Docking Study, Synthesis, and Anti-Inflammatory Potential of Some New Pyridopyrimidine-Derived Compounds

Background and Purpose Because of gastrointestinal irritation and kidney toxicity associated with non-steroidal anti-inflammatory drugs and the cardiovascular problems of Coxibs use, developing novel anti-inflammatory agents with reduced toxicity and improved selectivity remains a major challenge. Depending on our previous work, a novel series of pyridopyrimidinones IIIa-i has been synthesized via reaction of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (I) and phenyldiazenyl aromatic aldehydes (IIa-i). All the new constructed compounds were fully characterized by elemental and spectral analysis. Methods The target compounds IIIa–i were investigated for their potential towards COX inhibition, anti-inflammatory properties using carrageenan induced edema model in rat paw, and the ulcer indices of the most active members. Results The ethyl pyridopyrmidinone-benzoates IIIf, IIIg and IIIh showed superior inhibitory activity of carrageenan induced edema to celecoxib. Furthermore, the pyridopyrimidinones IIId, IIIf, IIIg, and IIIi exerted improved COX-2 inhibitory activity (IC50 = 0.67–1.02 µM) comparing to celecoxib (IC50 = 1.11 µM). Moreover, the gastric ulcerogenic potential assay of compounds IIIf–h revealed their lower ulcerogenic liability than indomethacin with comparable effect to celecoxib. Conclusion Virtual docking investigation of the most active candidates IIId, IIIf, IIIg and IIIi in the active site of COX-2 enzyme showed that these compounds implied interaction and binding motif similar to the cocrystallized ligand bromocelecoxib.


Introduction
Inflammation is a cellular reaction to any harmful stimuli and conditions like tissue damage and infection. [1][2][3] This physiological response includes the delivery of blood components to the local infection site or injury triggering vasodilation and increased vascular permeability. 4-7 NSAIDs (Non-steroidal anti-inflammatory drugs) are clinically indicated for relieving fever, pain, and inflammation by suppressing cyclooxygenase (COX) enzymes. 8,9 Cyclooxygenases (COXs) are key enzymes responsible for transforming arachidonic acid, which is released on affected tissues by the effect of phospholipase A2 to various prostaglandins. 10,11 The major two isoforms of cyclooxygenase are COX-1 and 2. 12 The constitutive COX-1 mediates the formation of diverse cytoprotective prostaglandins, which are responsible for lining the gastric mucosa, inducing platelet aggregation and preserving homeostasis. 13,14 However, the inducible COX-2 is accountable for synthesizing of pain and inflammatory mediating prostaglandins. 15,16 Conventional NSAIDs non-selectively inhibit COX-1 and 2 isozymes, therefore their administration is associated with gastrointestinal side effects. [17][18][19] Therefore, selective COX-2 inhibitors were developed to achieve enhanced safety profile on gastric mucosa. Nonetheless, certain members such as rofecoxib and valdecoxib have been associated with increased probability of myocardial infarction incidences as well as hypertensive actions. 20 In this regard, discovery of selective COX-2 blockers for management of pain and inflammation with diminished side actions emerged as an urgent medical need.

Results and Discussion Chemistry
The key building block, pyrimidine-4-one (I) and phenyldiazenyl aromatic aldehydes IIa-i, were chemically synthesized as shown in Scheme 1. Condensation of the ethyl cyanoacetate ester with thiourea in presence of sodium ethoxide as a strong base afforded compound I in quantitative yield. 45 On the other hand, phenyldiazenyl aldehydes IIa-i were achieved through diazotization of various anilines followed by treatment with aromatic aldehydes under basic conditions. [46][47][48] The synthesis of the target pyridopyrimidinones IIIa-i was accomplished in 66-93% yield by treatment of compound I with various phenyldiazenyl aromatic aldehydes in CH 3 OH containing a catalytic amount of HCl adopting the reported method. 34 In this reaction, the acidic polar solvent favors the reaction progress by the creation of a 6-imino that directs to higher nucleophilic character of carbon five, causing reaction to occur on the aromatic aldehydic carbonyl (Scheme 2). The chemical structures of newly prepared compounds IIIa-i have been elucidated via different spectroscopic and elemental analysis. 1 HNMR data for the novel compounds IIIa-i is shown in Table 1.

Assay of COX Inhibition
The newly prepared azo molecules IIIa-i were investigated for their COX blocking action -in terms of IC 50 -by enzyme immunoassay (EIA) utilizing ovine COX-1/2 assay kit. Furthermore, SI (selectivity index) was assessed as IC 50 against COX-1/IC 50 against COX-2 applying positive standard celecoxib. As shown in Table 2, the obtained data pointed out that the pyridopyrimidinones IIIa-i exhibited modest to moderate blocking activity to COX-1 (IC 50 = 3.25-11.23 µM), and favorable suppressing activity for COX-2 (IC 50 = 0.67-4.78 µM). While the pyridopyrimidinone IIId,

In vivo Anti-Inflammatory Action
Animal and ethics: we used in this study adult male Wister albino rats weighing 150-180g. Before any experimental study, rats are given 14 days to acclimate. The rats were kept in a controlled environment with access to water and food. All assays and practical animal studies had been done in Nahda university and adapting rules for care of animals in lab in

