Discovery of Novel Pyridazine-Based Cyclooxygenase-2 Inhibitors with a Promising Gastric Safety Profile

Cyclooxygenase-2 (COX-2) is implicated in the development of chronic inflammatory diseases. Recently, pyridazine derivatives have emerged as a novel prototype to develop COX-2 inhibitors. Accordingly, some pyridazine-based COX-2 inhibitors are reported herein. The reaction of aldehyde 3 and different hydrazines yielded the corresponding hydrazones. The hydrazones were further derivatized to the title compounds, which were assessed for COX-1 and COX-2 inhibitory action, gastric ulcerogenic effects, and lipid peroxidation properties. Molecular docking studies and determination of the physicochemical parameters were also carried out. The allocated structures of the reported compounds were coherent with their spectroscopic data. The compounds 9a (IC50 = 15.50 nM, 114.77%), 9b (IC50 = 17.50 nM, 101.65%), 12 (IC50 = 17.10 nM, 104.03%), 16b (IC50 = 16.90 nM, 105.26%), and 17 (IC50 = 17.70 nM, 100.5%) displayed better COX-2 inhibition than celecoxib (IC50 = 17.79 nM, 100%). These outcomes were harmonious with the molecular docking studies of 9a, 9b, 12, 16b, and 17. These compounds also displayed comparable onset and the duration of action concerning celecoxib and indomethacin in the in vivo studies. No ulcerogenic effects were observed for 9a and 12, whereas 9b, 16b, and 17 showed an insignificant ulcerogenic effect compared to celecoxib. The compounds 9a, 9b, 12, 16b, and 17 displayed a better lipid peroxidation profile than celecoxib and indomethacin. The compounds 9a (%ABS = 84.09), 9b (%ABS = 84.09), 12 (%ABS = 66.87), 16b (%ABS = 75.02), and 17 (%ABS = 81.42) also displayed appreciable calculated absorption compared to celecoxib (%ABS = 82.09). The compounds 9a, 9b, 11, 16b, and 17 have been recognized and postulated as non-ulcerogenic COX-2 inhibitors with promising physicochemical parameters and gastric safety profile. These compounds may be useful candidates to combat diseases caused by higher levels of COX-2.


