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. 2024 Jun 5;15(8):2692–2708. doi: 10.1039/d4md00135d

New pyrazole–pyridazine hybrids as selective COX-2 inhibitors: design, synthesis, molecular docking, in silico studies and investigation of their anti-inflammatory potential by evaluation of TNF-α, IL-6, PGE-2 and NO in LPS-induced RAW264.7 macrophages†

Eman O Osman a,✉, Nadia A Khalil a, Alaa Magdy a, Yara El-Dash a
PMCID: PMC11324043  PMID: 39149111

Abstract

Hybrid-based design has gained significant interest in the development of novel active substances with anti-inflammatory properties. In this study, two series of new pyrazole–pyridazine-based hybrids, 5a–f and 6a–f, were designed and synthesized. Molecules containing pyrazole and pyridazine pharmacophores in a single molecule, each with a unique mechanism of action and different pharmacological characteristics, are believed to exert higher biological activity. The cell viability of all compounds was evaluated using MTT assay in LPS-induced RAW264.7 macrophages. In vitro COX-1 and COX-2 inhibition assays were performed for the investigation of the anti-inflammatory activity of target compounds. Trimethoxy derivatives 5f and 6f were the most active candidates, demonstrating higher COX-2 inhibitory action than celecoxib, with IC50 values of 1.50 and 1.15 μM, respectively. Bromo derivative 6e demonstrated a COX-2 inhibitory activity comparable to celecoxib. Further, the ability of compounds 5f, 6e, and 6f to inhibit the generation of specific pro-inflammatory cytokines and mediators, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and prostaglandin-E2 (PGE-2), in RAW264.7 macrophages stimulated by LPS was also estimated. Compounds 5f and 6f demonstrated the most potent activity. Morover, according to the investigation using molecular modeling studies, derivatives 5f and 6f showed respectable binding affinity towards the COX-2 active site compared to the reference ligand. Moreover, the ADME parameters, physicochemical characteristics, pharmacokinetic characteristics, and l of the most potent compounds were also computed.

Trimethoxy derivatives 5f (IC50 = 1.50 μM) and 6f (IC50 = 1.15 μM) demonstrated higher COX-2 inhibitory activity than celecoxib. Also, they showed the highest inhibition of NO, TNF-α, IL-6, and PGE-2 production in LPS-induced RAW264.7 macrophages.graphic file with name d4md00135d-ga.jpg

1. Introduction

Physiologically, inflammation occurs when an organism is exposed to harmful stimuli such as microbial infection, tissue damage, and other toxic conditions.1 As a defense mechanism, inflammation enables the elimination of toxic stimuli and restoration of injured tissues. Persistent chronic inflammation is frequently linked to the development of osteoarthritis and cancer as well as autoimmune, neurological, and cardiovascular disorders.2,3 Innate immunity is the primary cause of inflammation, and immune cells, including mast cells, neutrophils, lymphocytes, dendritic cells, and macrophages, are important players in the inflammatory response.4

Nowadays, aspirin, ibuprofen, and indomethacin, as examples of non-steroidal anti-inflammatory drugs (NSAIDs), are frequently used as the primary treatment for inflammatory illnesses.5 The inhibition of cyclooxygenases (COXs), which catalyze the bioconversion of arachidonic acid (AA) to prostaglandins (PGs), is strongly linked to the clinical efficacy of the majority of NSAIDs.5–7 There are three different isoforms of the membrane-bound hemeprotein COX: COX-1, COX-2, and COX-3.8 COX-1 is a constitutive form that is responsible for thrombogenesis and homeostasis. Alternatively, the production of several pro-inflammatory mediators causes the release of the inducible enzyme COX-2. Nephrotoxicity, hemorrhage, and gastrointestinal discomfort are among the unfavorable side effects of NSAIDs that are linked to COX-1 inhibition.9–11 Thus, the promising concept of selective COX-2 inhibition have led to the development of numerous new potential anti-inflammatory candidates with improved therapeutic benefits and reduced side effects. The COX-3 isoform is isolated from the cerebral cortex and heart tissues, and it is more sensitive to paracetamol than other NSAIDs.12

Lipopolysaccharides (LPSs) on the outer membrane of Gram-negative bacteria are a strong inducer of macrophages and promote the release of pro-inflammatory mediators, such as iNOS, NO, PGE-2, TNF-α, and IL-6.13–15 Catalyzed by inducible nitric oxide synthase (iNOS), NO is produced from l-arginine.16 However, NO may be viewed as a two-edged sword because while it functions as an anti-inflammatory agent under physiological conditions, it also works as a pro-inflammatory mediator under pathological conditions.17–19 In chronic inflammation, the release of NO causes local vasodilation, leading to edema.17,20 In addition, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) are also believed to be key mediators in inflammatory episodes.21–24 TNF-α belongs to a certain class of cytokines that is secreted from variant immune cells and is expressed due to exposure to LPS of a pathogen.25 Alternatively, IL-6 belongs to a broad protein family that is essential in immune system regulation.26

The pyrazole ring is an integral part of many medications acting as anticancer,27–30 anti-androgenic,31 antimicrobial,28,32–34 antifungal,35 antiviral36 and anti-inflammatory agents37,38 (Fig. 1). A literature survey revealed that pyrazole is a promising scaffold that demonstrated selective COX-2 inhibition, leading to minimal risk of undesirable side effects.37 Among the selective COX-2 inhibitors that contain the pyrazole nucleus is celecoxib, which is considered a safe anti-inflammatory and analgesic agent (Fig. 1).38 Alternatively, the pyridazine scaffold serves as a crucial framework in medicinal chemistry. Compounds containing a pyridazine ring exhibit diverse biological activities such as antimicrobial,39 antitubercular,40 analgesic,41 anti-inflammatory,42,43 herbicidal,44 antiviral,45 anti-platelet,46 anticancer,47 cardiotonic,48 antihypertensive,49 anticonvulsant50 and antidiabetic.51 Nowadays, hybrid-based design has attracted great interest in the development of new anti-inflammatory active compounds. Pyridazine–pyrazole hybrids occupy an important place in the field of synthetic medicinal chemistry due to their valuable therapeutic and pharmacological potential. Recently, hybrid compounds including pyrazole and pyridazine pharmacophores have been described by several research teams to show significant anti-inflammatory activity.52–59 The majority of the hybrid compounds demonstrated favorable anti-inflammatory activity, particularly compound 6, which exhibited 84% inhibition of edema,56 and compound 9, demonstrating 78% inhibition of COX-2 activity (Fig. 2).59 Herein, the design of the novel compounds 5a–f and 6a–f was based on the combination of the pyridazine and pyrazole active pharmacophores in a single molecular framework. The bulkier pyrazole–pyridazine core was supposed to selectively accommodate the pocket in the COX-2 receptor without affecting COX-1 receptors. Moreover, the effect of various substituents on the 5-aminopyrazole ring was investigated to boost the formation of hydrogen bonding with the receptor. Besides, N-substitution by the bulky arylidene moiety is supposed to increase both the anti-inflammatory activity and selectivity.

Fig. 1. Chemical structures of some FDA-approved pyrazole-containing anti-inflammatory agents.

Fig. 1

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Fig. 2. Structures and activity of some anti-inflammatory compounds with a pyrazole–pyridazine core.

Fig. 2

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To find new and more potent anti-inflammatory medicines, this work constructed a novel series of pyridazine–pyrazole hybrid scaffolds based on the previously mentioned facts.

In the current study, based on previous work, the new compounds were designed considering the pharmacophoric structural features that were found to increase COX-2 inhibitory effects, as shown in Fig. 3.53,57–60 These pharmacophoric elements include the pyridazine moiety (position A),53 an aromatic ring in position B,57 and the incorporation of a pyrazolone moiety in position C, as reported in many FDA-approved anti-inflammatory drugs such as antipyrine, famprofazone and ramifenazone.61,62 Furthermore, novel COX-2/5-LOX inhibitors with an aminopyrazole scaffold were also reported in the literature.59 Based on this, pyridazine–pyrazole hybrids were designed with different arylidene moieties in position 3 to construct a template that may exert a synergistic effect on anti-inflammatory activity. Herein, the effect of all the newly synthesized derivatives was initially estimated on cell viability. Additionally, their ability to inhibit COX-1 and COX-2 isoenzymes was assessed. Furthermore, the inhibition of pro-inflammatory mediators and cytokines including TNF-α, PGE-2, IL-6, and NO was also inspected by the most active candidates. The structure–activity relationship (SAR) and the docking results were investigated to rationalize and correlate the structure with the biological results.

Fig. 3. Design of target compounds through modifications in different positions (A, B and C).

Fig. 3

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2. Results and discussion

2.1. Chemistry

Scheme 1 shows the synthetic pathway to target compounds 1–4, 5a–f, and 6a–f. Initially, the reaction of the commercially available 3,6-dichloropyridazine with ethyl cyanoacetate or malononitrile in ethanol containing sodium ethoxide afforded ethyl 2-(6-chloropyridazin-3-yl)-2-cyanoacetate (1) and 2-(6-chloropyridazin-3-yl)malononitrile (2), respectively. The physical and spectroscopic data of 1 and 2 were consistent with the literature.51,52 The reaction of key intermediates 1 and 2 with hydrazine hydrate resulted in compounds 3 and 4, respectively. The formation of the new intermediates 3 and 4 was interpreted by microanalyses and spectral data. The IR spectra of compounds 3 and 4 showed absorption bands corresponding to NH2 and NH str. in the range of 3545–3271 cm−1 and lacked an absorption band corresponding to the cyano group. Compound 3 revealed an absorption band corresponding to the carbonyl group at 1681 cm−1. The 1HNMR spectra of title compounds 3 and 4 revealed a singlet signal at δ 3.09 and 3.08 ppm, respectively, corresponding to a pyrazole CH proton. The 13CNMR spectra of 3 and 4 revealed the pyrazole carbon at δ 27.3 and 28.2 ppm, respectively, while the signal corresponding to C Created by potrace 1.16, written by Peter Selinger 2001-2019 O for compound 3 was observed at δ 160.3 ppm. N-Substituted benzylidene-4-(6-chloropyridazin-3-yl)-1H-pyrazol-5(4H)-ones (5a–f) and N-substituted benzylidene-4-(6-chloropyridazin-3-yl)-4H-pyrazole-3,5-diamines (6a–f) were prepared by reacting compound 3 or 4 with the appropriate substituted benzaldehydes under reflux temperature. The IR spectra of compounds 5a and 6a showed broad absorption bands in the range of 3500–3267 cm−1 due to the OH group. The 1HNMR spectra of compounds 5a–f and 6a–f showed no NH2 exchangeable singlet signal. Additionally, an increase in the number of aromatic protons was noted, suggesting that the reaction was successful. Moreover, a singlet signal in the range of δ 8.58–8.72 ppm was observed, corresponding to the benzylidene proton. Compounds 5a and 6a demonstrated an exchangeable singlet signal at δ 8.14 and 9.62 ppm, respectively, due to the OH function. In addition, compounds 5b and f and 6b and f exhibited a single signal corresponding to OCH3 protons in the range of δ 3.72–3.82 ppm. Besides, the 13CNMR spectra of compounds 5a–f and 6a–f displayed the characteristic benzylidene carbon in the range of δ 157.8–161.6 ppm. Moreover, compounds 5b and f and 6b and f demonstrated the appearance of methoxy group carbons in the range of δ 55.4–60.6 ppm.