Scheme 2
The reasonable mechanism for compound III formation. The pyridopyrimidinones were orally administrated in a dose of 50 mg/kg nearly earlier persuading inflammation through SC (subcutaneous injection) of carrageenan. Inhibition of carrageenan induced inflammation was estimated by measuring the changes of paw dimensions after 1, 3 and 5 h ( Table 3). The obtained findings showed that the pyridopyrimidinones IIIf and IIIh had superior anti-inflammatory properties than celecoxib at the three examined time intervals. In addition, the target compound IIIg exhibited higher anti-inflammatory potential than displayed by celecoxib after 3 and 5 hours. Close inspection of the results listed in Table 3 underscored that the pyridopyrimidinone derivatives substituted with ethyl ester IIIf (Anti-inflammatory (AI) % = 52-66) and IIIh (AI % = 57-67) displayed higher activity for inhibition of edema than those congeners containing either electron donating (IIId; AI % = 28-55) or electron withdrawing (IIIc; AI % = 24-54) groups, particularly after 1 and 3 hours. In harmony with the COX-2 inhibitory assessment, the 4-benzothiazol-2-yl containing pyridopyrimidinone IIIi exerted superior in vivo anti-inflammatory activity than its corresponding methoxy derivative IIIb. Moreover, introducing bromine on compound IIIg augmented the anti-inflammatory activity (3 h; AI % = 54, 5 h; AI % = 57).
The ethyl acetoxy group in compounds IIIf and IIIh has important role in vivo activity anti-inflammatory activity also bromine at for position 4 in compound IIIg. Also, the ethyl acetoxy group in IIIf and IIIh increases absorption so fast onset of action consequently high anti-inflammatory activities 52% and 67% respectively but bromine in IIIg delay onset of action firstly in compared with IIIf and IIIh.

Ulcerogenic Liability
The top three active pyridopyrimidinones IIIf-h were further evaluated for their gastric ulcerogenic liability in rats ( Table 4). The ulcerogenicity liability of the investigated pyridopyrimidinones was compared with both COX-1 inhibitor (indomethacin) and COX-2 inhibitor (celecoxib). Interestingly, compound IIIf had the lowest ulcerogenic effect, which might be attributed to its potential selectivity for COX-2 (SI = 11.82). Additionally, all of the tested candidates showed lower ulcerogenic action than the standard indomethacin.

Molecular Docking Studies
To acquire insights about the underlying mechanism of action of the newly investigated pyridopyrimidinones, virtual docking of the highly selective COX-2 inhibitors IIId, IIIf, IIIg and IIIi within the active binding site of COX-2 enzyme was studied. The co-crystal structure of COX-2 complex with SC-558, a selective COX-2 blocker, was acquired from PDB (protein data bank: 1CX2), 49 Table 5). Interestingly, compound IIIf showed profitable fitting with COX-2 with superior docking score (S = −13.89 kcal/mol) to bromocelecoxib (S = −11.93 kcal/mol). Furthermore, IIIf displayed two HB interactions through its carbonyl oxygen with Arg120 (2.65 A°) as well as azo moiety with His90 (3.25 A°) (Figure 3; Table 5). Also, the pyridopyrimidinone IIIg exhibited score energy (S) of −11.20 kcal/mol, and was able to form three HB interactions with Arg120, His90 and Tyr355 (Figure 3).

Conclusion
Novel derivatives of pyridopyrimidinones IIIa-i were prepared and assessed in vitro and in vivo for their COXs and carrageenan induced edema anti-inflammatory activities, respectively. Preliminary screening of the target compounds disclosed that the pyridopyrimidinone IIIf possessing ethyl acetate had the best activity with potent edema inhibition in percent = 52% after one hour, 59% after three hours and 66% after five hours. Moreover, certain members among this focused library were identified to be selective COX-2 inhibitors. Particularly, IIId, IIIf, IIIg and IIIi, exerted superior inhibition for COX-2 (IC 50 = 0.67-1.02 µM) than celecoxib (IC 50 = 1.11 µM). Ulcerogenic accountability of compounds IIIf-h exhibited their comparable activity to celecoxib along with less ulcerogenic effect than indomethacin. It was noted that the lipophilic group (ethyl ester or benzothiazol-2-yl) containing compounds IIIf, IIIh and IIIi elicited superior antiinflammatory effect and better selectivity for COX-2 than other derivatives. Overall, combining the privileged pyridodipyrimidinone scaffold with diphenylazo structural feature in single molecule with appropriate hydrophobic substitution pattern may represent a promising core structure for further design of potent anti-inflammatory agents with minimized gastric side effects. , a mixture of chloroform /methanol (9.5:0.5 mL) as eluent and visualized with UV lamp. All chemicals and reagents were commercially purchased and used directly without purification. 6-Aminopyrimidinone derivative I and the aldehyde derivatives IIa-i were prepared adopting the reported methods. 45

Pharmacological Activity Studies
All utilized procedures in the pharmacological evaluation were carried out as described earlier. Colorimetric assay of COXs, 20 anti-inflammatory activity (in-vivo), 40 ulcerogenic liability 52 were cited in the Supplementary Materials.

Molecular Docking
The virtual docking study was performed by utilizing the x-ray crystal structure of COX-2 enzyme (pdb code: 1CX2). 28 Ligand and protein preparation (3D protonation for the amino acid side chain of enzyme, addition of hydrogen atoms, and deletion of all water of crystallization away from the active site) was performed using MOE software (version 2010, Chemical Computing Group Inc., QC, Canada). The pyridopyrimidinones compounds were sketched in their threedimensional (3D) structures by Chemo-Draw, protonated, and subjected to energy minimization. Molecular docking of these compounds has been applied, amino acid interactions were examined, and the hydrogen bond lengths were recorded.

Statistical Analysis
The significant difference for groups was measured utilizing one-way ANOVA followed by Dunnett's test. Significant differences are at *P >0.05, **P > 0.01 and ***P >0.001, and GraphPad Prism software (version 5) was used for statistical tests (version 5).