Introduction
Inflammation is a self-protective reaction of human body tissues towards injurious stimuli like infection, irritants, poisonous substances, irradiation, and tissue injury [1]. The inflammation can be divided into acute inflammation and chronic inflammation. The symptoms of acute inflammation comprise of swelling, heat, immobility, redness, and pain, which may last for several days [2]. However, persistent inflammation for a longer time or chronic inflammation may lead to the development of diseases like gout, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, Alzheimer's disease, ulcerative colitis, depression, epilepsy, irritable bowel diseases, kidney injury, cancer, asthma, hepatitis, pancreatitis, and atherosclerosis [1,3]. According to the published data, 40% and 61% of the population of the age > 60 years in the UK and Saudi Arabia, respectively, are suffering from arthritis [4]. It is estimated that about 1/4th of the adults in the USA will be affected by osteoarthritis by 2030 [5]. Non-steroidal anti-inflammatory drugs (NSAIDs) are regularly prescribed for chronic inflammatory diseases. NSAIDs inhibit the cyclooxygenase enzyme (COX). COX is accountable for transforming arachidonic acid into prostaglandins, which initiate the inflammatory events in a cell [1,2]. The constitutive COX-1 is responsible for the maintenance functions of the cell, including the safety of the gastric mucosa, aggregation of platelets, and control of the renal blood flow [6,7]. COX-2 cannot be detected in healthy cells. However, this inducible enzyme is produced intracellularly after harmful stimuli. It is responsible for the development of inflammatory events in a cell, which ultimately lead to the development of inflammatory diseases [1,3]. The commonly used NSAIDs cause ulcerogenic effects after prolonged use because they inhibit both COX-2 and COX-1 [6,7]. The ulcerogenic effect of NSAIDs is credited to the inhibition of COX-1, and the anti-inflammatory action is credited to the inhibition of COX-2 [8,9]. Based on this understanding, celecoxib, rofecoxib, and etoricoxib were developed as specific COX-2 inhibitors [10][11][12]. However, cerebrovascular risk and cardiac toxicity have been reported as adverse effects of some particular COX-2 inhibitors at the standard dose, for example, rofecoxib [12,13]. Accordingly, medicinal chemists are looking forward to developing new anti-inflammatory agents, which lack the aforementioned adverse effects and have a promising gastric safety profile [14].
The cycloalkylation of the thiocarbamoyl group of 4b with the ethyl α-chloroacetate (5a) and ethyl α-chloropropionate (5b) in glacial acetic acid comprising a catalytic amount of the fused sodium acetate at the reflux temperature afforded the corresponding 4-thiazolidinone derivatives, 6a and 6b, respectively (Scheme 2). The formation of 6a and 6b is expected to proceed through the initial S-alkylation via the loss of sodium chloride followed by the intramolecular cyclization with the elimination of ethanol. The Hantzsch reaction of 4b with the ethyl α-chloro acetoacetate in acetic acid in the presence of sodium acetate led to the formation of 4-methyl-thiazole derivative 8 (Scheme 2). The possibility of compound 7 was excluded based on the spectral analyses. The treatment of 4b with 4-substituted phenacyl bromides in refluxing ethanol in the presence of anhydrous sodium acetate produced the corresponding thiazole derivatives, 9a and 9b (Scheme 2). The cyclization reaction of 4b with dimethyl acetylenedicarboxylate in methanol provided the 4-thiazolidinone derivative 10 (Scheme 2). The condensation of the active methylene group of 6a with electrophilic dimethylformamidedimethylacetal (DMF-DMA) in dry dioxane afforded 5-dimethylaminomethylidine derivative 11 (Scheme 3). Similarly, the treatment of 6a with isatin in dioxane comprising a catalytic amount of piperidine provided compound 12 (Scheme 3). The treatment of 6a with α-cinnamonitriles in dioxane containing a catalytic amount of piperidine furnished the benzylidene derivatives 16a,b, wherein other possible structures 15a,b were ruled out based on the spectral data. Another synthetic route of compounds 16a,b was achieved via the Knoevenagel condensation of compounds 6a with the corresponding aromatic aldehydes (Scheme 3).  There is a possibility of at least four or more geometrical isomers for some of the compounds, for example, 10, 11, 12, 16a, 16b, 17, and 18. However, these isomers may be able to interconvert. Therefore, we have not mentioned the E or Z configuration in the nomenclature of our compounds. The structures of 4a, 6a, 6b, 8, 9a, 9b, 10, 11, 12, 16a, 16b, 17, and 18 were proven on the basis of their spectroscopical data. The detailed spectroscopical data are provided in the experimental part.