Scheme 1. Synthesis of 4-(6-chloropyridazin-3-yl)-3-(substitutedbenzylidene)amino-1H-pyrazol-5(4H)-one (5a–f) and N-substituted benzylidene-4-(6-chloropyridazin-3-yl)-4H-pyrazole-3,5-diamines (6a–f).

Scheme 1

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Cell viability assay

The inhibitory properties of the compounds may be due to their toxic effect, which may lead to an erroneous conclusion. Therefore, before conducting further biological analyses, the impact of compounds 5a–f and 6a–f on the viability of RAW264.7 cells was assessed by adopting the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated with the samples at varying concentrations (100, 25, 6.3, 1.6, and 0.4 μM L−1) in the presence of 0.5 μg mL−1 lipopolysaccharide (LPS) for 24 h.63 The pharmacological results were displayed as a percentage of the respective control and the data are reported as the mean ± SD for each group, as summarized in Fig. 4. The data showed that the synthesized derivatives had relatively lower toxicity than the reference drug celecoxib. These results encouraged us to perform further pharmacological assays.

Fig. 4. Effect of the synthesized compounds and celecoxib on cell viability in RAW 264.7 cells stimulated by LPS.

Fig. 4

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

Synthesized derivatives 5a–f and 6a–f were also evaluated for their ability to inhibit the COX-1 and COX-2 isoenzymes in vitro using a solid-phase ELISA assay, and their concentrations that produce 50% inhibition (IC50) were determined.37,64,65 In addition, the selectivity indices (SI values) for the COX-2 enzyme were calculated and compared with the reference drugs indomethacin and celecoxib. The results are recorded in Table 1. It can be observed that the majority of the synthesized compounds showed notable, selective COX-2 inhibitory activities with mild COX-1 inhibition. A stronger COX-2 inhibitory effect was observed for compounds 5f and 6f with IC50 values of 1.50 and 1.15 μM, respectively, compared to celecoxib (IC50 value = 2.16 μM). They also showed superior selectivity over celecoxib (SI = 9.56, 8.31 for 5f and 6f, respectively, in contrast to SI = 2.51 for celecoxib). Compound 6e (IC50 value = 2.51 μM) showed comparable activity to celecoxib.

Results of in vitro inhibition of COX-1/COX-2 and the selectivity index (SI).

Cpd. no. COX-1 aIC50 COX-2 aIC50 bSI
5a 20.11 ± 1.06 5.40 ± 0.23 3.72
5b 4.58 ± 0.24 12.40 ± 0.53 0.37
5c 18.00 ± 0.95 9.04 ± 0.39 1.99
5d 4.15 ± 0.22 20.71 ± 0.89 0.20
5e 2.42 ± 0.13 3.92 ± 0.17 0.62
5f 14.38 ± 0.76 1.50 ± 0.06 9.56
6a 35.95 ± 1.89 7.76 ± 0.33 4.63
6b 19.29 ± 1.01 56.73 ± 2.43 0.34
6c 31.20 ± 1.64 3.85 ± 0.17 8.10
6d 42.91 ± 2.26 9.87 ± 0.42 4.35
6e 5.48 ± 0.29 2.51 ± 0.11 2.18
6f 9.61 ± 0.51 1.15 ± 0.05 8.31
Indomethacin 0.43 ± 0.02 1.22 ± 0.05 0.36
Celecoxib 5.43 ± 0.29 2.16 ± 0.09 2.51
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a

IC50 is the concentration in μM needed to inhibit 50% of COX-1/COX-2 enzymatic activity. All values are expressed as the mean of four replicates ± SD.

b

Selectivity index (SI) = [COX-1 IC50 (μM)/COX-2 IC50 (μM)].

Fig. 5 illustrates the most striking relationship between the structural alterations in the synthetic derivatives and their anti-inflammatory properties. It was observed that the compounds with the pyrazolone skeleton, 5a–f, showed more potent COX-2 inhibitory activity than that with the aminopyrazole scaffold, 6a–f, with IC50 in the range of 1.50–20.71 μM and 1.15–56.73 μM, respectively (Table 1). Concerning substituents on the benzylidene ring, it was observed that compounds 5a and 6a with a meta hydroxy group on the benzylidene moiety demonstrated unpromising COX-2 inhibitory activity. Also, para-chloro analogues 5c and 6c, meta-bromo analogues 5d and 6d, and para-methoxy analogues 5b and 6b revealed relatively poor results. Alternatively, substitution with a bromo substituent at the para position in compounds 5e and 6e prompted a slight improvement in activity compared to celecoxib. Interestingly, the trimethoxy groups in derivatives 5f and 6f resulted in about 1.5-fold and 2-fold increase in activity, respectively, compared to celecoxib.

Fig. 5. Structure–activity relationship of the synthesized pyridazine derivatives.

Fig. 5

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2.3. Evaluation of compounds 5f, 6e and 6f on the production of TNF-α, IL-6, and PGE-2

To inspect the anti-inflammatory activity, the most active compounds 5f, 6e, and 6f were evaluated for their TNF-α, IL-6, and PGE-2 levels in LPS/INF γ-stimulated RAW 264.7 macrophages compared to celecoxib as the reference compound.66 When LPS was applied alone, the RAW 264.7 cells produced significantly more cytokines than the control group (Fig. 6). However, pretreatment with compound 6f (50 μM) significantly decreased the TNF-α, IL-6, and PGE-2 production by 70%, 78%, and 64%, respectively, with a relatively similar extent to celecoxib (75% inhibition for TNF-α, 83% inhibition for IL-6 and 69% inhibition for PGE-2). Compound 6e exhibited a moderate inhibitory effect (60% inhibition for PGE-2, 65% inhibition for IL-6, and 65% inhibition for TNF-α) at 50 μM concentration. In contrast, incubation with compound 5f showed the least inhibitory effect (44% inhibition for PGE-2, 62% inhibition for IL-6, and 48% inhibition for TNF-α), as shown in Fig. 6.

Fig. 6. Bar diagram showing the inhibitory effect of compounds 5f, 6e, and 6f on TNF-α (blue), IL-6, and PGE-2 production in RAW264.7 macrophage cells stimulated with LPS using celecoxib as the positive control.

Fig. 6

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2.4. Inhibition of NO production on LPS-induced RAW264.7 macrophage cells

Despite being responsible for host defense mechanisms, a high NO concentration can lead to toxicity and host cell damage. Excessive NO production participates in the pathogenesis of inflammatory diseases such as atherosclerosis, vascular diseases, and septic shock.67–69 As a result, suppression of NO production may be a crucial target in the management of inflammation.20,70 Herein, the effect of compounds 5f, 6e, and 6f on NO production was evaluated on RAW264.7 macrophage cells stimulated by LPS using the Griess reagent.47,69,71,72 Among the synthesized derivatives, 6e and 6f exhibited more than 50% inhibition of NO production at 20 μM concentration with inhibition rates of 53% and 70%, respectively (Fig. 7).

Fig. 7. Effects of compounds 5f, 6e, and 6f on the levels of NO in RAW 264.7 macrophage cells stimulated by LPS using celecoxib as the positive control.

Fig. 7

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2.5. Molecular docking of compounds 5f, 6e and 6f in the active site of COX-2

To explore the possible potential interactions between the active synthesized derivatives and the crystal structure of the COX-2 enzyme and to rationalize the biological results, the promising compounds 5f, 6e, and 6f were docked into the active site of COX-2 (PDB ID: 3LN1, https://www.rcsb.org) using the Molecular Operating Environment (MOE version 2015.10) software. In this study, self-docking of celecoxib (co-crystallized ligand) in the COX-2 active site was conducted to validate the docking approach used (Fig. 8). The self-alignment of the lead compound with its best-fitted pose demonstrated RMSD = 0.4172 Å, indicating the good coincidence between them and the validity of the utilized docking approach. Within the active site of the enzyme, celecoxib showed five H-bonds via the sulfonamide group, three H-bonds between NH2 and the amino acid residues Leu338, Ser339, and Gln178, and two H-bonds between the sulfone oxygen atoms and the amino acid residues Arg499 and Phe504 with 3.08, 3.14, 3.08, 3.40 and 3.41 Å, respectively (Table 2), and with the affinity binding energy of −9.3344 kcal mol−1 (Fig. 8) (Table 2). The examined compounds showed similar binding patterns to that of celecoxib with Arg120, Gln178, Ser339, and Phe504, which are the key amino acids of the active site. In comparison to celecoxib, the newly synthesized analogue 5f demonstrated a positive binding affinity towards the intended enzyme, as shown by its ability to interact through H-bonds between Arg499 and Tyr341 and the carbonyl group with a binding score of −7.0722 kcal mol−1 (Fig. 9). Compound 6e showed one H-bond with Gln178 and one π–H stacking interaction with Ala513 with a binding score of −5.4147 kcal mol−1 (Fig. 10). Compound 6f exhibited three H bonds with Ser339, Arg499, and Phe504, with a binding score of −4.5505 kcal mol−1 (Fig. 11) (Table 2). The docking process of the active compounds showed satisfactory interaction with a distance in the range of 2.62–3.62 Å from the main residue, while celecoxib showed interaction with a distance in the range of 3.08–3.41 Å.

