, 51.78; H, 3.16; N, 13.72. Found: C, 51.95; H, 3.24; N, 13.88. HRMS (ESI): m/z cald C₂₂H₁₆BrN₅O₃S ([M+H] ⁺) 512.02299 (Br⁸¹), found 512.02244.

N-(4-(5-((2-((2-Chlorophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8g).

White crystals (91 % yield). M.p. 218–220 °C. IR: ν_max/cm⁻¹; 3256, 3010, 2921, 1685, 1666, 1313. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.76 (s, 1H, NH, exchangeable with D₂O), 10.01 (s, 1H, NH, exchangeable with D₂O), 9.13 (d, J = 1.5 Hz, 1H, C₂-H of pyridine), 8.79 (dd, J = 4.8, 1.5 Hz, 1H, C₆-H of pyridine), 8.31 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 8.03–7.97 (m, 4H, Ar-H), 7.74 (d, J= 7.7, 1H, Ar-H), 7.59 (dd, J = 8.0, 4.8 Hz, 1H, C₅-H of pyridine ), 7.51 (d, J = 7.7 Hz, 1H, Ar-H), 7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.22 (t, J = 7.3 Hz, 1H, Ar-H), 4.41 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 165.7 (CO), 165.0 (CO), 164.5 (C5-oxadiazole), 162.7 (C2-oxadiazole), 152.4 (CH, pyridine), 148.8 (CH, pyridine), 142.2 (Ar-C), 135.6 (CH, pyridine), 134.4 (Ar-C), 130.3 (C3 pyridine), 129.6 (Ar-CH), 127.5 (Ar-CH), 127.3 (Ar-CH), 126.7 (Ar-CH), 126.3 (Ar-C), 125.8 (Ar-CH), 123.5 (CH, pyridine), 120.4 (Ar-CH), 118.1 (Ar-C), 36.2 (CH₂). Analysis calcd. for C₂₂H₁₆ClN₅O₃S: C, 56.72; H, 3.46; N, 15.03. Found: C, 56.63; H, 3.49; N, 15.26. HRMS (ESI): m/z cald C₂₂H₁₆ClN₅O₃S ([M+H] ⁺) 466.07351 (Cl³⁵), found 466.07411.

N-(4-(4-amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl)nicotinamide (9).

Carbon disulphide (0.296 ml, 6 mmol) was added to a mixture of the hydrazide 4 (0.512 g, 2 mmol in 10 ml of absolute ethanol) and of KOH (0.112 g, 2 mmol, dissolved in 2 ml H₂O) and the reaction mixture was allowed to stirr at room temprature for 12 hrs. Diethylether was added to the resulted solution and the obtained potassium salt was collected by filteration and used directly without purification. A suspension of potassium dithiocarbazate 5 (00.74 g, 2 mmol) in dioxane (10 mL) and hydrazine hydrate (98%) (0.15 mL, 3 mmol) was refluxed for 20 hrs. The reaction mixture was cooled and concentrated under reduced pressure then diluted with water (40 mL). The reaction mixture was acidified with acetic acid to precipitate the target triazole 9. The precipitate was filtered off and washed with water and crystalyzed from methanol. Pale yellow crystals (77 % yield). M.p. 231–233 °C. IR: ν_max/cm⁻¹; 3300, 3070, 2912, 1671, 1651, 1591, 1523. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 13.89 (s, 1H, NH, exchangeable with D₂O ), 10.68 (s, 1H, NH, exchangeable with D₂O), 9.14 (app.s, 1H, C₂-H of pyridine), 8.79 (app.s, 1H, C₆-H of pyridine), 8.33 (d, J = 7.9 Hz, 1H, C₄-H of pyridine), 8.07 (d, J = 8.5 Hz, 2H, Ar-H), 7.94 (d, J = 8.5 Hz, 2H, Ar-H), 7.61– 7.58 (m, 1H, C₅-H of pyridine), 5.81 (s, 2H, NH₂, exchangeable with D₂O). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 167.2 (CS), 164.8 (CO), 152.8 (CH, pyridine), 149.5 (C3-triazole), 149.2 (CH, pyridine), 141.2 (Ar-C), 136.0 (CH, pyridine), 130.8 (C3 pyridine), 129.0 (Ar-CH), 124.0 (CH, pyridine), 121.5 (Ar-C), 120.3 (Ar-CH). Analysis calcd. for C₁₄H₁₂N₆OS: C, 53.83; H, 3.87; N, 26.91. Found: C, 53.62; H, 3.91; N, 26.73.

General procedure for the synthesis of N-(4-(4-(substitutedarylideneamino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl)nicotinamide (10a,b).

To a solution of the triazole 9 (0.62 g, 2 mmol) in glacial acetic acid (10 ml), the appropriate aldehyde (2 mmol) was added to the reaction mixture which was refluxed for 4–5 hrs. The solvent was concentrated in vacuum to obtain the preciptate which was filtered and washed several times with water to remove excess glacial acetic acid to give the titled compounds (10a,b).

N-(4-(4-((4-Methylbenzylidene)amino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl) nicotinamide (10a).

Pale yellow crystals (79 % yield). M.p. 320°C. IR: ν_max/cm⁻¹; 3256, 3065, 1668, 1598, 1521. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 14.17 (s, 1H, NH, exchangeable with D₂O ), 10.68 (s, 1H, NH, exchangeable with D₂O), 9.64 (s, 1H, N=CH), 9.11 (s, 1H, C₂-H of pyridine), 8.77 (app.s, 1H, C₆-H of pyridine), 8.30 (d, J = 7.9 Hz, 1H, C₄-H of pyridine), 7.94–7.88 (m, 4H, Ar-H), 7.81 (d, J = 7.9 Hz, 2H, Ar-H), 7.58 (dd, J = 7.9, 4.9 Hz,1H, C₅-H of pyridine), 7.39 (d, J = 7.9 Hz, 2H, Ar-H), 2.40 (s, 3H. CH₃). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 166.8 (N=CH), 164.4 (CS), 162.2 (CO), 152.3 (CH, pyridine), 148.7 (CH, pyridine), 148.3 (C3-triazole), 143.3 (Ar-C), 140.9 (Ar-C), 135.6 (CH, pyridine), 130.4 (C3 pyridine), 129.9 (Ar-CH), 129.3 (Ar-C), 128.9 (Ar-CH), 128.8 (Ar-CH), 123.5 (CH, pyridine), 120.7 (Ar-C), 119.9 (Ar-CH), 21.3 (CH₃). Analysis calcd. for C₂₂H₁₈N₆OS: C, 63.75; H, 4.38; N, 20.28. Found: C, 63.81; H, 4.68; N, 20.11. MS, m/z: 414.27 (M⁺).

N-(4-(4-(((1H-Indol-3-yl)methylene)amino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl) nicotinamide (10b).

Pale yellow crystals (85 % yield). M.p. 279–281°C. IR: ν_max/cm⁻¹; 3356, 3056, 1684, 1594, 1515. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 14.04 (s, 1H, NH, exchangeable with D₂O ), 12.09 (s, 1H, NH of indole, exchangeable with D₂O), 10.65 (s, 1H, NH, exchangeable with D₂O), 9.47 (s, 1H, N=CH), 9.10 (d, J = 1.8 Hz, 1H, C₂-H of pyridine), 8.77 (dd, J = 4.8, 1.8 Hz, 1H, C₆-H of pyridine), 8.29 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 8.17 (d, J = 2.9 Hz, 1H, indolyl-H), 8.12 (d, J = 7.8 Hz, 1H, indolyl-H), 8.01 (d, J = 8.8 Hz, 2H, Ar-H), 7.90 (d, J = 8.8 Hz, 2H, Ar-H), 7.65–7.50 (m, 2H, C₅-H of pyridine and indolyl-H), 7.29 (t, J = 7.5 Hz, 1H, indolyl-H), 7.22 (t, J = 7.5 Hz, 1H, indolyl-H). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 164.3 (CS), 163.9 (N=CH), 162.4 (CO), 152.3 (CH, pyridine), 148.6 (CH, pyridine), 147.9 (C3-triazole), 140.8 (Ar-C), 137.4 (C8-indole), 135.7 (CH, pyridine), 135.5 (C₂–H-indole), 130.3 (C3 pyridine), 128.7 (Ar-CH), 128.5 (C₆–H-indole), 124.3 (C9- indole), 123.5 (CH, pyridine), 123.3 (C₄–H-indole), 121.7 (C₅–H-indole), 121.0 (Ar-C), 119.8 (Ar-CH), 112.5 (C₇–H-indole), 109.9 (C3-indole). Analysis calcd. for C₂₃H₁₇N₇OS: C, 62.86; H, 3.90; N, 22.31. Found: C, 62.96; H, 4.15; N, 22.17. MS, m/z: 439.56 (M⁺).

General procedre for the synthesis of N-(4-(4-amino-5-((2-((4-substitutedphenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl)nicotinamide (11a-g)

To a solution of the triazole 9 (0.62 g, 2 mmol) and K₂CO₃ (0.55 g, 4 mmol) in 10 ml dry acetone, the appropriate 2-chloro-N-substituted phenylacetamide derivatives 7a-g (2 mmol) were added and heated under reflux for 4 hrs. The progress of the reaction was monitored by TLC, and after the completion of reaction, the reaction mixture was concentrated under reduced pressure and precipitate was washed with water then filtered and crystalized from methanol to get products 11a-g.