It is a well known fact that the inhibition of COX-1 is mainly responsible for the ulcerogenic effect of NSAIDs like indomethacin [6,7]. It is also well documented that specific COX-2 inhibitors like celecoxib are potent anti-inflammatory agents and possess a better gastric safety profile because they do not inhibit COX-1 [10]. Therefore, for a better comparison, the %COX-1 inhibition of indomethacin was normalized to 100% for COX-1, and the %COX-2 inhibition of celecoxib was normalized to 100% for COX-2 ( Table 1). The selectivity index of celecoxib was also normalized to 100%. All the compounds comprising celecoxib (IC 50 = 320 nM) displayed greater IC 50 against COX-1, when compared to indomethacin (IC 50 = 220 nM) (Table 1, Figure 1). This result points out that our compounds should have a better gastric safety profile than indomethacin [8,9]. Our belief is further strengthened by the fact that our compounds showed better inhibition of COX-2 in contrast to COX-1.  The novel pyridazine derivatives can be categorized as thiazole derivatives (8, 9a, and 9b) and 4-thiazolidinone derivatives (6a, 6b, 10, 11, 12, 16a, 16b, 17, and 18). It is apparent from Table 1 that the thiazole derivative 9a (4-phenyl thiazole group) was more potent than thiazole derivative 9b (4-bromophenyl thiazole group). This reflects that the incorporation of bromine in the structure of 9a decreases its COX-2 inhibitory potential. This result is in concurrence with the earlier report [22]. However, the COX-2 inhibitory potential of the corresponding chrolo, fluoro, iodo, and nitro derivatives of 9b should also be assessed for a better understanding of this observation. The thiazole derivative 8 (4-methyl-thiazole-5-carboxylate group) displayed a further decrease in the COX-2 inhibitory potential. This also indicates that the incorporation of methyl and carboxylate groups in the structure of 9a decreases its COX-2 inhibitory activity. In case of 4-thiazolidinone derivatives, the COX-2 inhibitory activity increases as 11 < 6a < 18 < 6b < 16a < 10 < celecoxib < 17 < 12 < 16b. The presence of the 5-dimethylaminomethylidene group (11), unsubstituted 4-thiazolidinone ring (6a), 5-methoxycarbonylmethylidene-3-phenyl group (18), methyl group (6b), 4-chlorophenylmethylidene group (16a), and 5-methoxycarbonylmethylidene group (10) at position 5 of the 4-thiazolidinone ring provides compounds with lesser or average COX-2 inhibitory action. However, when the chlorine of the 16a is replaced with a methoxy group, a potent COX-2 inhibitor 16b is obtained. This observation indicates that the presence of the electron donor group in these types of compounds increases the COX-2 inhibitory action. This reflection is also in concurrence with the earlier reports that the presence of the electron donor group may increase the COX-2 inhibitory action of thiazolidinone ring-bearing compounds [22,24]. The 2,3-disubstituted-4-thiazolidinone derivative (17) has been recognized as a potent inhibitor of COX-2. Some earlier reports also support this fact [26,28]. We also believe that this kind of other 2,3-disubstituted derivatives may provide potent COX-2 inhibitors. The isatin-bearing compound 12 also provided a potent inhibitor of COX-2. The incorporation of the isatin moiety is reported to potentiate the COX-2 inhibitory activity of a compound [33]. Recently, we reported the isomeric 4-thiazolidinone-bearing pyridazine derivatives, which had a methylidine linker joining the phenyl ring and the 4-thiazolidinone ring [30]. The presently reported 4-thiazolidinone-bearing pyridazine derivatives contain a methylidene hydrazinyl linker between the phenyl ring and the 4-thiazolidinone ring. A comparison of the COX-2 inhibitory activity among these isomeric compounds reveals that the incorporation of a methylidene hydrazinyl linker provides potent COX-2 inhibitors. This observation is in concurrence with our earlier report that states that compounds bearing a hydrazine moiety display higher COX-2 inhibition [30].