Fig. 8. 2D interactions of the redocked co-crystallized celecoxib in the active site of COX-2 (PDB 3LN1) (left), and 3D interactions of the redocked co-crystallized celecoxib in the active site of COX-2 (PDB 3LN1) showing five H-bonds with Gln138, Ser339, Arg499, Leu338, and Phe504 (right). The arrows represent hydrogen bonds with numbers indicating bond distances with an RMSD value of 0.4172 Å.

Fig. 8

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Docking scores, H bonds, and π–H stacking of compounds 5f, 6e, 6f, and celecoxib within the human COX-2 active site (PDB entry 3LN1).

Cpd. ID No. of bonds Binding score (kcal mol−1) Amino acids (bond length Å) Type of bond
Celecoxib 5 −9.3344 Phe504 (3.41) H-bond
Gln178 (3.08) H-bond
Leu338 (3.08) H-bond
Ser339 (3.14) H-bond
Arg499 (3.40) H-bond
5f 2 −7.0722 Arg499 (3.08) H-bond
Tyr341 (2.62) H-bond
6e 2 −5.4147 Gln178 (3.11) H-bond
Ala513 (3.62) π–H stacking
6f 3 −4.5505 Ser339 (3.37) H-bond
Phe504 (3.35) H-bond
Arg499 (2.88) H-bond
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Fig. 9. 2D representation of 5f interactions with the active site of COX-2 (PDB 3LN1) (left), and 3D representation of 5f interactions showing two H-bonds with Arg499 and Tyr341 (right). The arrows represent hydrogen bonds with numbers indicating bond distances.

Fig. 9

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Fig. 10. 2D representation of 6e interactions with the active site of COX-2 (PDB 3LN1) (left), and 3D representation of 6e interactions showing one H-bond with Gln178 and one π–H stacking interaction with Ala 513 (right). The arrows represent hydrogen bonds with numbers indicating bond distances.

Fig. 10

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Fig. 11. 2D representation of 6f interactions with the active site of COX-2 (PDB 3LN1) (left), and 3D representation of 6f interactions showing three H bonds with Ser339, Arg499 and Phe504 (right). The arrows represent to hydrogen bonds with numbers indicating bond distances.

Fig. 11

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The correlation of the biological activity with the molecular docking studies revealed that the inhibitory activity of derivative 6f (IC50 = 1.15 μM) may be potentiated through an additional interaction between the amino moiety of the pyrazole ring and the essential amino acid Phe504. In contrast, derivative 6e lacked H-bonding with the backbone of Arg499, which may reflect its lower inhibitory effect (IC50 = 2.51 μM) compared to compound 6f. Furthermore, the presence of H-bond interaction between the pyrazole moiety and Tyr341 in derivative 5f may contribute to its slightly higher inhibitory activity (IC50 = 1.50 μM) over compound 6e.

Interestingly, it can be concluded that pyrazole has a significant effect on the binding interactions with the enzyme. The pyrazole nitrogen atom of compound 6f was involved in binding with Arg499, whereas the amino group interacts with Phe504 in the target protein. Additionally, the carbonyl oxygen atom in pyrazolone derivative 5f was involved in the interaction with Arg499 and Tyr341. Based on these facts, we can assume that the presence of the pyrazole ring can enhance the interaction with COX-2 receptors.

Additionally, it was noticed that compounds 5f and 6f containing three bulky methoxy groups in the meta and para positions showed better interaction and fitting into the binding site than compound 6e having a less bulky p-bromo substituent in its phenyl ring, respectively.

In conclusion, the docking studies suggested that drugs containing a central pyrazole core, together with trimethoxy groups in the meta and para positions may be potential targets for the further development of therapeutic agents for disorders associated with inflammation.

2.6. Physicochemical, ADME, and pharmacokinetic properties in silico prediction

Using the SwissADME online web tool (http://www.swissadme.ch) supplied by the Swiss Institute of Bioinformatics (SIB), compounds 5f, 6e, and 6f, as the most potent synthesized derivatives, were drawn on the molecular sketcher on the submission page, and then their physicochemical descriptors, ADME parameters, pharmacokinetic characteristics, and drug-like nature profile together with celecoxib were calculated.73

The summary of the prediction is presented in Table 3. It is interesting to note that despite the poor water solubility exhibited by celecoxib, as predicted from its log O/W, it showed high predicted GIT absorption. However, it failed to inhibit any of the three CYP isoforms, CYP2D6, CYP3A4, and CYP2C19. Conversely, the predicted log P O/W values of the tested compounds 5f, 6e, and 6f were in the range of 1.96 to 2.71, suggesting their poor to moderate water solubility and predicted high absorption through the gastrointestinal tract. Compounds 5f and 6f inhibited only two CYP isoforms, namely, CYP1A2 and CYP3A4, whereas compound 6e inhibited CYP1A2 and CYP2C19.

Molecular characteristics of celecoxib and compounds 5f, 6e and 6f using the SwissADME website.

Parameters Celecoxib Compound 5f Compound 6e Compound 6f
Consensus log P 3.40 1.96 2.71 1.96
Water solubility (ESOL) Poorly soluble Moderately soluble Poorly soluble Moderately soluble
GI absorption High High High High
BBB permeant No No No No
P-gp substrate No No No No
CYP1A2 inhibitor Yes Yes Yes Yes
CYP2C19 inhibitor No No Yes No
CYP2C9 inhibitor Yes No No No
CYP2D6 inhibitor No No No No
CYP3A4 inhibitor No Yes No Yes
Lipinski Yes Yes Yes Yes
Ghose No Yes Yes Yes
Veber Yes Yes Yes Yes
Egan Yes Yes Yes Yes
Muegge Yes Yes Yes Yes
Bioavailability score 0.55 0.55 0.55 0.55
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The BOILED-Egg graphical representation of the WLOGP vs. topological polar surface area for the investigated compounds is mentioned in the ESI.† Celecoxib, as well as compounds 5f, 6e, and 6f are in the human GIT absorption zone with the absence of BBB permeability, which reduces the possibility of expected CNS side effects. In addition, neither celecoxib nor compounds 5f, 6e, and 6f violate any Lipinski (Pfizer),74 Ghose,75 Veber (GSK),76 Egan (Pharmacia),77 or Muegge (Bayer)78 filters established by the major pharmaceutical corporations. Furthermore, similar to celecoxib, target compounds 5f, 6e, and 6f showed a high bioavailability score (0.55). Therefore, it can be concluded that these molecules possess potentially attractive pharmacokinetic and physicochemical features together with promising biological activity.

3. Experimental

3.1. Chemistry

3.1.1. General

A Griffin apparatus was used to measure the melting points, which were uncorrected. Carbon, hydrogen, and nitrogen microanalyses were conducted at the Regional Center for Mycology and Biotechnology, Faculty of Pharmacy, Al-Azhar University. IR spectra were recorded utilizing a Shimadzu IR 435 spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of Pharmacy, Cairo University, Cairo, Egypt, and the values are presented in cm−1. 1HNMR and 13CNMR spectra were recorded on Bruker 400 MHz and 100 MHz spectrophotometers, respectively (Bruker Corp., Billerica, MA, USA), Faculty of Pharmacy, Cairo University, Cairo, Egypt, using tetramethylsilane (TMS) as an internal standard. Chemical shifts were recorded on the δ scale and expressed in ppm. Coupling constants (J) are given in Hz. Thin-layer chromatography (TLC) was used to monitor the progress of the reactions and was performed on silica gel 60 F254 TLC plates (purchased from Merck). A UV lamp was used was used to visualize the spots. The selected solvent system for TLC was benzene : methanol : chloroform : TEA [9 : 3 : 1.5 : 0.1]. Compounds 1 and 2 were prepared following reported procedures.63,64

3.1.2. 3-Amino-4-(6-chloropyridazin-3-yl)-1H-pyrazol-5(4H)-one (3)

To a solution of compound 1 (0.01 mol, 2.50 g) in ethanol (20 mL), hydrazine hydrate (0.02 mol, 1 g) was added, and then the reaction mixture was heated under reflux for 24 h. After cooling, the solid product was filtered, dried, and crystallized from ethanol.

Yield 75%, mp 109–111 °C., IR (KBr) cm−1: 3545–3271 (NH2, NH), 3066 (CH aromatic), 2954 (CH aliphatic), 1681 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.09 (s, 1H, CH), 4.01 (s, 2H, NH2, D2O exchangeable), 6.96 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.50 (d, 1H, J = 9.84 Hz, pyridazine-H), 8.04 (s, 1H, NH, D2O exchangeable). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 27.3 (CH), 118.9, 132.3, 135.2, 138.2, 156.3 (aromatic-Cs), 160.3 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). Anal. calcd. (%) for C7H6ClIN5O (211.50): C, 39.73; H, 2.86; N, 33.10. Found: C, 40.02; H, 2.98; N, 33.37.

3.1.3. 4-(6-Chloropyridazin-3-yl)-4H-pyrazole-3,5-diamine (4)

A solution of compound 2 (0.011 mol, 2 g) was dissolved in ethanol (20 mL), and then hydrazine hydrate (0.022 mol, 2.2 g) was added, followed by 24 h of heating under reflux. The solid product was filtered, dried, and crystallized from ethanol after cooling.