N-(4-(5-((2-((4-Acetylphenyl)amino)-2-oxoethyl)thio)-4-amino-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11a).

Yellow crystals (80 % yield). M.p. 254–256 °C. IR: ν_max/cm⁻¹; 3377, 3303, 3103, 2912, 1671, 1651, 1321. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.73 (s, 1H, NH, exchangeable with D₂O), 10.65 (s, 1H, NH, exchangeable with D₂O), 9.14 (s, 1H, C₂-H of pyridine), 8.79 (d, J = 4.8 Hz, 1H, C₆-H of pyridine), 8.33 (d, J = 7.9 Hz, 1H, C₄-H of pyridine), 8.02 (d, J = 8.0 Hz, 2H, Ar-H), 7.99 – 7.88 (m, 4H, Ar-H), 7.74 (d, J = 8.0 Hz, 2H, Ar-H), 7.60 (dd, J = 7.9, 4.8 Hz, 1H, C₅-H of pyridine), 6.23 (s, 2H, NH₂, exchangeable with D₂O), 4.22 (s, 2H, CH₂), 2.54 (s, 3H, CH₃). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 196.5 (CO), 166.7 (CO), 164.3 (CO), 153.8 (C5-triazole), 153.1 (C3-triazole), 152.3 (CH, pyridine), 148.7 (CH, pyridine), 143.1 (Ar-C), 140.1 (Ar-C), 135.5 (CH, pyridine), 131.9 (Ar-C), 130.4 (C3 pyridine), 129.6 (Ar-CH), 128.2 (Ar-CH), 123.5 (CH, pyridine), 122.2 (Ar-C), 119.9 (Ar-CH), 118.4 (Ar-CH), 36.2 (CH₂), 26.4 (CH₃). Analysis calcd. for C₂₄H₂₁N₇O₃S: C, 59.13; H, 4.34; N, 20.11. Found: C, 59.26; H, 4.21; N, 20.08. HRMS (ESI): m/z cald C₂₄H₂₁N₇O₃S ([M+H] ⁺) 488.14993, found 488.15028.

N-(4-(4-Amino-5-((2-((4-nitrophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11b).

White crystals (79 % yield). M.p. 270–272 °C. IR: ν_max/cm⁻¹; 3330, 3109, 2920, 1696, 1591, 1336. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.99 (s, 1H, NH, exchangeable with D₂O), 10.64 (s, 1H, NH, exchangeable with D₂O), 9.13 (s, 1H, C₂-H of pyridine), 8.78 (d, J = 4.8 Hz, 1H, C₆-H of pyridine), 8.32 (d, J = 7.9 Hz, 1H, C₄-H of pyridine), 8.24 (d, J = 9.0 Hz, 2H, Ar-H), 8.01 (d, J = 8.6 Hz, 2H, Ar-H), 7.92 (d, J = 8.6 Hz, 2H, Ar-H), 7.85 (d, J = 9.0 Hz, 2H, Ar-H), 7.59 (dd, J = 7.9, 4.8 Hz, 1H, C₅-H of pyridine), 6.23 (s, 2H, NH₂, exchangeable with D₂O), 4.23 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 167.2 (CO), 164.3 (CO), 153.9 (C5-triazole), 153.1 (C3-triazole), 152.3 (CH, pyridine), 148.7 (CH, pyridine), 145.0 (Ar-C), 142.4 (Ar-C), 140.1 (Ar-C), 135.5 (CH, pyridine), 130.4 (C3 pyridine), 128.2 (Ar-CH), 125.1 (Ar-CH), 123.5 (CH, pyridine), 122.2 (Ar-C), 120.0 (Ar-CH), 118.9 (Ar-CH), 36.1 (CH₂). Analysis calcd. for C₂₂H₁₈N₈O₄S: C, 53.87; H, 3.70; N, 22.85. Found: C, 53.98; H, 3.88; N, 22.56. HRMS (ESI): m/z cald C₂₂H₁₈N₈O₄S ([M+H] ⁺) 491.12444, found 491.12491.

N-(4-(4-Amino-5-((2-((4-methylphenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11c).

White crystals (72 % yield). M.p. 215–217 °C. IR: ν_max/cm⁻¹; 3306, 3206, 2927, 1670, 1590, 1316.¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.64 (s, 1H, NH, exchangeable with D₂O), 10.27 (s, 1H, NH, exchangeable with D₂O), 9.13 (d, J = 1.6 Hz, 1H, C₂-H of pyridine), 8.78 (dd, J = 4.8, 1.6 Hz, 1H, C₆-H of pyridine), 8.32 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 8.01 (d, J = 8.9 Hz, 2H, Ar-H), 7.92 (d, J = 8.9 Hz, 2H, Ar-H), 7.59 (dd, J = 8.0, 4.8 Hz, 1H, C₅-H of pyridine), 7.47 (d, J = 8.4 Hz, 2H, Ar-H), 7.12 (d, J = 8.4 Hz, 2H, Ar-H), 6.21 (s, 2H, NH₂, exchangeable with D₂O), 4.13 (s, 2H, CH₂), 2.25 (s, 3H, CH₃). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 165.8 (CO), 164.3 (CO), 153.7 (C5-triazole), 153.1 (C3-triazole), 152.3 (CH, pyridine), 148.7 (CH, pyridine), 140.1 (Ar-C), 136.3 (Ar-C), 135.5 (CH, pyridine), 132.4 (Ar-C), 130.4 (C3 pyridine), 129.2 (Ar-CH), 128.2 (Ar-CH), 123.5 (CH, pyridine), 122.2 (ArC), 119.9 (Ar-CH), 119.1 (Ar-CH), 36.3 (CH₂), 20.4 (CH₃). Analysis calcd. for C₂₃H₂₁N₇O₂S : C, 60.12; H, 4.61; N, 21.34; Found: C, 60.49; H, 4.83; N, 21.61. HRMS (ESI): m/z cald: C₂₃H₂₁N₇O₂S ([M+H] ⁺) 460.15502, found 460.15600.

N-(4-(4-Amino-5-((2-((2,5-dimethylphenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11d).

Yellow crystals (69 % yield). M.p. 240–242 °C. IR: ν_max/cm⁻¹; 3271, 3061, 2918, 666, 1644, 1587.¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.65 (s, 1H, NH, exchangeable with D₂O), 9.66 (s, 1H, NH, exchangeable with D₂O), 9.13 (d, J = 1.5 Hz, 1H, C₂-H of pyridine), 8.78 (dd, J = 4.8, 1.5 Hz, 1H, C₆-H of pyridine), 8.32 (d, J = 7.9 Hz, 1H, C₄-H of pyridine), 8.03 (d, J = 8.8 Hz, 2H, Ar-H), 7.92 (d, J = 8.8 Hz, 2H, Ar-H), 7.59 (dd, J = 7.9, 4.8 Hz, 1H, C₅-H of pyridine), 7.28 (s, 1H, Ar-H), 7.08 (d, J = 7.7 Hz, 1H, Ar-H), 6.89 (d, J = 7.7 Hz, 1H, Ar-H), 6.21 (s, 2H, NH₂, exchangeable with D₂O), 4.14 (s, 2H, CH₂), 2.23 (s, 3H, CH₃), 2.14 (s, 3H, CH₃). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 166.8 (CO), 164.8 (CO), 154.3 (C5-triazole), 153.6 (C3-triazole), 152.8 (CH, pyridine), 149.2 (CH, pyridine), 140.6 (Ar-C), 136.3 (Ar-C), 136.0 (CH, pyridine), 135.5 (Ar-C), 130.9 (C3 pyridine), 130.6 (Ar-CH), 128.7 (Ar-CH), 128.6 (Ar-C), 126.4 (Ar-CH), 125.3 (Ar-CH), 124.0 (CH, pyridine), 122.7 (Ar-C), 120.4 (Ar-CH), 36.3 (CH₂), 21.1 (CH₃), 17.8 (CH₃). Analysis calcd. for C₂₄H₂₃N₇O₂S: C, 60.87; H, 4.90; N, 20.70. Found: C, 60.63; H, 5.03; N, 20.48. HRMS (ESI): m/z cald : C₂₄H₂₃N₇O₂S ([M+H] ⁺) 474.17066, found 474.17149.

N-(4-(4-Amino-5-((2-((4-fluorophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11e).