Ulcerogenic Activity
The compromising gastric safety profile of the existing NSAIDs is a concern [6,7]. The ulcerogenic effects are more pronounced in non-specific COX inhibitors like indomethacin, whereas specific COX-2 inhibitors like celecoxib lack this side effect [6,10]. Accordingly, 9a, 9b, 12, 16b, and 17 were evaluated for their gastric safety profile. It was performed by the indomethacin-induced gastric erosion method [30]. A total of eight groups of rats were utilized, wherein each group comprised of six rats. The compounds were administered orally (10 mg/kg) (Table 3, Figure 3).  The compounds 9a and 12 did not produce any ulcerogenic impact. The compounds 9b, 16b, and 17 also exhibited insignificant ulcerogenic effects. The negligible ulcerogenic effect produced by 9a, 9b, 12, 16b, and 17 is attributed to their higher COX-2 inhibitory potential plus their lesser potential to inhibit COX-1 (Table 1) [8,9]. It is well established that the presence of the -COOH group in NSAIDs aids in their ulcerogenic effect [6]. Another prospect of the insignificant ulcerogenic effect produced by 9a, 9b, 12, 16b, and 17 may be the absence of the -COOH moiety in their structure.

Lipid Peroxidation Studies
The lipid peroxidation inhibitory activity of a compound makes it less ulcerogenic [35,36]. The lipid peroxidation in tissue increases the malondialdehyde (MDA) content in the tissue [35]. A decrease in the MDA content is a measure to assess the non-ulcerogenic effect of a compound. Accordingly, the compounds 9a, 9b, 12, 16b, and 17 were evaluated for their lipid peroxidation effects [35,36] (Table 3, Figure 3) . The compounds 9a, 9b, 12, 16b, and 17 displayed a better lipid peroxidation profile than celecoxib and indomethacin, which provides concurrence to the ulcerogenic activity data (Table 3).

Physicochemical Parameters
The molecular properties of 9a, 9b, 12, 16b, and 17 were determined by the available online software, Molinspiration [40]. The calculated absorption (%ABS), number of H-bond acceptors (nON), number of H-bond donors (nOHNH), number of rotatable bonds (nrotb), and the topological polar surface area (tPSA) are provided in Table 5. The compounds 9a, 12, 16b, and 17 passed Lipinski's rule [40,41]. This rule defines the molecular properties of a drug, which governs its pharmacokinetic performance, for example, the absorption of a drug. It also helps to evaluate a compound's ability to be orally active based on its molecular properties [40,41], which are mentioned in Table 5. To pass Lipinski's rule, a compound should not have more than one violation of the calculated properties. As per a recent report, Lipinski's rule of five does not have any significant deficiency in defining the druggability of a compound and is still useful today [42]. It can be observed that compound 9a displayed a better calculated absorption (%ABS = 84.09) than celecoxib (%ABS = 82.09) (Figure 4). The compound 9b (%ABS = 84.09) also displayed a better calculated absorption than celecoxib. The compound 9b did not pass Lipinski's rule. However, there are many clinically used natural compounds and drugs that violate Lipinski's rule [42]. The compound 12 (%ABS = 66.87), compound 16b (%ABS = 75.02), and compound 17 (%ABS = 81.42) also exhibited appreciable calculated absorption. These observations revealed that the compounds 9a, 9b, 12, 16b, and 17 possess not only better COX-2 inhibitory action than indomethacin and celecoxib but also possess a promising pharmacokinetic/physicochemical profile.

In Vitro COX-1 and COX-2 Inhibition Assay
The compounds 4a, 6a, 6b, 8, 9a, 9b, 10, 11, 12, 16a, 16b, 17, and 18 were exposed to their COX-1/COX-2 inhibitory action by a 10-fold dilution strategy (1-10 −4 µg/mL) utilizing dimethylsulfoxide (DMSO) [30]. The test packs of the human COX-1/COX-2 were obtained from Cayman Chemicals (560131, Ann Arbor, MI, USA). The supplier's instructions were followed to prepare the reagents as well as to perform the experiment. Briefly, samples (20 µL), the COX-1 and COX-2 enzyme (10 µL), and heme (10 µL) were mixed with the buffer solution (160 µL), which was supplied with the kits. The resultant combination was incubated at 37 • C in a water bath for 10 min, and arachidonic acid (10 µL) was added to initiate the COX reaction. A saturated solution of stannous chloride (30 µL) was added after 2 min to halt the COX reaction. The resultant mixture was incubated at ambient temperature for 5 min. The Prostaglandin-2α (PGF2α) developed after the COX reaction was measured through ELISA. The samples were shifted to a 96-well plate and incubated for 18 h at 25 • C. The plate was washed to get rid of the unbound components. The Ellman's reagent (200 µL), containing the acetylcholine substrate, was mixed and further incubated at 25 • C for 1-1.5 h till the absorbance (410 nm) of the B o well was in the range of 0.3 to 0.8 A.U. The plate was read through the ELISA reader. The IC 50 values of COX-1 and COX-2 were determined by the regression analysis.