Yield 70%, mp 134–136 °C, IR (KBr) cm−1: 3417–3332 (NH2), 3074 (CH aromatic), 2970 (CH aliphatic). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.08 (s, 1H, CH), 6.96 (d, 1H, J = 9.88 Hz, pyridazine-H), 7.50 (d, 1H, J = 9.88 Hz, pyridazine-H), 8.04 (s, 4H, 2NH2, D2O exchangeable). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 28.2 (CH), 128.7, 132.6, 134.7, 159.6, 161.7 (aromatic-Cs). Anal. calcd. (%) for C7H7ClN6 (210.50): C, 39.92; H, 3.35; N, 39.90. Found: C, 40.21; H, 3.49; N, 39.76.

3.1.4. 4-(6-chloropyridazin-3-yl)-3-(substitutedbenzylidene)amino-1H-pyrazol-5(4H)-one (5a–f)

To a solution of compound 3 (0.02 mol, 4 g) and the appropriate substituted benzaldehyde (0.02 mol) in ethanol (20 mL), five drops of glacial acetic acid were added. The reaction mixture was heated under reflux for 15 h. After cooling to room temperature, the resulting solid product was filtered, dried, and crystallized from ethanol to afford compounds 5a–f.

3.1.4.1. 4-(6-chloropyridazin-3-yl)-3-[(3-hydroxybenzylidene)amino]-1H-pyrazol-5(4H)-one (5a)

Yield 65%, mp >300 °C, IR (KBr) cm−1: 3417–3267 (OH, NH), 3055 (CH aromatic), 2927 (CH aliphatic), 1674 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.42 (s, 1H, CH), 6.80 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.07 (d, 1H, J = 7.84, aromatic-H), 7.20 (dd, 1H, J = 7.84, 8.08 Hz, aromatic-H), 7.31 (s, 1H, aromatic-H), 7.58 (d, 1H, J = 8.08 Hz, aromatic-H), 7.66 (d, 1H, J = 9.84 Hz, pyridazine-H), 8.07 (s, 2H, NH, OH, D2O exchangeable), 8.57 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 18.6 (CH), 112.5, 114.2, 115.9, 117.8, 120.2, 129.9, 135.1, 136.0, 142.4, 147.4, 157. (aromatic-Cs), 158.7 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 161.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). Anal. calcd. for C14H10ClN5O2 (315.50): C, 53.26, H, 3.19, N 22.18. Found: C, 53.47, H, 3.35, N 22.06.

3.1.4.2. 4-(6-chloropyridazin-3-yl)-3-((4-methoxybenzylidene)amino)-1H-pyrazol-5(4H)-one (5b)

Yield 50%, mp 184–186 °C, IR (KBr) cm−1: 3417 (NH), 3035 (CH aromatic), 2927 (CH aliphatic), 1680 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.78 (s, 1H, CH), 3.82 (s, 3H, OCH3), 6.97 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.04 (d, 2H, J = 8.68 Hz, aromatic-H), 7.63 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.80 (d, 2H, J = 8.68 Hz, aromatic-H), 8.09 (s, 1H, NH, D2O exchangeable). 8.62 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 24.7 (CH), 55.4 (OCH3), 114.3, 115.9 (2 C), 127.4, 128.0, 130.0 (2 C), 142.2, 147.0, 158.9, 160.2 (aromatic-Cs), 160.5 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 161.7 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). Anal. calcd. for C15H12ClN5O2 (329.50): C, 54.64, H, 3.67, N 21.24. Found: C, 54.88, H, 3.79, N 21.40.

3.1.4.3. 4-(6-chloropyridazin-3-yl)-3-[(4-chlorobenzylidene)amino]-1H-pyrazol-5(4H)-one (5c)

Yield 85%, mp 224–226 °C, IR (KBr) cm−1: 3441 (NH), 3032 (CH aromatic), 2943 (CH aliphatic), 1680 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.87 (s, 1H, CH), 7.47 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.58 (d, 2H, J = 8.04 Hz, aromatic-H), 7.73 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.89 (d, 2H, J = 8.04 Hz, aromatic-H), 8.10 (1 s, 1H, NH, D2O exchangeable). 8.71 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 24.6 (CH), 128.1 (2 C), 128.9, 129.2, 130.1 (2 C), 132.6, 133.7, 134.9, 136.2, 140.9 (aromatic-Cs), 160.7 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 165.7 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). Anal. calcd. for C14H9Cl2N5O (334.00): C, 50.32, H, 2.71, N, 20.96. Found: C, 50.49, H, 2.87, N, 20.85.

3.1.4.4. 4-(6-chloropyridazin-3-yl)-3-[(3-bromobenzylidene)amino]-1H-pyrazol-5(4H)-one (5d)

Yield 85%, mp 169–171 °C, IR (KBr) cm−1: 3441 (NH), 3055 (CH aromatic), 2951 (CH aliphatic), 1680 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.87 (s, 1H, CH), 6.97 (d, 1H, J = 9.64 Hz, pyridazine-H), 7.47 (dd, 1H, J = 8.24, 8.16 Hz, aromatic-H), 7.65 (d, 1H, J = 9.64 Hz, pyridazine-H), 7.72 (d, 1H, J = 8.24 Hz, aromatic-H), 7.88 (d, 1H, J = 8.24 Hz, aromatic-H), 8.06 (s, 1H, aromatic-H), 8.11 (s, 1H, NH, D2O exchangeable), 8.69 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 24.6 (CH), 116.2, 122.2, 125.6, 125.6, 127.3, 128.3, 130.0, 130.8, 131.6, 137.1, 140.2 (aromatic-Cs), 158.7 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 160.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). Anal. calcd. for C14H9BrClN5O (378.50): C, 44.41, H, 2.40, N, 18.50. Found: C, 44.63, H, 2.63, N, 18.72.

3.1.4.5. 4-(6-chloropyridazin-3-yl)-3-[(4-bromobenzylidene)amino]-1H-pyrazol-5(4H)-one (5e)

Yield 70%, mp 194–196 °C, IR (KBr) cm−1: 3417 (NH), 3028 (CH aromatic), 2939 (CH aliphatic), 1674 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.87 (s, 1H, CH), 6.96 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.50 (d, 2H, J = 8.48 Hz, aromatic-H), 7.59 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.65 (d, 2H, J = 8.48 Hz, aromatic-H), 8.11 (s, 1H, NH, D2O exchangeable). 8.68 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 24.9 (CH), 115.9, 118.7, 128.6 (2 C), 131.8 (2 C), 132.0, 132.8, 134.2, 135.2, 138.0 (aromatic-Cs), 160.2 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 165.9 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). Anal. calcd. for C14H9BrClN5O (378.50): C, 44.41, H, 2.40, N, 18.50. Found: C, 44.67, H, 2.62, N, 18.69.

3.1.4.6. 4-(6-chloropyridazin-3-yl)-3-[(3,4,5-trimethoxybenzylidene)amino]-1H-pyrazol-5(4H)-one (5f)

Yield 75%, mp 239–241 °C, IR (KBr) cm−1: 3425 (NH), 3035 (CH aromatic), 2935 (CH aliphatic), 1674 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.73 (s, 3H, OCH3), 3.76 (s, 1H, CH), 3.84 (s, 6H, 2OCH3), 6.99 (d, 1H, J = 9.80 Hz, pyridazine-H), 7.21 (s, 2H, aromatic-H), 7.67 (d, 1H, J = 9.80 Hz, pyridazine-H), 8.05 (1 s, 1H, NH, D2O exchangeable). 8.65 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 23.5 (CH), 55.9 (2 OCH3), 60.1 (OCH3), 102.3, 103.5 (2 C), 116.0, 129.9, 132.5, 134.7, 137.3, 141.8, 153.1 (2 C) (aromatic-Cs), 158.7 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 159.6 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). Anal. calcd. for C17H16ClN5O4 (389.50): C, 52.38, H, 4.14, N, 17.97. Found: C, 52.60, H, 4.29, N, 18.18.

3.1.5. N-Substituted benzylidene-4-(6-chloropyridazin-3-yl)-4H-pyrazole-3,5-diamines (6a–f)

The appropriate substituted benzaldehyde (0.02 mol, 4 g) and compound 4 (0.02 mol, 4 g) were dissolved in ethanol (20 mL) with 5 drops of glacial acetic acid added. The reaction mixture was heated under reflux for 15 h. The resultant solid product was filtered, dried, and crystallized from ethanol after cooling to room temperature to afford compounds 6a–f.

3.1.5.1. N-(3-Hydroxybenzylidene)-4-(6-chloropyridazin-3-yl)-4H-pyrazol-3,5-diamine (6a)

Yield 60%, mp 179–181 °C, IR (KBr) cm−1: 3500–3267 (OH, NH2), 3055 (CH aromatic), 2947 (CH aliphatic). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.44 (s, 1H, CH), 6.79 (d, 1H, J = 9.84 Hz, pyridazine-H), 6.94 (d, 1H, J = 7.84, aromatic-H), 7.13 (dd, 1H, J = 7.84, 7.80 Hz, aromatic-H), 7.27 (s, 1H, aromatic-H), 7.59 (d, 1H, J = 7.84 Hz, aromatic-H), 7.66 (d, 1H, J = 9.84, pyridazine-H), 8.06 (s, 2H, NH2, D2O exchangeable), 8.58 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 9.66 (s, 1H, OH, D2O exchangeable). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 25.5 (CH), 114.2, 115.9, 117.8, 118.7, 119.9, 129.9, 132.6, 134.8, 135.1, 135.9, 142.4, 147.4 (aromatic-Cs), 157.8 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). Anal. calcd. for C14H11ClN6O (314.50): C, 53.43, H 3.52, N, 26.70. Found: C, 53.70, H 3.71, N 26.58.

3.1.5.2. N-(4-Methoxybenzylidene)-4-(6-chloropyridazin-3-yl)-4H-pyrazol-3,5-diamine (6b)

Yield 65%, mp 159–161 °C, IR (KBr) cm−1: 3305–3190 (NH2), 3070 (CH aromatic), 2962 (CH aliphatic). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.78 (s, 1H, CH), 3.82 (s, 3H, OCH3), 6.97 (d, 1H, J = 9.92 Hz, pyridazine-H), 7.04 (d, 2H, J = 8.68 Hz, aromatic-H), 7.63 (d, 1H, J = 9.92 Hz, pyridazine-H), 7.80 (d, 2H, J = 8.68 Hz, aromatic-H), 8.09 (s, 2H, NH2, D2O exchangeable), 8.62 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 24.0 (CH), 55.4 (OCH3), 114.3 (2 C), 126.5, 127.3, 127.9, 129.9 (2 C), 131.8, 134.7, 142.0, 158.8, 160.4 (aromatic-Cs), 161.6 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). Anal. calcd. for C15H13ClN6O (328.50): C, 54.80, H, 3.99, N, 25.56. Found: C, 55.07, H, 4.16, N, 25.49.