White crystals (70 % yield). M.p. 252–254 °C. IR: ν_max/cm⁻¹; 3337, 3063, 1661, 1538, 1508. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.64 (s, 1H, NH, exchangeable with D₂O), 10.43 (s, 1H, NH, exchangeable with D₂O), 9.13 (d, J = 1.7 Hz 1H, C₂-H of pyridine), 8.78 (dd, J = 4.8, 1.7 Hz, 1H, C₆-H of pyridine), 8.33 – 8.30 (m, C₄-H of pyridine), 8.01 (d, J = 8.8 Hz, 2H, Ar-H), 7.92 (d, J = 8.8 Hz, 2H, Ar-H), 7.61 – 7.59 (m, 3H, C₅-H of pyridine and 2Ar-H ), 7.18 – 7.14 (m, 2H, Ar-H ), 6.21 (s, 2H, NH₂, exchangeable with D₂O), 4.15 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 166.0 (CO), 164.3 (CO), 159.3 (d, J = 238 Hz, Ar-C-F), 153.8 (C5-triazole), 153.1 (C3-triazole), 152.3 (CH, pyridine), 148.7 (CH, pyridine), 140.1 (Ar-C), 135.5 (CH, pyridine), 135.2 (Ar-C), 130.4 (C3 pyridine), 128.2 (Ar-CH), 123.5 (CH, pyridine), 122.2 (Ar-C), 120.9 (d, J = 7.8 Hz, Ar-CH), 119.9 (Ar-CH), 115.4 (d, J = 22.3 Hz, Ar-CH), 36.1 (CH₂). Analysis calcd. for C₂₂H₁₈FN₇O₂S: C, 57.01; H, 3.91; N, 21.15. Found: C, 56.97; H, 4.11; N, 21.36. HRMS (ESI): m/z cald : C₂₂H₁₈FN₇O₂S ([M+H] ⁺) 464.12994, found 464.13013.

N-(4-(4-Amino-5-((2-((3-bromophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11f).

Pale yellow crystals (73 % yield). M.p. 225–227°C. IR: ν_max/cm⁻¹; 3269, 3061, 1662, 1592, 1538. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.65 (s, 1H, NH, exchangeable with D₂O), 10.54 (s, 1H, NH, exchangeable with D₂O), 9.13 (app. s, 1H, C₂-H of pyridine), 8.78 (app. d, J = 4.7 Hz, 1H, C₆-H of pyridine), 8.32 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 8.01 (d, J = 8.7 Hz, 2H, Ar-H), 7.94 – 7.57 (m, 3H, Ar-H), 7.60 – 7.57 (m, 1H, C₅-H of pyridine), 7.49 (d, J = 7.4 Hz, 1H, Ar-H), 7.32 – 7.25 (m, 2H, Ar-H), 6.22 (s, 2H, NH₂, exchangeable with D₂O), 4.16 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 166.5 (CO), 164.3 (CO), 153.8 (C5-triazole), 153.0 (C3-triazole), 152.3 (CH, pyridine), 148.7 (CH, pyridine), 140.4 (Ar-C), 140.1 (Ar-C), 135.5 (CH, pyridine), 130.8 (Ar-CH), 130.4 (C3 pyridine), 128.2 (Ar-CH), 126.1 (Ar-CH), 123.5 (CH, pyridine), 122.2 (Ar-C), 121.6 (Ar-C), 121.4 (Ar-CH), 119.9 (Ar-CH), 117.9 (Ar-CH), 36.1 (CH₂). Analysis calcd. for C₂₂H₁₈BrN₇O₂S : C, 50.39; H, 3.46; N, 18.70. Found: C, 50.63; H, 3.81; N, 18.52. HRMS (ESI): m/z cald : C₂₂H₁₈BrN₇O₂S ([M+H] ⁺) 526.04988 (Br⁸¹), found 526.0484.

N-(4-(4-Amino-5-((2-((2-chlorophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11g).

Yellow crystals (70 % yield). M.p. 247–249 °C. IR: ν_max/cm⁻¹; 3280, 3081, 1663, 1593, 1537. ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.66 (s, 1H, NH, exchangeable with D₂O), 9.99 (s, 1H, NH, exchangeable with D₂O), 9.14 (d, J = 1.7 Hz, 1H, C₂-H of pyridine), 8.79 (dd, J = 4.7, 1.7 Hz, 1H, C₆-H of pyridine), 8.33 (d, J = 8.0 Hz, 1H, C₄-H of pyridine), 8.04 (d, J = 8.7 Hz, 2H, Ar-H), 7.94 (d, J = 8.7 Hz, 2H, Ar-H), 7.84 (d, J = 7.9 Hz, 1H, Ar-H), 7.60 (dd, J = 8.0, 4.7 Hz, 1H, C₅-H of pyridine), 7.50 (d, J = 7.9 Hz, 1H, Ar-H), 7.35 (t, J = 7.7 Hz, 1H, Ar-H), 7.20 (t, J = 7.7 Hz, 1H, Ar-H), 6.23 (s, 2H, NH₂, exchangeable with D₂O), 4.21 (s, 2H, CH₂). ¹³C APT NMR(100 MHz, DMSO-d₆) δ ppm: 166.8 (CO), 164.3 (CO), 153.9 (C5-triazole), 153.0 (C3-triazole), 152.3 (CH, pyridine), 148.7 (CH, pyridine), 140.2 (Ar-C), 135.6 (CH, pyridine), 134.7 (Ar-C), 133.6 (Ar-C), 130.4 (C3 pyridine), 129.5 (Ar-CH), 128.2 (Ar-CH), 127.5 (Ar-CH), 126.3 (Ar-CH), 125.1 (Ar-CH), 123.5 (CH, pyridine), 122.2 (Ar-C), 120.0 (Ar-CH), 35.7 (CH₂). Analysis calcd. for C₂₂H₁₈ClN₇O₂S : C, 55.06; H, 3.78; N, 20.43; Found C, 55.20; H, 3.98; N, 20.19. HRMS (ESI): m/z cald: C₂₂H₁₈ClN₇O₂S ([M+H] ⁺) 480.10039 (Cl³⁵), found 480.10063.

4.2. Biological activity

4.2.1. In vitro COX-1 and COX-2 inhibition assay

The inhibitory potency of the compounds against COX-1/COX-2 was measured using the colorimetric COX (ovine) inhibitor screening assay kit (Catalog No. 560131) supplied by Cayman Chemicals (Ann Arbor, MI, USA) according to the manufacturer’s instructions [49,50].

4.2.2. NO production in LPS-activated RAW 264.7 macrophages

The fluorescent probe 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM Diacetate; Cayman Chemical, MI, USA) was used to detect NO as detailed in our previous studies [51]. Briefly, RAW 264.7 cells were incubated with the individual test compounds, celecoxib, or diclofenac at different concentrations (12.5, 25, 50 and 100 μM) for 2 hr at 37 °C. Lipopolysaccharides (LPS) from Escherichia coli O111:B4 (Sigma Aldrich. Saint Louis, MO, USA) was then added at a final concentration of 1 μg/mL for an additional 20 hrs following by the incubation with DAF-FM. The fluorescence intensity, which is directly proportional to NO levels, was quantified, as detailed in our previous study [52]. The dose-response curves for the tested compounds were used to calculate the IC₅₀ values.

4.2.3. ROS production in LPS-activated RAW 264.7 macrophages

The ability of the test compounds to protect against LPS-induced ROS production in RAW 264.7 was investigated using the probe of oxidative species 2,7-dichlorofluorescein diacetate (DCFH-DA) (Cayman Chemical, MI, USA). Following 2 hr incubation of the cells with the different test compounds (12.5, 25, 50 and 100 μM), the induction of the cells was started using LPS (1 μg/mL, 20 hr). The cells were then incubated with DCFH-DA (25 μM). The fluorescence intensity, which is directly proportional to intracellular ROS levels, was measured as detailed in our previous report [52]. Individual IC₅₀ values were calculated from the dose-response curves.

4.2.4. TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages

The ability of the test compounds to inhibit LPS-induced inflammation by attenuating TNF-α and IL-6 cytokines production was investigated as detailed in our previous study [53]. Briefly, following 2 hr incubation of RAW 264.7 cells with the different compounds (12.5, 25, and 50 μM), LPS (1 μg/mL) was added for an additional 20 hr. Commercially available ELISA kits were used to measure TNF-α (catalog No. DY410, R&D Systems, Inc., MN, USA) and IL-6 (catalog No. DY406, R&D Systems, Inc., MN, USA), in the cell culture supernatants according to the manufacturer’s instructions.

4.3. In-silico study

4.3.1. Molecular Modelling

All the molecular modelling studies were carried out on Dell precision T3600 workstation with Intel Xeon^® CPU-1650.0 @ 3.20 GHz and Windows 7 operating system using Molecular Operating Environment (MOE 2020.09, Chemical Computing Group, Canada) as the computational software. MOE (Molecular Operating Environment) software was used for docking studies performance using Cox-2 enzyme co-crystallized with ligand SC-558 as a template (PDB ID: 1CX2 ) [41]. The protein preparation was included by removing other chains, non-reactive water molecules, and ligand molecules other than crystal ligand. The MOE builder was used to create the synthesized molecules. Structural preparation and 3D protonation of the co-crystallized enzyme was performed. The active site was selected using the MOE atom selector. Triangle matcher placement was used for docking. The London dG scoring function calculates the ligand’s free binding energy from a particular pose. The model was utilized to predict ligand-enzyme interactions at the active site and docking poses were selected according to the scoring functions along with binding interactions formed with the surrounding amino acids, and the relative orientation of the docked compounds compared to the co-crystallized ligand.