In Vivo Anti-Inflammatory Activity
The compounds 9a, 9b, 12, 16b, and 17 were evaluated for anti-inflammatory action utilizing Wistar rats (130-150 g) by following the rat paw edema method [30,34]. The animal approval (IAEC/KSOP/E/18/12) was obtained from the CPCSEA. A total of eight groups of rats were utilized, wherein each group comprised of 6 rats. The compounds (test group), celecoxib (standard group), and indomethacin (standard group) were administered orally (10 mg/kg) as a 10% Tween-80 solution. Saline solution (1 mL) was given to the control group. The carrageenan solution (1%, 0.1 mL) was administered by injection after 1 h in the right hind paw of the rats in the test, standard, and the control groups. The volume of the paws was calculated after the carrageenan injection at 0, 1, 2, 3, and 4 h by a plethysmometer. The % edema was calculated as follows: paw diameter a f ter carrageenan − paw diameter be f ore carrageenan paw diameter be f ore carrageenan × 100.

Gastric Ulcerogenic Activity
The eight groups of rats were fasted for 18 h, wherein each group consisted of 6 rats. The compounds (9a, 9b, 12, 16b, and 17), celecoxib, and indomethacin were given orally (10 mg/kg) as 10% Tween-80 solution. Saline solution (1 mL) was given to the control group. The animals were sacrificed after 4 h to isolate their stomachs. A longitudinal cut was made along the greater curvature, and the presence of ulcers was evaluated. The counting of ulcers was marked as 0 (no ulcer) to 5 (≥3 ulcers) [30].

Lipid Peroxidation Inhibitory Activity
It was carried out on compounds 9a, 9b, 12, 16b, 17, celecoxib, and indomethacin after the ulcerogenic activity [35,36]. The gastric mucosa (100 mg) was scraped with two glass slides and homogenized in ice-cold potassium chloride (1.8 mL of 1.15% KCl). Sodium dodecyl sulphate (0.2 mL of 8.1% SDS), acetate buffer (pH = 3.5, 1.5 mL), and thiobarbituric acid (1.5 mL of 0.8% TBA) was mixed with the homogenate, and the mixture was heated to 95 • C for 1 h. The obtained mixture was extracted with a 5-mL mixture (15:1 v/v) of n-butanol and pyridine and centrifuged at 4000 rpm for 10 min. The supernatant liquid was separated, and its absorbance was measured at 532 nm using a spectrophotometer. A standard linear curve (absorbance vs. concentration in nM) was prepared using MDA tetrabutylammonium salt. The lipid peroxidation was calculated from the standard curve. The results are expressed as nmol MDA 100 mg −1 tissues.

Molecular Docking
The molecular docking of 9a, 9b, 12, 16b, 17, and celecoxib was performed by the method reported in our earlier report [30]. This study was conducted on the HP ® computer system (Intel ® core i5-4570 CPU, 3.20 GHz, Window 10). ChemDraw was used to design the compounds, wherein ChemBio3D Ultra was used to generate 3-D conformations. The 3-D structure of COX-2 protein (PDB entry 5KIR) was retrieved from PDB [37][38][39]. The Auto Dock and the Auto Dock Vina were utilized to generate the data. The detailed procedure was demonstrated in our earlier report [30].

Statistical Analysis
It was performed by the SPSS software. The p values, N values, mean values, and standard deviation values are mentioned at the desirable places of the manuscript.

Conclusions
Five compounds, 9a, 9b, 12, 16b, and 17, demonstrated superior COX-2 inhibition than celecoxib. These compounds had a similar onset/duration of action to celecoxib. The compounds 9a and 12 were devoid of any ulcerogenic effect, whereas 9b, 16b, and 17 showed insignificant ulcerogenic effects. The compounds 9a, 9b, 12, 16b, and 17 also displayed a better lipid peroxidation profile than indomethacin and celecoxib. They also demonstrated a considerable calculated absorption. The compounds 9a, 9b, 11, 16b, and 17 are thus recognized and postulated as non-ulcerogenic COX-2 inhibitors with promising physicochemical parameters and gastric safety profile. These compounds may be useful candidates to combat diseases caused by higher levels of COX-2 like gout, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, Alzheimer's disease, ulcerative colitis, depression, epilepsy, irritable bowel diseases, kidney injury, cancer, asthma, hepatitis, pancreatitis, and atherosclerosis. Funding: This study received no external funding.