3.1.5.3. N-(4-Chlorobenzylidene)-4-(6-chloropyridazin-3-yl)-4H-pyrazol-3,5-diamine (6c)

Yield 90%, mp >300 °C, IR (KBr) cm−1: 3302–3194 (NH2), 3097 (CH aromatic), 2943 (CH aliphatic). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.41 (s, 1H, CH), 6.96 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.46 (d, 2H, J = 8.40 Hz, aromatic-H), 7.72 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.89 (d, 2H, J = 8.40 Hz, aromatic-H), 8.13 (s, 2H, NH2, D2O exchangeable), 8.70 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 27.4 (CH), 116.1, 128.0 (2 C), 128.7, 129.0, 129.4, 130.1 (2 C), 131.3, 132.7, 134.9, 137.6 (aromatic-Cs), 159.8 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). Anal. calcd. for C14H10Cl2N6 (333.00): C, 50.47, H, 3.03, N, 25.22. Found: C, 50.76, H, 2.97, N, 25.43.

3.1.5.4. N-(3-Bromobenzylidene)-4-(6-chloropyridazin-3-yl)-4H-pyrazol-3,5-diamine (6d)

Yield 80%, mp 259–261 °C, IR (KBr) cm−1: 3313–3194 (NH2), 3055 (CH aromatic), 2951 (CH aliphatic). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.87 (s, 1H, CH), 7.35 (d, 1H, J = 9.92 Hz, pyridazine-H), 7.48 (dd, 1H, J = 7.84, 8.20, aromatic-H), 7.54 (d, 1H, J = 9.92 Hz, pyridazine-H), 7.72 (d, 1H, J = 8.32 Hz, aromatic-H), 7.88 (d, 1H, J = 8.32 Hz, aromatic-H), 8.05 (s, 1H, aromatic-H), 8.07 (s, 2H, NH2, D2O exchangeable), 8.69 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 18.5 (CH), 116.3, 122.2, 125.6, 127.3, 128.2, 130.1, 130.7, 131.6, 132.6, 134.7, 137.2, 147.6 (aromatic-Cs), 160.5 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). Anal. calcd. for C14H10BrClN6 (377.50): C, 44.53, H, 2.67, N, 22.25. Found: C, 44.71, H, 2.89, N, 22.48.

3.1.5.5. N-(4-Bromobenzylidene)-4-(6-chloropyridazin-3-yl)-4H-pyrazol-3,5-diamine (6e)

Yield 80%, mp. 289–291 °C, IR (KBr) cm−1: 3329–3205 (NH2), 3047 (CH aromatic), 2935 (CH aliphatic).1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.43 (s, 1H, CH), 6.96 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.59 (d, 2H, J = 8.44 Hz, aromatic-H), 7.65 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.82 (d, 2H, J = 8.44 Hz, aromatic-H), 8.11 (s, 2H, NH2, D2O exchangeable), 8.69 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 18.9 (CH), 116.4, 122.6, 128.6 (2 C), 130.5 (2 C), 131.7, 132.1, 132.7, 134.4, 135.2, 148.0 (aromatic-Cs), 159.1 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). Anal. calcd. for C14H10BrClN6 (377.50): C, 44.53, H 2.67, N, 22.25. Found: C, 44.71, H, 2.80, N, 22.49.

3.1.5.6. N-(3,4,5-Trimethoxybenzylidene)-4-(6-chloropyridazin-3-yl)-4H-pyrazol-3,5-diamine (6f)

Yield 70%, mp 224–226 °C, IR (KBr) cm−1: 3340–3201 (NH2), 3008 (CH aromatic), 2947 (CH aliphatic). 1HNMR (DMSO-d6, 400 MHz, δ ppm): 3.72 (s, 3H, OCH3), 3.81 (s, 1H, CH), 3.84 (s, 6H, 2 OCH3), 6.98 (d, 1H, J = 9.84 Hz, pyridazine-H), 7.24 (s, 2H, aromatic-H), 7.63 (d, 1H, J = 9.84 Hz, pyridazine-H), 8.05 (s, 2H, NH2, D2O exchangeable), 8.64 (s, 1H, N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). 13CNMR (DMSO-d6, 100 MHz, δ ppm): 18.9 (CH), 56.5 (2 OCH3), 60.6 (OCH3), 104.0 (2 C), 106.0, 107.1, 132.0, 135.1, 135.2, 142.4, 143.2, 153.6 (2 C), 153.7 (aromatic-Cs), 161.6 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). Anal. calcd. for C17H17ClN6O3 (388.50): C, 52.51, H, 4.41, N, 21.61. Found: C, 52.74, H 4.62, N, 21.89.

3.2. Cell viability assay

RAW264.7 cells (6 × 103 cells per per) were placed in a 96-well plate and kept for 24 h. Five different concentrations of the investigated compounds were used (100, 25, 6.3, 1.6, and 0.4 μM L−1). Firstly, the cells were subjected to various concentrations for 1 h, and then incubated with LPS (0.5 μg mL−1) for 24 h. After that, MTT solution (5 mg mL−1) was added to each well, and then kept for 4 h at 37 °C. The medium containing MTT was removed, and then treated with 150 μL of DMSO. A microplate reader detected the absorbance at 500–600 nm.63

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

In this assay, celecoxib and indomethacin were used as reference medications to investigate the ability of the new compounds to suppress human COX-1 and COX-2 using serial dilutions (1, 0.1, 0.01, 0.001 μg mL−1) (Table 1). This was achieved using human COX-1 and COX-2 inhibitor screening kits supplied by Cayman Chemicals (catalog numbers 701070 and 701080, respectively, Ann Arbor, MI, USA). Dimethylsulfoxide (DMSO) was used to solubilize the new derivatives. Briefly, a mixture containing COX-1 or COX-2 enzyme (10 μL), heme (10 μL), and DMSO solutions of the tested compounds samples (20 μL) was added to the supplied reaction buffer solution [160 μL, 0.1 M Tris-HCl, pH 8 containing 5 mM ethylenediamine tetra acetate (EDTA) and 2 mM phenol], and then incubated for 10 min at 37 °C. Meanwhile, arachidonic acid was added (10 μL). To initiate the reaction, the final reaction mixture concentration was 100 μM. Later, stannous chloride (30 μL) was added to stop the COX reactions, and then incubated at an ambient temperature for 5 min. After that, quantification of PGF2α produced in the samples by COX reactions was carried out by adopting an enzyme-linked immunosorbent assay (ELISA). The samples were transferred to a 96-well plate, and then incubated at room temperature for 18 h. Any unbound reagent was removed by washing. Ellman's reagent (200 μL) was added and incubated for 60–90 min at room temperature. Ultimately, an ELISA plate reader was used to read the plate, and then calculation of the IC50 values for the inhibition of the COX-1 and COX-2 enzymes was carried out.37,64,65,79

3.4. Inhibition of TNF-α, IL-6, and PGE-2 expression in LPS-induced RAW264.7 cells

RAW 264.7 cells were incubated in 96 well-plates (5 × 103 cells per well) in 5% CO2 at 37 °C for 24 h. Thereafter, the cells were treated with the tested compounds (50 μM) in the presence of LPS (1 μg mL−1). The untreated cells acted as the blank control, whereas the negative control group (LPS group) was represented by the treated cells with LPS + DMSO (final concentration of 0.1% (v/v). TNF-α, PGE-2, and IL-6 were quantified 24 h after treatment, utilizing specified ELISA kits.66,80,81

3.5. Inhibitory effect of 5f, 6e and 6f on NO expression

Aiming to measure the NO expression in the culture medium, RAW264.7 cells pretreated with the test compounds for 2 h were treated with LPS (1 μg mL−1) for 24 h. Griess reagent (100 L) (Sigma-Aldrich, St. Louis, MO, USA) was added to 100 L of culture medium, and then kept at room temperature for 10 min. An ELISA reader at 540 nm was used to measure the optical density (OD) as an indicator of NO.69,70

3.6. Molecular docking of compounds 5f, 6e and 6f in the active site of COX-2

The Molecular Operating Environment (MOE version 2015.10) software was used to study the molecular modeling of the new active compounds 5f, 6e, and 6f. The COX-2 enzyme X-ray crystal structure (PDB entry 3LN1) was downloaded from the RCSB protein data bank website (http://www.rcsb.org). Aiming to prepare the protein structure, the repeating chains were removed from the enzyme active site. Water molecules that do not contribute to binding were removed from the pocket. A protonate 3D process was applied to add hydrogen atoms to the atoms of the receptor using the default settings. The determined pocket was isolated, and the partial charges were calculated. Hiding backbone was performed to give the final prepared protein. In addition, all compounds were outlined using ChemDraw Office (ChemDraw Office version 12.0.2) and saved as mol extension, and then 3D protonated. Then, partial charges were automatically calculated, and energy was minimized with Merck Molecular force field (MMFF94x) until a root mean square deviation (RMSD) gradient of 0.05 kcal mol−1 Å−1 was performed. The co-crystalline ligand was redocked to validate the docking technique. The RMSD was 0.4172 Å with a docking score (S) of −9.3344 kcal mol−1. Docking into the active site was achieved using the MOE Dock option. The triangle matcher placement was the method of displacement in molecular modeling, and the main scoring function was London dG (Table 2 and Fig. 8–11).

Statistical analysis

All data are expressed as means ± SEM. Statistical significance was determined by P values less than 0.05. Tukey's multiple comparison test was applied to analyze the data after a one-way analysis of variance (ANOVA). GraphPad Prism (version 6; GraphPad Software, Inc., San Diego, CA, USA) was utilized for data analysis.