4.3.2. Molecular dynamic simulations

Desmond v. 2.2 software was used for performing MDS experiments [54–56]. This software applies the OPLS-2005 force field. Protein system was built using the System Builder Option, where the protein structure was checked for any missing hydrogens, the co-crystalized water molecules were removed and the protonation states of the amino acid residues were set at (pH = 7.4). Thereafter, the complex was solvated in a in 20 Å orthorhombic box of TIP3P and neutralized with 0.15 M Na⁺ and Cl⁻ ions of solvent buffer. Afterward, the prepared system was energy minimized and equilibrated for 10 ns. For protein-ligand complex, the top-scoring pose was used as a starting points for simulation. Desmond software automatically parameterizes inputted ligands during the system building step according to the OPLS force field. Absolute binding free energy (ΔG_binding) was determined via simulations performed by NAMD [57], the protein structures were built and optimized by using the QwikMD toolkit of the VMD software. The parameters and topologies of the compound were calculated by using the VMD plugin Force Field Toolkit (ffTK). Afterward, the generated topology files and parameters were loaded to VMD to readily read the protein–ligand complexes without errors and then conduct the simulation steps. Binding free energy calculations (ΔG) were performed by the free energy perturbation (FEP) method using NAMD software, this method was described in detail in the recent article by Kim and coworkers [58].

4.3.3. In-silico prediction of physicochemical properties and pharmacokinetic profile.

Prediction of oral bioavailability and pharmacokinetic parameters of the designed compounds was performed using Molinspiration Chemoinformatics server [46] and PreADMET calculator [47].

4.4. Statistical analysis

Data are presented as the mean ± SEM. Statistical differences were considered significant at p < 0.05. GraphPad Prism 8 software (GraphPad Software, San Diego, California) was used to analyze the data. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, was used to state significance between groups.

Supplementary Material

NIHMS1968898-supplement-1.docx^{(3.4MB, docx)}

Acknowledgements

We are grateful to the efforts of Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt, for the purchase of MOE license version 2020.09 (Chemical Computing Group, Montreal, CA). We appreciate Dr. Ahmed M. Sayed, Department of Pharmacognosy, faculty of pharmacy, Nahda University Beni-Suef (NUB) for assistance with molecular dynamics simulation.We appreciate Dr. Waleed Ali’s contributions to in vitro COX-1/COX-2 enzyme assays from the Cairo General Hospital’s Biochemistry lab.

Funding

The biological work in this study was financed by a grant number P20GM121334 (D.G.R.) from National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health. The authors are entirely responsible for the content, which does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Declaration of interests

The authors state that they have no financial, conflict-of-interest, or personal relationships that could obstruct or influence this study.

Declaration of interests

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.