Conclusion

This study demonstrated the synthesis of new pyrazole–pyridazine hybrids 5a–f and 6a–f. Based on the cell viability assay, all tested compounds showed no cytotoxicity to vital cells. Additionally, COX-2 inhibition and selectivity were tested for all synthesized compounds. Among the tested compounds, 5f and 6f displayed more potent COX-2 enzyme inhibitory activity than celecoxib, while compound 6e emerged as a promising COX-2 inhibitory candidate with an IC50 value comparable to that of celecoxib. Consequently, the effects of these compounds on pro-inflammatory mediators and cytokines, namely, PGE-2, TNF-α, IL-6, and NO, were evaluated in LPS-induced RAW264.7 macrophage cells. Compound 6f exhibited the most potent inhibition of NO production (70%) in comparison to celecoxib (83%). Moreover, compounds 6e and 6f exhibited promising inhibition of the pro-inflammatory mediators TNF-α, IL-6, and PGE-2 in vitro. The docking results of the most active compounds 5f, 6e, and 6f achieved a binding mode with H-bond and π–H stacking interactions within the COX-2 active site. Based on the in vitro biological investigations and molecular docking studies, some pyridazine–pyrazole hybrids may provide insight into more potent and safer anti-inflammatory candidates. The bulkier structure of the pyridazine–pyrazole hybrids is supposed to selectively target the bulkier COX-2 enzyme pocket, imparting safety, which remains a future perspective in the management of inflammation. In the following work, our research group aims to synthesize bulky pyridazine–pyrazole hybrids that include different pharmacophoric functional groups to aid in better interactions with the binding site as a trial to improve the activity and selectivity towards COX-2 inhibition.

Author contributions

Eman O. Osman: formal analysis, funding acquisition, investigation, methodology, supervision, writing (original draft) and writing (review and editing). Nadia A. Khalil: formal analysis, funding acquisition, investigation, methodology, supervision, writing (original draft) and writing (review and editing). Alaa Magdy: funding acquisition, investigation, writing (original draft) and writing (review and editing). Yara El-Dash: formal analysis, funding acquisition, investigation, methodology, supervision, writing (original draft) and writing (review and editing).

Conflicts of interest

The authors declare that they have no competing interests. The content and writing of this manuscript are solely the authors' responsibility.

Supplementary Material

MD-015-D4MD00135D-s001

Acknowledgments

The authors are thankful to the associates of the confirmatory diagnostic unit VACSERA-EGYPT, for performing in vitro COX-1 and COX-2 inhibition assays and inhibitory assays of TNF-α, IL-6, and PGE-2 mediators in LPS-induced RAW264.7 cells.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4md00135d

References

  1. Zhang H. Tsao R. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr. Opin. Food Sci. 2016;8:33–42. doi: 10.1016/j.cofs.2016.02.002. [DOI] [Google Scholar]
  2. Alonazi M. A. Jemel I. Moubayed N. Alwhibi M. El-Sayed N. N. E. Ben B. A. Evaluation of the in vitro anti-inflammatory and cytotoxic potential of ethanolic and aqueous extracts of Origanum syriacum and Salvia lanigera leaves. Environ. Sci. Pollut. Res. 2021;28(16):19890–19900. doi: 10.1007/s11356-020-11961-z. [DOI] [PubMed] [Google Scholar]
  3. Zhong J. Shi G. Regulation of inflammation in chronic disease. Front. Immunol. 2019;10:737–738. doi: 10.3389/fimmu.2019.00737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Akira S. Uematsu S. Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124(4):783–801. doi: 10.1016/j.cell.2006.02.015. [DOI] [PubMed] [Google Scholar]
  5. Panchal N. K. Sabina E. P. Non-steroidal anti-inflammatory drugs (NSAIDs): A current insight into its molecular mechanism eliciting organ toxicities. Food Chem. Toxicol. 2023;172:113598. doi: 10.1016/j.fct.2022.113598. [DOI] [PubMed] [Google Scholar]
  6. Vane J. R. Botting R. M. Anti-inflammatory drugs and their mechanism of action. Inflammation Res. 1998;47:78–87. doi: 10.1007/s000110050284. [DOI] [PubMed] [Google Scholar]
  7. Isaza-Guzmán D. M. Teller-Carrero C. B. Laberry-Bermúdez M. P. González-Pérez L. V. Tobón-Arroyave S. I. Assessment of clinicopathological characteristics and immunoexpression of COX-2 and IL-10 in oral pyogenic granuloma. Arch. Oral Biol. 2012;57(5):503–512. doi: 10.1016/j.archoralbio.2011.11.004. [DOI] [PubMed] [Google Scholar]
  8. Julémont F. De Leval X. Michaux C. Renard J. F. Winum J. Y. Montero J. L. et al., Design, synthesis, and pharmacological evaluation of pyridinic analogues of nimesulide as cyclooxygenase-2 selective inhibitors. J. Med. Chem. 2004;47(27):6749–6759. doi: 10.1021/jm049480l. [DOI] [PubMed] [Google Scholar]
  9. Hijos-mallada G. Sostres C. Gomollón F. NSAIDs, gastrointestinal toxicity and inflammatory bowel disease. J. Gastroenterol. Hepatol. 2022;45:215–222. doi: 10.1016/j.gastrohep.2021.06.003. [DOI] [PubMed] [Google Scholar]
  10. Wirth T. Lafforgue P. Pham T. NSAID : Current limits to prescription. Jt., Bone, Spine. 2024;91(4):105685. doi: 10.1016/j.jbspin.2023.105685. [DOI] [PubMed] [Google Scholar]
  11. Qiu K. M. Yan R. Xing M. Wang H. H. Cui H. E. Bin G. H. et al., Synthesis, biological evaluation and molecular modeling of dihydro-pyrazolyl-thiazolinone derivatives as potential COX-2 inhibitors. Bioorg. Med. Chem. 2012;20:6648–6654. doi: 10.1016/j.bmc.2012.09.021. [DOI] [PubMed] [Google Scholar]
  12. Botting R. Ayoub S. S. COX-3 and the mechanism of action of paracetamol / acetaminophen. Prostaglandins, Leukotrienes Essent. Fatty Acids. 2005;72:85–87. doi: 10.1016/j.plefa.2004.10.005. [DOI] [PubMed] [Google Scholar]
  13. Chang Y. Chao Y. Jean W. Lin T. Lu C. Nitric oxide and tumor necrosis factor-α levels are negatively correlated in endotoxin tolerance recovery in vitro. Nitric Oxide. 2024;147:1–5. doi: 10.1016/j.niox.2024.03.005. [DOI] [PubMed] [Google Scholar]
  14. Li H. Yoon J. H. Won H. J. Ji H. S. Yuk H. J. Park K. H. et al., Isotrifoliol inhibits pro-inflammatory mediators by suppression of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 cells. Int. Immunopharmacol. 2017;45:110–119. doi: 10.1016/j.intimp.2017.01.033. [DOI] [PubMed] [Google Scholar]
  15. Wang F. Liu L. Zhu Z. Akber H. Xin X. Anti-inflammatory effect and mechanism of active parts of Artemisia mongolica in LPS-induced Raw264. 7 cells based on network pharmacology analysis. J. Ethnopharmacol. 2024;321:117509. doi: 10.1016/j.jep.2023.117509. [DOI] [PubMed] [Google Scholar]
  16. Sharma J. N. Al-Omran A. Parvathy S. S. Role of nitric oxide in inflammatory diseases. Inflammopharmacology. 2007;15:252–259. doi: 10.1007/s10787-007-0013-x. [DOI] [PubMed] [Google Scholar]
  17. Gamal El-Din M. M. El-Gamal M. I. Abdel-Maksoud M. S. Lee H. Choi J. Kim T. W. et al., Inhibitory effects of triarylpyrazole derivatives on LPS-induced nitric oxide and PGE2 productions in murine RAW 264.7 macrophages. Bioorg. Med. Chem. Lett. 2020;30(4):68–84. doi: 10.1016/j.bmcl.2019.126884. [DOI] [PubMed] [Google Scholar]
  18. Bhardwaj A. Batchu S. N. Kaur J. Huang Z. Seubert J. M. Knaus E. E. Cardiovascular properties of a nitric oxide releasing rofecoxib analogue: Beneficial anti-hypertensive activity and enhanced recovery in an ischemic reperfusion injury model. ChemMedChem. 2012;7(8):1365–1368. doi: 10.1002/cmdc.201200234. [DOI] [PubMed] [Google Scholar]
  19. Kaur J. Bhardwaj A. Huang Z. Knaus E. E. Aspirin analogues as dual cyclooxygenase-2/5-lipoxygenase inhibitors: Synthesis, nitric oxide release, molecular modeling, and biological evaluation as anti-inflammatory agents. ChemMedChem. 2012;7(1):144–150. doi: 10.1002/cmdc.201100460. [DOI] [PubMed] [Google Scholar]
  20. Emam S. H. Sonousi A. Osman E. O. Hwang D. Kim G. Hassan R. A. 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. 2021;107:104630. doi: 10.1016/j.bioorg.2021.104630. [DOI] [PubMed] [Google Scholar]
  21. Carter P. H. Scherle P. A. Muckelbauer J. A. Voss M. E. Liu R. Q. Thompson L. A. et al., Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-α. Proc. Natl. Acad. Sci. U. S. A. 2001;98(21):11879–11884. doi: 10.1073/pnas.211178398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Bandgar B. P. Gawande S. S. Bodade R. G. Gawande N. M. Khobragade C. N. Synthesis and biological evaluation of a novel series of pyrazole chalcones as anti-inflammatory, antioxidant and antimicrobial agents. Bioorg. Med. Chem. 2009;17(24):8168–8173. doi: 10.1016/j.bmc.2009.10.035. [DOI] [PubMed] [Google Scholar]
  23. Raj D. S. C. Role of Interleukin-6 in the anemia of chronic disease. Semin. Arthritis Rheum. 2009;38(5):382–388. doi: 10.1016/j.semarthrit.2008.01.006. [DOI] [PubMed] [Google Scholar]
  24. Soares C. L. R. Wilairatana P. Silva L. R. Moreira P. S. Barbosa N. M. M. V. Silva P. R. et al., Biochemical aspects of the inflammatory process : A narrative review. Biomed. Pharmacother. 2023;168:115764. doi: 10.1016/j.biopha.2023.115764. [DOI] [PubMed] [Google Scholar]
  25. Salim T. Sershen C. L. May E. E. Investigating the role of TNF-α and IFN- γ activation on the dynamics of iNOS gene expression in LPS stimulated macrophages. PLoS One. 2016;11:53–89. doi: 10.1371/journal.pone.0153289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tanaka T. Narazaki M. Kishimoto T. IL-6 in Inflammation, immunity, and disease. Cold Spring Harbor Perspect. Biol. 2014;6:62–95. doi: 10.1101/cshperspect.a016295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sayed A. R. Gomha S. M. Abdelrazek F. M. Farghaly M. S. Hassan S. A. Metz P. Design, efficient synthesis and molecular docking of some novel thiazolyl - pyrazole derivatives as anticancer agents. BMC Chem. 2019;13:116–128. doi: 10.1186/s13065-019-0632-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Edrees M. M. Melha S. A. Saad A. M. Kheder N. A. Gomha S. M. Muhammad Z. A. Eco-friendly synthesis, characterization and biological evaluation of some novel pyrazolines containing thiazole moiety as potential anticancer and antimicrobial agents. Molecules. 2018;23:2970–2982. doi: 10.3390/molecules23112970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Abdallah M. A. Gomha S. M. Abbas I. M. Kazem M. S. H. Alterary S. S. Mabkhot Y. N. An efficient synthesis of novel pyrazole-based heterocycles as potential antitumor agents. Appl. Sci. 2017;7:785–797. doi: 10.3390/app7080785. [DOI] [Google Scholar]
  30. Gomha S. M. Abdallah M. A. Abbas I. M. Kazem M. S. H. Synthesis, cytotoxicity evaluation, molecular docking and utility of novel chalcones as precursors for heterocycles incorporating pyrazole moiety. Med. Chem. 2018;14(4):344–355. doi: 10.2174/1573406413666171020114105. [DOI] [PubMed] [Google Scholar]
  31. Gomha S. M. Abdel-aziz H. M. Badrey M. G. Abdulla M. M. Efficient synthesis of some new 1,3,4-thiadiazoles and 1,2,4-triazoles linked to pyrazolylcoumarin ring system as potent 5α-reductase inhibitors. J. Heterocyclic Chem. 2019;56:1275–1282. doi: 10.1002/jhet.3487. [DOI] [Google Scholar]
  32. Abdelhamid A. O. Gomha S. M. El-Enany W. A. M. A. Efficient synthesis and antimicrobial evaluation of new azolopyrimidines-bearing pyrazole moiety. J. Heterocyclic Chem. 2019;56:2487–2493. doi: 10.1002/jhet.3638. [DOI] [Google Scholar]
  33. Abbas I. M. Abdallah M. A. Gomha S. M. Kazem M. S. H. Synthesis and antimicrobial activity of novel azolopyrimidines and pyrido-triazolo-pyrimidinones incorporating pyrazole moiety. J. Heterocyclic Chem. 2017;54:3447–3457. doi: 10.1002/jhet.2968. [DOI] [Google Scholar]
  34. Gomha S. M. Dawood K. M. Synthetic utility of pyridinium bromide : synthesis and antimicrobial activity of novel 2,4,6-trisubstituted pyridines having pyrazole moiety. J. Heterocyclic Chem. 2017;54:1943–1948. doi: 10.1002/jhet.2790. [DOI] [Google Scholar]
  35. Gomha S. M. Hassaneen H. M. E. Synthesis and antimicrobial activity of some new pyrazoles, fused pyrazolo[3,4-d]-pyrimidine and 1,2-dihydroimidazo-[2,1-c][1,2,4]triazin-6-one derivatives. Molecules. 2011;16:6549–6560. doi: 10.3390/molecules16086549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Abolibda T. Z. Fathalla M. Aljohani G. F. Zayed E. M. Gomha S. M. Synthesis and in silico antiviral activity of novel bioactive thiobarbituric acid based hydrazones and pyrazoles against SARS-CoV-2 main protease (Mpro) Polycyclic Aromat. Compd. 2023;8:7635–7650. doi: 10.1080/10406638.2022.2138922. [DOI] [Google Scholar]
  37. El-Sayed M. A. A. Abdel-Aziz N. I. Abdel-Aziz A. A. M. El-Azab A. S. Asiri Y. A. Eltahir K. E. H. Design, synthesis, and biological evaluation of substituted hydrazone and pyrazole derivatives as selective COX-2 inhibitors: Molecular docking study. Bioorg. Med. Chem. 2011;19(11):3416–3424. doi: 10.1016/j.bmc.2011.04.027. [DOI] [PubMed] [Google Scholar]
  38. Palomer A. Cabré F. Pascual J. Campos J. Trujillo M. A. Entrena A. et al., Identification of novel cyclooxygenase-2 selective inhibitors using pharmacophore models. J. Med. Chem. 2002;45(7):1402–1411. doi: 10.1021/jm010458r. [DOI] [PubMed] [Google Scholar]
  39. Alang G. Kauyr R. Singh A. Budhlakoti P. Singh A. Synthesis, characterization and anti-bacterial activity of certain 2,3,4,5-tetrahydropyridazinone analogues. Turk. J. Pharm. Sci. 2011;8(1):71–80. [Google Scholar]
  40. Husain A. Ahmad A. Bhandari A. Ram V. Synthesis and antitubercular activity of pyridazinone derivatives. J. Chil. Chem. Soc. 2011;56(3):778–780. doi: 10.4067/S0717-97072011000300013. [DOI] [Google Scholar]
  41. Asif M. Singh D. Singh A. Analgesic activity of some 6-phenyl-4-substituted benzylidene tetrahydro pyridazin-3(2H)-ones. Global J. Pharmacol. 2011;5(1):18–22. [Google Scholar]