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References

[1].Hyde CAC, Missailidis S, Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis, Int. Immunopharmacol. 9 (2009) 701–715. 10.1016/j.intimp.2009.02.003. [DOI] [PubMed] [Google Scholar]
[2].Nathan C, Points of control in inflammation, Nature. 420 (2002) 846–852. 10.1038/nature01320. [DOI] [PubMed] [Google Scholar]
[3].Hunter TM, Boytsov NN, Zhang X, Schroeder K, Michaud K, Araujo AB, Prevalence of rheumatoid arthritis in the United States adult population in healthcare claims databases, 2004–2014, Rheumatol. Int. 37 (2017) 1551–1557. 10.1007/s00296-017-3726-1. [DOI] [PubMed] [Google Scholar]
[4].Abdelrahman MH, Youssif BGM, abdelgawad MA, Abdelazeem AH, Ibrahim HM, Moustafa AEGA, Treamblu L, Bukhari SNA, Synthesis, biological evaluation, docking study and ulcerogenicity profiling of some novel quinoline-2-carboxamides as dual COXs/LOX inhibitors endowed with anti-inflammatory activity, Eur. J. Med. Chem. 127 (2017) 972–985. 10.1016/j.ejmech.2016.11.006. [DOI] [PubMed] [Google Scholar]
[5].Li Z, Wang ZC, Li X, Abbas M, Wu SY, Ren SZ, Liu QX, Liu Y, Chen PW, Duan YT, Lv PC, Zhu HL, Design, synthesis and evaluation of novel diaryl-1,5-diazoles derivatives bearing morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents, Eur. J. Med. Chem. 169 (2019) 168–184. 10.1016/j.ejmech.2019.03.008. [DOI] [PubMed] [Google Scholar]
[6].Morita I, Distinct functions of COX-1 and COX-2, Prostaglandins Other Lipid Mediat. 68–69 (2002) 165–175. 10.1016/S0090-6980(02)00029-1. [DOI] [PubMed] [Google Scholar]
[7].Gandhi J, Khera L, Gaur N, Paul C, Kaul R, Role of modulator of inflammation cyclooxygenase-2 in gammaherpesvirus mediated tumorigenesis, Front. Microbiol. 8 (2017) 538. 10.3389/fmicb.2017.00538. [DOI] [PMC free article] [PubMed] [Google Scholar]
[8].Arora M, Choudhary S, Singh PK, Sapra B, Silakari O, Structural investigation on the selective COX-2 inhibitors mediated cardiotoxicity: A review, Life Sci. 251 (2020) 117631. 10.1016/j.lfs.2020.117631. [DOI] [PubMed] [Google Scholar]
[9].Dogné JM, Supuran CT, Pratico D, Adverse cardiovascular effects of the coxibs, J. Med. Chem. 48 (2005) 2251–2257. 10.1021/jm0402059. [DOI] [PubMed] [Google Scholar]
[10].Catella-Lawson F, Crofford LJ, Cyclooxygenase inhibition and thrombogenicity, Am. J. Med. 110 (2001) 28–32. 10.1016/s0002-9343(00)00683-5. [DOI] [PubMed] [Google Scholar]
[11].Zafarullah M, Li WQ, Sylvester J, Ahmad M, Molecular mechanisms of N-acetylcysteine actions, Cell. Mol. Life Sci. 60 (2003) 6–20. 10.1007/s000180300001. [DOI] [PMC free article] [PubMed] [Google Scholar]
[12].Adibhatla RM, Hatcher JF, Phospholipase A 2, reactive oxygen species, and lipid peroxidation in cerebral ischemia, Free Radic. Biol. Med. 40 (2006) 376–387. 10.1016/j.freeradbiomed.2005.08.044. [DOI] [PubMed] [Google Scholar]
[13].Fylaktakidou K, Hadjipavlou-Litina D, Litinas K, Nicolaides D, Natural and Synthetic Coumarin Derivatives with Anti-Inflammatory / Antioxidant Activities, Curr. Pharm. Des. 10 (2005) 3813–3833. 10.2174/1381612043382710. [DOI] [PubMed] [Google Scholar]
[14].Emam SH, Sonousi A, Osman EO, Hwang D, Do Kim G, Hassan RA, Design and synthesis of methoxyphenyl- and coumarin-based chalcone derivatives as anti-inflammatory agents by inhibition of NO production and down-regulation of NF-κB in LPS-induced RAW264.7 macrophage cells, Bioorg. Chem. 107 (2021) 1–9. 10.1016/j.bioorg.2021.104630. [DOI] [PubMed] [Google Scholar]
[15].Fan X, Li J, Deng X, Lu Y, Feng Y, Ma S, Wen H, Zhao Q, Tan W, Shi T, Wang Z, Design, synthesis and bioactivity study of N-salicyloyl tryptamine derivatives as multifunctional agents for the treatment of neuroinflammation, Eur. J. Med. Chem. 193 (2020) 112217. 10.1016/j.ejmech.2020.112217. [DOI] [PubMed] [Google Scholar]
[16].Grover J, Bhatt N, Kumar V, Patel NK, Gondaliya BJ, Sobhia ME, Bhutani KK, Jachak SM, 2,5-Diaryl-1,3,4-oxadiazoles as selective COX-2 inhibitors and anti-inflammatory agents, RSC Adv. 5 (2015) 45535–45544. 10.1039/c5ra01428j. [DOI] [Google Scholar]
[17].Abdelazeem AH, El-Din AGS, Arab HH, El-Saadi MT, El-Moghazy SM, Amin NH, Design, synthesis and anti-inflammatory/analgesic evaluation of novel di-substituted urea derivatives bearing diaryl-1,2,4-triazole with dual COX-2/sEH inhibitory activities, J. Mol. Struct. 1240 (2021) 130565. 10.1016/j.molstruc.2021.130565. [DOI] [Google Scholar]
[18].Jiang B, Zeng Y, Li MJ, Xu JY, Zhang YN, Wang QJ, Sun NY, Lu T, Wu XM, Design, synthesis, and biological evaluation of 1,5-diaryl-1,2,4-triazole derivatives as selective cyclooxygenase-2 inhibitors, Arch. Pharm. (Weinheim). 343 (2010) 500–508. 10.1002/ardp.200900227. [DOI] [PubMed] [Google Scholar]
[19].Uddin MJ, Rao PNP, Knaus EE, Design and synthesis of novel celecoxib analogues as selective cyclooxygenase-2 (COX-2) inhibitors: Replacement of the sulfonamide pharmacophore by a sulfonylazide bioisostere, Bioorganic Med. Chem. 11 (2003) 5273–5280. 10.1016/j.bmc.2003.07.005. [DOI] [PubMed] [Google Scholar]
[20].Yoon SH, Cho DY, Choi SR, Lee JY, Choi DK, Kim E, Park JY, Synthesis and biological evaluation of salicylic acid analogues of celecoxib as a new class of selective cyclooxygenase-1 inhibitor, Biol. Pharm. Bull. 44 (2021) 1230–1238. 10.1248/bpb.b20-00991. [DOI] [PubMed] [Google Scholar]
[21].Abd-ellah HS, Abdel-aziz M, Shoman ME, Beshr EAM, Kaoud TS, Ahmed AFF, Novel 1, 3, 4-oxadiazole / oxime hybrids : Synthesis, docking studies and investigation of anti-inflammatory, ulcerogenic liability and analgesic activities, Bioorg. Chem. 69 (2016) 48–63. 10.1016/j.bioorg.2016.09.005. [DOI] [PubMed] [Google Scholar]
[22].Mohassab AM, Hassan HA, Abdelhamid D, Gouda AM, Gomaa HAM, Youssif BGM, 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, Elsevier; B.V., 2021. 10.1016/j.molstruc.2021.130948. [DOI] [Google Scholar]
[23].El-dash Y, Khalil NA, Ahmed EM, Hassan MSA, Synthesis and biological evaluation of new nicotinate derivatives as potential anti-inflammatory agents targeting COX-2 enzyme, Bioorg. Chem. 107 (2021) 104610. 10.1016/j.bioorg.2020.104610. [DOI] [PubMed] [Google Scholar]
[24].Lu X, Zhang H, Li X, Chen G, Li QS, Luo Y, Ruan BF, Chen XW, Zhu HL, Design, synthesis and biological evaluation of pyridine acyl sulfonamide derivatives as novel COX-2 inhibitors, Bioorganic Med. Chem. 19 (2011) 6827–6832. 10.1016/j.bmc.2011.09.034. [DOI] [PubMed] [Google Scholar]
[25].Abouzid K, Abouzid M, Khalil A, Ahmed M, Zaitone SA, Synthesis and Biological Evaluation of New Heteroaryl Carboxylic Acid Derivatives as Anti-inflammatory-Analgesic Agents, Chem. Pharm. Bull. 61 (2013) 222–228. 10.1248/cpb.c12-00949. [DOI] [PubMed] [Google Scholar]
[26].Hamoud MMS, Pulya S, Osman NA, Bobde Y, Hassan AEA, Abdel-fattah HA, Ghosh B, Ghanim AM, Design, synthesis, and biological evaluation, New J. Chem. 44 (2020) 9671–9683. 10.1039/D0NJ01274B. [DOI] [Google Scholar]
[27].Nicotera P, Melino G, Regulation of the apoptosis-necrosis switch, Oncogene. 23 (2004) 2757–2765. 10.1038/sj.onc.1207559. [DOI] [PubMed] [Google Scholar]
[28].Perkins DJ, Kniss DA, Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages, J. Leukoc. Biol. 65 (1999) 792–799. 10.1002/jlb.65.6.792. [DOI] [PubMed] [Google Scholar]
[29].Daiber A, Kröller-schön S, Oelze M, Hahad O, Li H, Schulz R, Steven S, Münzel T, Oxidative stress and in flammation contribute to traffic noise-induced vascular and cerebral dysfunction via uncoupling of nitric oxide synthases, Redox Biol. 34 (2020) 101506. 10.1016/j.redox.2020.101506. [DOI] [PMC free article] [PubMed] [Google Scholar]
[30].Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB, Reactive Oxygen Species in Inflammation and Tissue Injury, Antioxid. Redox Signal. 20 (2014) 1126–1167. 10.1089/ars.2012.5149. [DOI] [PMC free article] [PubMed] [Google Scholar]
[31].Nesaragi AR, Kamble RR, Dixit S, Kodasi B, Hoolageri SR, Bayannavar PK, Dasappa JP, Vootla S, Joshi SD, Kumbar VM, Green synthesis of therapeutically active 1,3,4-oxadiazoles as antioxidants, selective COX-2 inhibitors and their in silico studies, Bioorganic Med. Chem. Lett. 43 (2021) 1–7. 10.1016/j.bmcl.2021.128112. [DOI] [PubMed] [Google Scholar]
[32].Kotaiah Y, Harikrishna N, Nagaraju K, Venkata Rao C, Synthesis and antioxidant activity of 1,3,4-oxadiazole tagged thieno[2,3-d]pyrimidine derivatives, Eur. J. Med. Chem. 58 (2012) 340–345. 10.1016/j.ejmech.2012.10.007. [DOI] [PubMed] [Google Scholar]
[33].Ma L, Xiao Y, Li C, Xie ZL, Li DD, Wang YT, Ma HT, Zhu HL, Wang MH, Ye YH, Synthesis and antioxidant activity of novel Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan, Bioorganic Med. Chem. 21 (2013) 6763–6770. 10.1016/j.bmc.2013.08.002. [DOI] [PubMed] [Google Scholar]
[34].Jain A, Piplani P, Design, synthesis and biological evaluation of triazole-oxadiazole conjugates for the management of cognitive dysfunction, Bioorg. Chem. 103 (2020) 104151. 10.1016/j.bioorg.2020.104151. [DOI] [PubMed] [Google Scholar]
[35].Cocolas AH, Parks EL, Ressler AJ, Havasi MH, Seeram NP, Henry GE, Heterocyclic β-keto sulfide derivatives of carvacrol: Synthesis and copper (II) ion reducing capacity, Bioorganic Med. Chem. Lett. 29 (2019) 126636. 10.1016/j.bmcl.2019.126636. [DOI] [PubMed] [Google Scholar]
[36].Patil P, Yadav A, Bavkar L, Nippu BN, Satyanarayan ND, Mane A, Gurav A, Hangirgekar S, Sankpal S, [ MerDABCO-SO 3 H ] Cl catalyzed synthesis, antimicrobial and antioxidant evaluation and molecular docking study of pyrazolopyranopyrimidines, J. Mol. Struct. 1242 (2021) 130672. 10.1016/j.molstruc.2021.130672. [DOI] [Google Scholar]
[37].Kareem HS, Nordin N, Heidelberg T, Abdul-aziz A, Ariffin A, Conjugated Oligo-Aromatic Compounds Bearing a 3,4,5-Trimethoxy Moiety: Investigation of Their Antioxidant Activity Correlated with a DFT Study, Molecules. 21 (2016) 224. 10.3390/molecules21020224. [DOI] [PMC free article] [PubMed] [Google Scholar]
[38].Soromou LW, Zhang Z, Li R, Chen N, Guo W, Huo M, Guan S, Lu J, Deng X, Regulation of inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 murine macrophage by 7-O-methyl-naringenin, Molecules. 17 (2012) 3574–3585. 10.3390/molecules17033574. [DOI] [PMC free article] [PubMed] [Google Scholar]
[39].Debnath M, Berk M, Maes M, Translational evidence for the Inflammatory Response System (IRS)/Compensatory Immune Response System (CIRS) and neuroprogression theory of major depression, Prog. Neuro-Psychopharmacology Biol. Psychiatry. 111 (2021) 110343. 10.1016/j.pnpbp.2021.110343. [DOI] [PubMed] [Google Scholar]
[40].Westacott CI, Barakat AF, Wood L, Perry MJ, Neison P, Bisbinas I, Armstrong L, Millar AB, Elson CJ, Tumor necrosis factor alpha can contribute to focal loss of cartilage in osteoarthritis, Osteoarthr. Cartil. 8 (2000) 213–221. 10.1053/joca.1999.0292. [DOI] [PubMed] [Google Scholar]
[41].Kurumbail RG, Stevens AM, Gierse JK, Mcdonald JJ, Stegeman RA, Pak JY, Gildehaus D, Miyashiro JM, Penning TD, Seibert K, Isakson PC, Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents, Lett. to Nat. Nat. 384 (1996) 644–648. 10.1038/384644a0. [DOI] [PubMed] [Google Scholar]
[42].Rayar A-M, Lagarde N, Ferroud C, Zagury J-F, Montes M, Sylla-Iyarreta Veitia M, Update on COX-2 Selective Inhibitors: Chemical Classification, Side Effects and their Use in Cancers and Neuronal Diseases, Curr. Top. Med. Chem. 17 (2017) 2935–2956. 10.2174/1568026617666170821124947. [DOI] [PubMed] [Google Scholar]
[43].Soliva R, Almansa C, Kalko SG, Luque FJ, Orozco M, Theoretical studies on the inhibition mechanism of cyclooxygenase-2. Is there a unique recognition site?, J. Med. Chem. 46 (2003) 1372–1382. 10.1021/jm0209376. [DOI] [PubMed] [Google Scholar]
[44].Blobaum AL, Marnett LJ, Structural and functional basis of cyclooxygenase inhibition, J. Med. Chem. 50 (2007) 1425–1441. 10.1021/jm0613166. [DOI] [PubMed] [Google Scholar]
[45].Alam MJ, Alam O, Naim MJ, Islamuddin M, Deara GS, Docking Studies of Hybrid Pyrazole Analogues, Drug Des. Devel. Ther. 10 (2016) 3529–3543. 10.2147/DDDT.S118297. [DOI] [PMC free article] [PubMed] [Google Scholar]
[46].Molinspiration Cheminformatics, (n.d.). www.molinspiration.com (accessed September 7, 2021).
[47].PreADMET, (n.d.). https://preadmet.bmdrc.kr. (accessed September 7, 2021).
[48].Mao R, Shao J, Zhu K, Zhang Y, Ding H, Zhang C, Shi Z, Jiang H, Sun D, Duan W, Luo C, Potent, Selective, and Cell Active Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor Developed by Structure-Based Virtual Screening and Hit Optimization, J. Med. Chem. 60 (2017) 6289–6304. 10.1021/acs.jmedchem.7b00587. [DOI] [PubMed] [Google Scholar]
[49].Roschek B, Fink RC, Li D, McMichael M, Tower CM, Smith RD, Alberte RS, Pro-inflammatory enzymes, cyclooxygenase 1, cyclooxygenase 2, and 5-lipooxygenase, inhibited by stabilized rice bran extracts, J. Med. Food. 12 (2009) 615–623. 10.1089/jmf.2008.0133. [DOI] [PubMed] [Google Scholar]
[50].Abdu-Allah HHM, Abdelmoez AAB, Tarazi H, El-Shorbagi ANA, El-Awady R, Conjugation of 4-aminosalicylate with thiazolinones afforded non-cytotoxic potent in vitro and in vivo anti-inflammatory hybrids, Bioorg. Chem. 94 (2020) 103378. 10.1016/j.bioorg.2019.103378. [DOI] [PubMed] [Google Scholar]
[51].Sakr A, Rezq S, Ibrahim SM, Soliman E, Baraka MM, Romero DG, Kothayer H, Design and synthesis of novel quinazolinones conjugated ibuprofen, indole acetamide, or thioacetohydrazide as selective COX-2 inhibitors: anti-inflammatory, analgesic and anticancer activities, J. Enzyme Inhib. Med. Chem. 36 (2021) 1810–1828. 10.1080/14756366.2021.1956912. [DOI] [PMC free article] [PubMed] [Google Scholar]
[52].Rezq S, Abdel-Rahman AA, Central GPR109A activation mediates glutamate-dependent pressor response in conscious ratss, J. Pharmacol. Exp. Ther. 356 (2016) 456–465. 10.1124/jpet.115.229146. [DOI] [PMC free article] [PubMed] [Google Scholar]
[53].Ghanim AM, Rezq S, Ibrahim TS, Romero DG, Kothayer H, Novel 1,2,4-triazine-quinoline hybrids: The privileged scaffolds as potent multi-target inhibitors of LPS-induced inflammatory response via dual COX-2 and 15-LOX inhibition, Eur. J. Med. Chem. 219 (2021) 113457. 10.1016/j.ejmech.2021.113457. [DOI] [PMC free article] [PubMed] [Google Scholar]
[54].Bowers DE; K. J.; Chow E; Xu H; Dror RO; Eastwood MP; Gregersen BA; Klepeis JL; Kolossvary I; Moraes MA; Sacerdoti FD; Salmon JK; Shan Y; Shaw, Scalable algorithms for molecular dynamics simulations on commodity clusters, in: Proc. 2006 ACM/IEEE Conf. Supercomput. Tampa, FL, USA, 11–17 Novemb. 2006; IEEE New York, NY, USA, 2006: p. 43. [Google Scholar]
[55].Release, S. 3: Desmond Molecular Dynamics System, DE Shaw Research, New York, NY, 2017; Maestro-Desmond Interoperability Tools, Schrödinger: New York, NY, USA, (2017). [Google Scholar]
[56].Schrodinger LLC. Maestro, Version 9.0; Schrodinger LLC: New York, NY, USA, (2009). [Google Scholar]
[57].Phillips K, J.C.; Braun R; Wang W; Gumbart J; Tajkhorshid E; Villa E; Chipot C; Skeel RD; Kalé L; Schulten, Scalable molecular dynamics with NAMD, J. Comput. Chem. 26 (2005) 1781–1802. 10.1002/jcc.20289. [DOI] [PMC free article] [PubMed] [Google Scholar]
[58].Kim S, Oshima H, Zhang H, Kern NR, Re S, Lee J, Roux B, Sugita Y, Jiang W, Im W, CHARMM-GUI Free Energy Calculator for Absolute and Relative Ligand Solvation and Binding Free Energy Simulations, J. Chem. Theory Comput. 16 (2020) 7207–7218. 10.1021/acs.jctc.0c00884. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[R1] [1].Hyde CAC, Missailidis S, Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis, Int. Immunopharmacol. 9 (2009) 701–715. 10.1016/j.intimp.2009.02.003. [DOI] [PubMed] [Google Scholar]