  42. Asif M. General study of pyridazine compounds against cyclooxygenase enzyme and their relation with analgesic, anti-inflammatory and anti-arthritic activities. Chron. Young Sci. 2010;1(3):3–9. [Google Scholar]
  43. Osman E. O. Khalil N. A. Magdy A. El-Dash Y. Pyridazine and pyridazinone derivatives: Synthesis and in vitro investigation of their anti-inflammatory potential in LPS-induced RAW264. 7 macrophages. Drug Dev. Res. 2024;85(2):e22173. doi: 10.1002/ddr.22173. [DOI] [PubMed] [Google Scholar]
  44. Xu H. Hu X. Zou X. Liu B. Zhu Y. Wang Y. et al., Synthesis and herbicidal activities of novel 3-N-substituted amino-6-methyl-4-(3-trifluromethylphenyl)pyridazine derivatives. J. Agric. Food Chem. 2008;56(15):6567–6572. doi: 10.1021/jf800900h. [DOI] [PubMed] [Google Scholar]
  45. Abu-Melha S. Edrees M. M. Riyadh S. M. Abdelaziz M. R. ElfikY A. A. Gomha S. M. Clean grinding technique: A facile synthesis and in silico antiviral activity of hydrazones, pyrazoles, and pyrazines bearing thiazole moiety against SARS-CoV-2 main protease (Mpro) Molecules. 2020;25:4565–4577. doi: 10.3390/molecules25194565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Costas T. Costas-Lago M. C. Vila N. Besada P. Cano E. Terán C. New platelet aggregation inhibitors based on pyridazinone moiety. Eur. J. Med. Chem. 2015;94:113–122. doi: 10.1016/j.ejmech.2015.02.061. [DOI] [PubMed] [Google Scholar]
  47. El-Ghaffar N. F. A. Mohamed M. K. Kadah M. S. Radwan A. M. Said G. H. et al., Synthesis and anti-tumor activities of some new pyridazinones containing the 2-phenyl-1H-indolyl moiety. J. Chem. Pharm. Res. 2011;3(3):248–259. [Google Scholar]
  48. Wang T. Dong Y. Wang L. C. Chen Z. Synthesis and bioactivity of 6-phenyl-4,5-dihydro-3(2H)-pyridazinone derivatives. Arzneim. Forsch. 2007;57(10):641–646. doi: 10.1055/s-0031-1296662. [DOI] [PubMed] [Google Scholar]
  49. Siddiqui A. A. Mishra R. Shaharyar M. Synthesis, characterization and antihypertensive activity of pyridazinone derivatives. Eur. J. Med. Chem. 2010;45:2283–2290. doi: 10.1016/j.ejmech.2010.02.003. [DOI] [PubMed] [Google Scholar]
  50. Banerjee P. S. Sharma P. K. Nema R. K. Synthesis and anticonvulsant activity of pyridazinone derivatives. Int. J. ChemTech Res. 2009;1(3):522–525. [Google Scholar]
  51. Rathish I. G. Javed K. Bano S. Ahmad S. Alam M. S. Pillai K. K. Synthesis and blood glucose lowering effect of novel pyridazinone substituted benzenesulfonylurea derivatives. Eur. J. Med. Chem. 2009;44:2673–2678. doi: 10.1016/j.ejmech.2008.12.013. [DOI] [PubMed] [Google Scholar]
  52. Khalil N. A. Ahmed E. M. Mohamed K. O. Nissan Y. M. Zaitone S. A. Synthesis and biological evaluation of new pyrazolone–pyridazine conjugates as anti-inflammatory and analgesic agents. Bioorg. Med. Chem. 2014;22(7):2080–2089. doi: 10.1016/j.bmc.2014.02.042. [DOI] [PubMed] [Google Scholar]
  53. Aggarwal R. Swati S. Kumar V. Singh R. Kajal A. Saini D. Design, synthesis, and biological evaluation of N-[1-(6′-chloropyridazin-3′-yl)-3-(4′′-substitutedphenyl)-1H-pyrazole-5-yl]alkanamides as anti-inflammatory agents. Drug Dev. Res. 2022;83(3):811–822. [Google Scholar]
  54. Banoglua E. Akoglua C. Ünlüa S. Ergüna B. C. Küpelib E. Yesiladab E. Sahin M. F. Synthesis of amide derivatives of [6-(3,5-dimethylpyrazol-1-yl)-3(2H)-pyridazinone- 2-yl]acetic acid and their analgesic and anti-inflammatory properties. Arzneim. Forsch. 2005;3(9):520–527. doi: 10.1055/s-0031-1296899. [DOI] [PubMed] [Google Scholar]
  55. Sukuroglu M. Ergun B. C. Unlu S. Sahin F. Kupeli E. Yesilada E. et al., Synthesis, analgesic, and anti-inflammatory activities of [6-(3,5-dimethyl-4-chloropyrazole-l-yl)-3(2H)-pyridazinon-2- yl]acetamides. Arch. Pharmacal Res. 2005;28(5):509–517. doi: 10.1007/BF02977751. [DOI] [PubMed] [Google Scholar]
  56. Ibrahim T. H. Loksha Y. M. Elshihawy H. A. Khodeer D. M. Said M. M. Synthesis of some novel 2,6-disubstituted pyridazin-3(2H)-one derivatives as analgesic, anti-inflammatory, and non-ulcerogenic agents. Arch. Pharm. 2017;350(9):70–93. doi: 10.1002/ardp.201700093. [DOI] [PubMed] [Google Scholar]
  57. Baytas S. İnceler N. Mavaneh K. F. Uluda M. O. Abacioglu N. Gokce M. Synthesis of antipyrine/pyridazinone hybrids and investigation of their in vivo analgesic and anti-inflammatory activities. Turk. J. Chem. 2012;36(5):734–748. [Google Scholar]
  58. Belal A. Abdelgawad M. A. New benzothiazole/benzoxazole-pyrazole hybrids with potential as COX inhibitors: design, synthesis and anticancer activity evaluation. Res. Chem. Intermed. 2017;43(7):3859–3872. doi: 10.1007/s11164-016-2851-x. [DOI] [Google Scholar]
  59. Ünlü S. Banoglu E. Ito S. Niiya T. Eren G. Ökçelik B. et al., Synthesis , characterization and preliminary screening of regioisomeric 1- ( 3-pyridazinyl ) -3- derivatives towards cyclooxygenase inhibition. J. Enzyme Inhib. Med. Chem. 2007;22(3):351–361. doi: 10.1080/14756360601073468. [DOI] [PubMed] [Google Scholar]
  60. Abdelgawad M. A. Labib M. B. Abdel-Latif M. Pyrazole-hydrazone derivatives as anti-inflammatory agents: Design, synthesis, biological evaluation, COX-1,2/5-LOX inhibition and docking study. Bioorg. Chem. 2017;74:212–220. doi: 10.1016/j.bioorg.2017.08.014. [DOI] [PubMed] [Google Scholar]
  61. Abdelgawad M. A. Labib M. B. Ali W. A. M. Kamel G. Azouz A. A. EL-Nahass E. S. Design, synthesis, analgesic, anti-inflammatory activity of novel pyrazolones possessing aminosulfonyl pharmacophore as inhibitors of COX-2/5-LOX enzymes: Histopathological and docking studies. Bioorg. Chem. 2018;78:103–114. doi: 10.1016/j.bioorg.2018.03.011. [DOI] [PubMed] [Google Scholar]
  62. Bandgar B. P. Chavan H. V. Adsul L. K. Thakare V. N. Shringare S. N. Shaikh R. et al., Design, synthesis, characterization and biological evaluation of novel pyrazole integrated benzophenones. Bioorg. Med. Chem. Lett. 2013;23(3):912–916. doi: 10.1016/j.bmcl.2012.10.031. [DOI] [PubMed] [Google Scholar]
  63. Pollak A. Stanovnik S. Tišler M. Venetic-Fortuna J. Heterocycles, CXXX.: Reactions of some halogenated heterocycles and N-oxides with reactive methylene compound. Monatsh. Chem. 1975;106:473–781. doi: 10.1007/BF01150528. [DOI] [Google Scholar]
  64. Petrenko O. P. Lapachev V. V. Mamaev V. P. Tautomerism of 3-pyridazinylmethane derivatives. Russ. Chem. Bull. 1984;32(6):1687–1688. [Google Scholar]
  65. Stockert J. C. Horobin R. W. Colombo L. L. Blázquez-Castro A. Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem. 2018;120(3):159–167. doi: 10.1016/j.acthis.2018.02.005. [DOI] [PubMed] [Google Scholar]
  66. Hwang D. Son B. W. Shin P. G. Choi J. S. Seo Y. B. Kim G. D. Toluhydroquinone from Aspergillus sp. suppress inflammatory mediators via nuclear factor-κB and mitogen-activated protein kinases pathways in lipopolysaccharide-induced RAW264.7 cells. J. Pharm. Pharmacol. 2015;67(9):1297–1305. doi: 10.1111/jphp.12421. [DOI] [PubMed] [Google Scholar]
  67. Abdel-Aziz A. A. M. Eltahir K. E. H. Asiri Y. A. Synthesis, anti-inflammatory activity and COX-1/COX-2 inhibition of novel substituted cyclic imides. Part 1: Molecular docking study. Eur. J. Med. Chem. 2011;46(5):1648–1655. doi: 10.1016/j.ejmech.2011.02.013. [DOI] [PubMed] [Google Scholar]
  68. Liao J. C. Deng J. S. Chiu C. S. Hou W. C. Huang S. S. Shie P. H. et al., Anti-inflammatory activities of Cinnamomum cassia constituents in vitro and in vivo. J. Evidence-Based Complementary Altern. Med. 2012;2012:1–12. doi: 10.1155/2012/429320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Vuolteenaho K. Moilanen T. Knowles R. G. Moilanen E. The role of nitric oxide in osteoarthritis. Scand. J. Rheumatol. 2007;36(4):247–258. doi: 10.1080/03009740701483014. [DOI] [PubMed] [Google Scholar]
  70. Boscá L. Zeini M. Través P. G. Hortelano S. Nitric oxide and cell viability in inflammatory cells: A role for NO in macrophage function and fate. Toxicology. 2005;208(2):249–258. doi: 10.1016/j.tox.2004.11.035. [DOI] [PubMed] [Google Scholar]
  71. Sun H. Cai W. Wang X. Liu Y. Hou B. Zhu X. et al., Vaccaria hypaphorine alleviates lipopolysaccharide-induced inflammation via inactivation of NF κB and ERK pathways in Raw 264.7 cells. BMC Complementary Altern. Med. 2017;17:120–131. doi: 10.1186/s12906-017-1635-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Kim T. W. Shin J. S. Chung K. S. Lee Y. G. Baek N. I. Lee K. T. Anti-inflammatory mechanisms of koreanaside A, a lignan isolated from the flower of forsythia koreana, against LPS-induced macrophage activation and DSS-induced colitis mice: The crucial role of AP-1, NF-κB, and JAK/STAT signaling. Cells. 2019;8(10):1163–1182. doi: 10.3390/cells8101163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Antypenko O. Antypenko L. Kalnysh D. Kovalenko S. ADME prperties prediction of 5-phenyl-5,6-dihydrotetrazolo[1,5-c] quinazolines. Grail of Science. 2022;13(12–13):684–692. doi: 10.36074/grail-of-science.29.04.2022.124. [DOI] [Google Scholar]
  74. Lipinski C. A. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discovery Today: Technol. 2004;1(4):337–341. doi: 10.1016/j.ddtec.2004.11.007. [DOI] [PubMed] [Google Scholar]
  75. Ghose A. K. Viswanadhan V. N. Wendoloski J. J. Prediction of hydrophobic (lipophilic) properties of small organic molecules using fragmental methods: An analysis of ALOGP and CLOGP methods. J. Phys. Chem. A. 1998;102(21):3762–3772. doi: 10.1021/jp980230o. [DOI] [Google Scholar]
  76. Veber D. F. Johnson S. R. Cheng H. Y. Smith B. R. Ward K. W. Kopple K. D. Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 2002;45:2615–2623. doi: 10.1021/jm020017n. [DOI] [PubMed] [Google Scholar]
  77. Egan W. J. Merz K. M. Baldwin J. J. Prediction of drug absorption using multivariate statistics. J. Med. Chem. 2000;43(21):3867–3877. doi: 10.1021/jm000292e. [DOI] [PubMed] [Google Scholar]
  78. Muegge I. Heald S. L. Brittelli D. Simple selection criteria for drug-like chemical matter. J. Med. Chem. 2001;44:1841–1846. doi: 10.1021/jm015507e. [DOI] [PubMed] [Google Scholar]
  79. Abdellatif K. R. A. Abdelall E. K. A. Elshemy H. A. H. Lamie P. F. Elnahaas E. Amin D. M. E. Design, synthesis of new anti-inflammatory agents with a pyrazole core: COX-1/COX-2 inhibition assays, anti-inflammatory, ulcerogenic, histopathological, molecular modeling, and ADME studies. Tetrahedron. 2021;1240:130554. [Google Scholar]
  80. Abd El-Karim S. S. Mohamed H. S. Abdelhameed M. F. Amr A. E. Almehizia A. A. Nossier E. S. Design, synthesis and molecular docking of new pyrazole-thiazolidinones as potent anti-inflammatory and analgesic agents with TNF-α inhibitory activity. Bioorg. Chem. 2021;111:104827. doi: 10.1016/j.bioorg.2021.104827. [DOI] [PubMed] [Google Scholar]
  81. Tale R. H. Rodge A. H. Hatnapure G. D. Keche A. P. The novel 3,4-dihydropyrimidin-2(1H)-one urea derivatives of N-aryl urea: Synthesis, anti-inflammatory, antibacterial and antifungal activity evaluation. Bioorg. Med. Chem. Lett. 2011;21:4648–4651. doi: 10.1016/j.bmcl.2011.03.062. [DOI] [PubMed] [Google Scholar]

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