[R2] [2].Nathan C, Points of control in inflammation, Nature. 420 (2002) 846–852. 10.1038/nature01320. [DOI] [PubMed] [Google Scholar]

[R3] [3].Hunter TM, Boytsov NN, Zhang X, Schroeder K, Michaud K, Araujo AB, Prevalence of rheumatoid arthritis in the United States adult population in healthcare claims databases, 2004–2014, Rheumatol. Int. 37 (2017) 1551–1557. 10.1007/s00296-017-3726-1. [DOI] [PubMed] [Google Scholar]

[R4] [4].Abdelrahman MH, Youssif BGM, abdelgawad MA, Abdelazeem AH, Ibrahim HM, Moustafa AEGA, Treamblu L, Bukhari SNA, Synthesis, biological evaluation, docking study and ulcerogenicity profiling of some novel quinoline-2-carboxamides as dual COXs/LOX inhibitors endowed with anti-inflammatory activity, Eur. J. Med. Chem. 127 (2017) 972–985. 10.1016/j.ejmech.2016.11.006. [DOI] [PubMed] [Google Scholar]

[R5] [5].Li Z, Wang ZC, Li X, Abbas M, Wu SY, Ren SZ, Liu QX, Liu Y, Chen PW, Duan YT, Lv PC, Zhu HL, Design, synthesis and evaluation of novel diaryl-1,5-diazoles derivatives bearing morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents, Eur. J. Med. Chem. 169 (2019) 168–184. 10.1016/j.ejmech.2019.03.008. [DOI] [PubMed] [Google Scholar]

[R6] [6].Morita I, Distinct functions of COX-1 and COX-2, Prostaglandins Other Lipid Mediat. 68–69 (2002) 165–175. 10.1016/S0090-6980(02)00029-1. [DOI] [PubMed] [Google Scholar]

[R7] [7].Gandhi J, Khera L, Gaur N, Paul C, Kaul R, Role of modulator of inflammation cyclooxygenase-2 in gammaherpesvirus mediated tumorigenesis, Front. Microbiol. 8 (2017) 538. 10.3389/fmicb.2017.00538. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] [8].Arora M, Choudhary S, Singh PK, Sapra B, Silakari O, Structural investigation on the selective COX-2 inhibitors mediated cardiotoxicity: A review, Life Sci. 251 (2020) 117631. 10.1016/j.lfs.2020.117631. [DOI] [PubMed] [Google Scholar]

[R9] [9].Dogné JM, Supuran CT, Pratico D, Adverse cardiovascular effects of the coxibs, J. Med. Chem. 48 (2005) 2251–2257. 10.1021/jm0402059. [DOI] [PubMed] [Google Scholar]

[R10] [10].Catella-Lawson F, Crofford LJ, Cyclooxygenase inhibition and thrombogenicity, Am. J. Med. 110 (2001) 28–32. 10.1016/s0002-9343(00)00683-5. [DOI] [PubMed] [Google Scholar]

[R11] [11].Zafarullah M, Li WQ, Sylvester J, Ahmad M, Molecular mechanisms of N-acetylcysteine actions, Cell. Mol. Life Sci. 60 (2003) 6–20. 10.1007/s000180300001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] [12].Adibhatla RM, Hatcher JF, Phospholipase A 2, reactive oxygen species, and lipid peroxidation in cerebral ischemia, Free Radic. Biol. Med. 40 (2006) 376–387. 10.1016/j.freeradbiomed.2005.08.044. [DOI] [PubMed] [Google Scholar]

[R13] [13].Fylaktakidou K, Hadjipavlou-Litina D, Litinas K, Nicolaides D, Natural and Synthetic Coumarin Derivatives with Anti-Inflammatory / Antioxidant Activities, Curr. Pharm. Des. 10 (2005) 3813–3833. 10.2174/1381612043382710. [DOI] [PubMed] [Google Scholar]

[R14] [14].Emam SH, Sonousi A, Osman EO, Hwang D, Do Kim G, Hassan RA, Design and synthesis of methoxyphenyl- and coumarin-based chalcone derivatives as anti-inflammatory agents by inhibition of NO production and down-regulation of NF-κB in LPS-induced RAW264.7 macrophage cells, Bioorg. Chem. 107 (2021) 1–9. 10.1016/j.bioorg.2021.104630. [DOI] [PubMed] [Google Scholar]

[R15] [15].Fan X, Li J, Deng X, Lu Y, Feng Y, Ma S, Wen H, Zhao Q, Tan W, Shi T, Wang Z, Design, synthesis and bioactivity study of N-salicyloyl tryptamine derivatives as multifunctional agents for the treatment of neuroinflammation, Eur. J. Med. Chem. 193 (2020) 112217. 10.1016/j.ejmech.2020.112217. [DOI] [PubMed] [Google Scholar]

[R16] [16].Grover J, Bhatt N, Kumar V, Patel NK, Gondaliya BJ, Sobhia ME, Bhutani KK, Jachak SM, 2,5-Diaryl-1,3,4-oxadiazoles as selective COX-2 inhibitors and anti-inflammatory agents, RSC Adv. 5 (2015) 45535–45544. 10.1039/c5ra01428j. [DOI] [Google Scholar]

[R17] [17].Abdelazeem AH, El-Din AGS, Arab HH, El-Saadi MT, El-Moghazy SM, Amin NH, Design, synthesis and anti-inflammatory/analgesic evaluation of novel di-substituted urea derivatives bearing diaryl-1,2,4-triazole with dual COX-2/sEH inhibitory activities, J. Mol. Struct. 1240 (2021) 130565. 10.1016/j.molstruc.2021.130565. [DOI] [Google Scholar]

[R18] [18].Jiang B, Zeng Y, Li MJ, Xu JY, Zhang YN, Wang QJ, Sun NY, Lu T, Wu XM, Design, synthesis, and biological evaluation of 1,5-diaryl-1,2,4-triazole derivatives as selective cyclooxygenase-2 inhibitors, Arch. Pharm. (Weinheim). 343 (2010) 500–508. 10.1002/ardp.200900227. [DOI] [PubMed] [Google Scholar]

[R19] [19].Uddin MJ, Rao PNP, Knaus EE, Design and synthesis of novel celecoxib analogues as selective cyclooxygenase-2 (COX-2) inhibitors: Replacement of the sulfonamide pharmacophore by a sulfonylazide bioisostere, Bioorganic Med. Chem. 11 (2003) 5273–5280. 10.1016/j.bmc.2003.07.005. [DOI] [PubMed] [Google Scholar]

[R20] [20].Yoon SH, Cho DY, Choi SR, Lee JY, Choi DK, Kim E, Park JY, Synthesis and biological evaluation of salicylic acid analogues of celecoxib as a new class of selective cyclooxygenase-1 inhibitor, Biol. Pharm. Bull. 44 (2021) 1230–1238. 10.1248/bpb.b20-00991. [DOI] [PubMed] [Google Scholar]

[R21] [21].Abd-ellah HS, Abdel-aziz M, Shoman ME, Beshr EAM, Kaoud TS, Ahmed AFF, Novel 1, 3, 4-oxadiazole / oxime hybrids : Synthesis, docking studies and investigation of anti-inflammatory, ulcerogenic liability and analgesic activities, Bioorg. Chem. 69 (2016) 48–63. 10.1016/j.bioorg.2016.09.005. [DOI] [PubMed] [Google Scholar]

[R22] [22].Mohassab AM, Hassan HA, Abdelhamid D, Gouda AM, Gomaa HAM, Youssif BGM, 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, Elsevier; B.V., 2021. 10.1016/j.molstruc.2021.130948. [DOI] [Google Scholar]

[R23] [23].El-dash Y, Khalil NA, Ahmed EM, Hassan MSA, Synthesis and biological evaluation of new nicotinate derivatives as potential anti-inflammatory agents targeting COX-2 enzyme, Bioorg. Chem. 107 (2021) 104610. 10.1016/j.bioorg.2020.104610. [DOI] [PubMed] [Google Scholar]

[R24] [24].Lu X, Zhang H, Li X, Chen G, Li QS, Luo Y, Ruan BF, Chen XW, Zhu HL, Design, synthesis and biological evaluation of pyridine acyl sulfonamide derivatives as novel COX-2 inhibitors, Bioorganic Med. Chem. 19 (2011) 6827–6832. 10.1016/j.bmc.2011.09.034. [DOI] [PubMed] [Google Scholar]

[R25] [25].Abouzid K, Abouzid M, Khalil A, Ahmed M, Zaitone SA, Synthesis and Biological Evaluation of New Heteroaryl Carboxylic Acid Derivatives as Anti-inflammatory-Analgesic Agents, Chem. Pharm. Bull. 61 (2013) 222–228. 10.1248/cpb.c12-00949. [DOI] [PubMed] [Google Scholar]

[R26] [26].Hamoud MMS, Pulya S, Osman NA, Bobde Y, Hassan AEA, Abdel-fattah HA, Ghosh B, Ghanim AM, Design, synthesis, and biological evaluation, New J. Chem. 44 (2020) 9671–9683. 10.1039/D0NJ01274B. [DOI] [Google Scholar]

[R27] [27].Nicotera P, Melino G, Regulation of the apoptosis-necrosis switch, Oncogene. 23 (2004) 2757–2765. 10.1038/sj.onc.1207559. [DOI] [PubMed] [Google Scholar]

[R28] [28].Perkins DJ, Kniss DA, Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages, J. Leukoc. Biol. 65 (1999) 792–799. 10.1002/jlb.65.6.792. [DOI] [PubMed] [Google Scholar]

[R29] [29].Daiber A, Kröller-schön S, Oelze M, Hahad O, Li H, Schulz R, Steven S, Münzel T, Oxidative stress and in flammation contribute to traffic noise-induced vascular and cerebral dysfunction via uncoupling of nitric oxide synthases, Redox Biol. 34 (2020) 101506. 10.1016/j.redox.2020.101506. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] [30].Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB, Reactive Oxygen Species in Inflammation and Tissue Injury, Antioxid. Redox Signal. 20 (2014) 1126–1167. 10.1089/ars.2012.5149. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] [31].Nesaragi AR, Kamble RR, Dixit S, Kodasi B, Hoolageri SR, Bayannavar PK, Dasappa JP, Vootla S, Joshi SD, Kumbar VM, Green synthesis of therapeutically active 1,3,4-oxadiazoles as antioxidants, selective COX-2 inhibitors and their in silico studies, Bioorganic Med. Chem. Lett. 43 (2021) 1–7. 10.1016/j.bmcl.2021.128112. [DOI] [PubMed] [Google Scholar]

[R32] [32].Kotaiah Y, Harikrishna N, Nagaraju K, Venkata Rao C, Synthesis and antioxidant activity of 1,3,4-oxadiazole tagged thieno[2,3-d]pyrimidine derivatives, Eur. J. Med. Chem. 58 (2012) 340–345. 10.1016/j.ejmech.2012.10.007. [DOI] [PubMed] [Google Scholar]

[R33] [33].Ma L, Xiao Y, Li C, Xie ZL, Li DD, Wang YT, Ma HT, Zhu HL, Wang MH, Ye YH, Synthesis and antioxidant activity of novel Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan, Bioorganic Med. Chem. 21 (2013) 6763–6770. 10.1016/j.bmc.2013.08.002. [DOI] [PubMed] [Google Scholar]

[R34] [34].Jain A, Piplani P, Design, synthesis and biological evaluation of triazole-oxadiazole conjugates for the management of cognitive dysfunction, Bioorg. Chem. 103 (2020) 104151. 10.1016/j.bioorg.2020.104151. [DOI] [PubMed] [Google Scholar]

[R35] [35].Cocolas AH, Parks EL, Ressler AJ, Havasi MH, Seeram NP, Henry GE, Heterocyclic β-keto sulfide derivatives of carvacrol: Synthesis and copper (II) ion reducing capacity, Bioorganic Med. Chem. Lett. 29 (2019) 126636. 10.1016/j.bmcl.2019.126636. [DOI] [PubMed] [Google Scholar]

[R36] [36].Patil P, Yadav A, Bavkar L, Nippu BN, Satyanarayan ND, Mane A, Gurav A, Hangirgekar S, Sankpal S, [ MerDABCO-SO 3 H ] Cl catalyzed synthesis, antimicrobial and antioxidant evaluation and molecular docking study of pyrazolopyranopyrimidines, J. Mol. Struct. 1242 (2021) 130672. 10.1016/j.molstruc.2021.130672. [DOI] [Google Scholar]

[R37] [37].Kareem HS, Nordin N, Heidelberg T, Abdul-aziz A, Ariffin A, Conjugated Oligo-Aromatic Compounds Bearing a 3,4,5-Trimethoxy Moiety: Investigation of Their Antioxidant Activity Correlated with a DFT Study, Molecules. 21 (2016) 224. 10.3390/molecules21020224. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] [38].Soromou LW, Zhang Z, Li R, Chen N, Guo W, Huo M, Guan S, Lu J, Deng X, Regulation of inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 murine macrophage by 7-O-methyl-naringenin, Molecules. 17 (2012) 3574–3585. 10.3390/molecules17033574. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] [39].Debnath M, Berk M, Maes M, Translational evidence for the Inflammatory Response System (IRS)/Compensatory Immune Response System (CIRS) and neuroprogression theory of major depression, Prog. Neuro-Psychopharmacology Biol. Psychiatry. 111 (2021) 110343. 10.1016/j.pnpbp.2021.110343. [DOI] [PubMed] [Google Scholar]

[R40] [40].Westacott CI, Barakat AF, Wood L, Perry MJ, Neison P, Bisbinas I, Armstrong L, Millar AB, Elson CJ, Tumor necrosis factor alpha can contribute to focal loss of cartilage in osteoarthritis, Osteoarthr. Cartil. 8 (2000) 213–221. 10.1053/joca.1999.0292. [DOI] [PubMed] [Google Scholar]

[R41] [41].Kurumbail RG, Stevens AM, Gierse JK, Mcdonald JJ, Stegeman RA, Pak JY, Gildehaus D, Miyashiro JM, Penning TD, Seibert K, Isakson PC, Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents, Lett. to Nat. Nat. 384 (1996) 644–648. 10.1038/384644a0. [DOI] [PubMed] [Google Scholar]

[R42] [42].Rayar A-M, Lagarde N, Ferroud C, Zagury J-F, Montes M, Sylla-Iyarreta Veitia M, Update on COX-2 Selective Inhibitors: Chemical Classification, Side Effects and their Use in Cancers and Neuronal Diseases, Curr. Top. Med. Chem. 17 (2017) 2935–2956. 10.2174/1568026617666170821124947. [DOI] [PubMed] [Google Scholar]

[R43] [43].Soliva R, Almansa C, Kalko SG, Luque FJ, Orozco M, Theoretical studies on the inhibition mechanism of cyclooxygenase-2. Is there a unique recognition site?, J. Med. Chem. 46 (2003) 1372–1382. 10.1021/jm0209376. [DOI] [PubMed] [Google Scholar]

[R44] [44].Blobaum AL, Marnett LJ, Structural and functional basis of cyclooxygenase inhibition, J. Med. Chem. 50 (2007) 1425–1441. 10.1021/jm0613166. [DOI] [PubMed] [Google Scholar]

[R45] [45].Alam MJ, Alam O, Naim MJ, Islamuddin M, Deara GS, Docking Studies of Hybrid Pyrazole Analogues, Drug Des. Devel. Ther. 10 (2016) 3529–3543. 10.2147/DDDT.S118297. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R46] [46].Molinspiration Cheminformatics, (n.d.). www.molinspiration.com (accessed September 7, 2021).

[R47] [47].PreADMET, (n.d.). https://preadmet.bmdrc.kr. (accessed September 7, 2021).

[R48] [48].Mao R, Shao J, Zhu K, Zhang Y, Ding H, Zhang C, Shi Z, Jiang H, Sun D, Duan W, Luo C, Potent, Selective, and Cell Active Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor Developed by Structure-Based Virtual Screening and Hit Optimization, J. Med. Chem. 60 (2017) 6289–6304. 10.1021/acs.jmedchem.7b00587. [DOI] [PubMed] [Google Scholar]

[R49] [49].Roschek B, Fink RC, Li D, McMichael M, Tower CM, Smith RD, Alberte RS, Pro-inflammatory enzymes, cyclooxygenase 1, cyclooxygenase 2, and 5-lipooxygenase, inhibited by stabilized rice bran extracts, J. Med. Food. 12 (2009) 615–623. 10.1089/jmf.2008.0133. [DOI] [PubMed] [Google Scholar]

[R50] [50].Abdu-Allah HHM, Abdelmoez AAB, Tarazi H, El-Shorbagi ANA, El-Awady R, Conjugation of 4-aminosalicylate with thiazolinones afforded non-cytotoxic potent in vitro and in vivo anti-inflammatory hybrids, Bioorg. Chem. 94 (2020) 103378. 10.1016/j.bioorg.2019.103378. [DOI] [PubMed] [Google Scholar]

[R51] [51].Sakr A, Rezq S, Ibrahim SM, Soliman E, Baraka MM, Romero DG, Kothayer H, Design and synthesis of novel quinazolinones conjugated ibuprofen, indole acetamide, or thioacetohydrazide as selective COX-2 inhibitors: anti-inflammatory, analgesic and anticancer activities, J. Enzyme Inhib. Med. Chem. 36 (2021) 1810–1828. 10.1080/14756366.2021.1956912. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R52] [52].Rezq S, Abdel-Rahman AA, Central GPR109A activation mediates glutamate-dependent pressor response in conscious ratss, J. Pharmacol. Exp. Ther. 356 (2016) 456–465. 10.1124/jpet.115.229146. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R53] [53].Ghanim AM, Rezq S, Ibrahim TS, Romero DG, Kothayer H, Novel 1,2,4-triazine-quinoline hybrids: The privileged scaffolds as potent multi-target inhibitors of LPS-induced inflammatory response via dual COX-2 and 15-LOX inhibition, Eur. J. Med. Chem. 219 (2021) 113457. 10.1016/j.ejmech.2021.113457. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R54] [54].Bowers DE; K. J.; Chow E; Xu H; Dror RO; Eastwood MP; Gregersen BA; Klepeis JL; Kolossvary I; Moraes MA; Sacerdoti FD; Salmon JK; Shan Y; Shaw, Scalable algorithms for molecular dynamics simulations on commodity clusters, in: Proc. 2006 ACM/IEEE Conf. Supercomput. Tampa, FL, USA, 11–17 Novemb. 2006; IEEE New York, NY, USA, 2006: p. 43. [Google Scholar]

[R55] [55].Release, S. 3: Desmond Molecular Dynamics System, DE Shaw Research, New York, NY, 2017; Maestro-Desmond Interoperability Tools, Schrödinger: New York, NY, USA, (2017). [Google Scholar]

[R56] [56].Schrodinger LLC. Maestro, Version 9.0; Schrodinger LLC: New York, NY, USA, (2009). [Google Scholar]

[R57] [57].Phillips K, J.C.; Braun R; Wang W; Gumbart J; Tajkhorshid E; Villa E; Chipot C; Skeel RD; Kalé L; Schulten, Scalable molecular dynamics with NAMD, J. Comput. Chem. 26 (2005) 1781–1802. 10.1002/jcc.20289. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R58] [58].Kim S, Oshima H, Zhang H, Kern NR, Re S, Lee J, Roux B, Sugita Y, Jiang W, Im W, CHARMM-GUI Free Energy Calculator for Absolute and Relative Ligand Solvation and Binding Free Energy Simulations, J. Chem. Theory Comput. 16 (2020) 7207–7218. 10.1021/acs.jctc.0c00884. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Design and Synthesis of Novel 1,3,4-Oxadiazole and 1,2,4-Triazole Derivatives as Cyclooxygenase-2 Inhibitors with Anti-inflammatory and Antioxidant Activity in LPS-stimulated RAW264.7 macrophages

Mohamed MS Hamoud

Nermine A Osman

Samar Rezq

Hend A A Abd El-wahab

Abdalla E A Hassan

Hanan A Abdel-Fattah

Damian G Romero

Amany M Ghanim

Abstract

Graphical abstract

1. Introduction

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

2. Results and Discussion

2.1. Chemistry

Scheme 1:

Scheme 2.

2.2. Biological activity

2.2.1. In vitro COX-1 and COX-2 inhibitory assay

Table 1:

2.2.2. Inhibitory activity against NO and ROS production in LPS-activated RAW 264.7 macrophages.

Table 2:

Fig. 5.

Fig. 6.

2.2.3. Inhibitory activity against TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages.

Table 3:

2.3. In-silico study

2.3.1. Molecular Modelling

Fig. 7.

2.3.2. Molecular dynamic simulation

Fig. 8.

Fig. 9.

2.3.3. In-silico prediction of physicochemical properties and pharmacokinetic profile.

3. Conclusion

4. Experimental

4.1. Chemistry

N-(4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl) phenyl)nicotinamide (6).

General procedure for the synthesis of N-(4-(5-((2-((substituted phenyl)amino)-2-oxoethyl) thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8a-g)

N-(4-(5-((2-((4-Acetylphenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8a).

N-(4-(5-((2-((4-Nitrophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8b)

N-(4-(5-((2-((4-Methylphenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8c).

N-(4-(5-((2-((2,5-Dimethylphenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl) nicotinamide (8d).

N-(4-(5-((2-((4-Fluorophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8e).

N-(4-(5-((2-((3-Bromophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8f).

N-(4-(5-((2-((2-Chlorophenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)phenyl)nicotinamide (8g).

N-(4-(4-amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl)nicotinamide (9).

General procedure for the synthesis of N-(4-(4-(substitutedarylideneamino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl)nicotinamide (10a,b).

N-(4-(4-((4-Methylbenzylidene)amino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl) nicotinamide (10a).

N-(4-(4-(((1H-Indol-3-yl)methylene)amino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl) nicotinamide (10b).

General procedre for the synthesis of N-(4-(4-amino-5-((2-((4-substitutedphenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl)nicotinamide (11a-g)

N-(4-(5-((2-((4-Acetylphenyl)amino)-2-oxoethyl)thio)-4-amino-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11a).

N-(4-(4-Amino-5-((2-((4-nitrophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11b).

N-(4-(4-Amino-5-((2-((4-methylphenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11c).

N-(4-(4-Amino-5-((2-((2,5-dimethylphenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11d).

N-(4-(4-Amino-5-((2-((4-fluorophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11e).

N-(4-(4-Amino-5-((2-((3-bromophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11f).

N-(4-(4-Amino-5-((2-((2-chlorophenyl)amino)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)phenyl) nicotinamide (11g).

4.2. Biological activity

4.2.1. In vitro COX-1 and COX-2 inhibition assay

4.2.2. NO production in LPS-activated RAW 264.7 macrophages

4.2.3. ROS production in LPS-activated RAW 264.7 macrophages

4.2.4. TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages

4.3. In-silico study

4.3.1. Molecular Modelling

4.3.2. Molecular dynamic simulations

4.3.3. In-silico prediction of physicochemical properties and pharmacokinetic profile.

4.4. Statistical analysis

Supplementary Material

Acknowledgements

Funding

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

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