Synthesis of Pyrroles and Condensed Pyrroles as Anti-Inflammatory Agents with Multiple Activities and Their Molecular Docking Study

We herein disclose a series of novel pyrrole derivatives as well as fused pyrrolopyridines 6a,b and 7a,b, pyrrolopyrazoles 8a, b, pyrrolo[2,3-d]pyrimidine derivatives 10a-d, 12a,b, 14a,b, 18a,b, 20a,b, 21a,b, 22a,b, 23a,b, 24a,b, 31a,b, 36a,b, 40a,b, pyrrolo[1,2,6]thiadiazine derivatives 19a,b, pyrrolotriazolopyrimidines 25a,b, 26a,b, 27a,b and 28a,b, pyrrolo[2,3-d][1,2,3]triazine derivatives 32a,b and pyrrolo[2,3-d][1,3]oxazine derivatives 39a,b as novel compounds. All compounds were evaluated for their anti-inflammatory, analgesic (compared to the reference drug Indomethacin) and antimicrobial activities (compared to the reference drug Ampicillin and Fluconazole). Compounds 4d, 5b-d, 6a,b, 9c,d, 10d, 12ab, 13b, 19a,b, 21b, 23b, 31a,b, 38b and 40a were found to be the most active anti-inflammatory drugs exhibiting potency ranging from 1 1.01 compared to the reference drug indomethacin. In addition to docking study of these highly active twenty compounds against the active site of cyclooxygenase-2 enzyme (COX-2), among the tested compounds, compounds 5d, 9d, 11b, 12a, 13b and 32a showed multiple activities; anti-inflammatory, analgesic and anti-bacterial activities.


Introduction
Nitrogen heterocycles are of special interest as they constitute an important class of natural and non-natural products as they occupy a key position in the area of drugs and pharmaceuticals [1] [2].Pyrroles have drawn considerable attention due to their synthetic importance and useful biological activities that are extensively used in drug discovery [3].Pyrrole derivatives exhibited a vital role in many pharmacological activities including anti-inflammatory [4]- [13], anti-microbial [14]- [19], anti-fungal [20]- [22], antiviral [23]- [25] and anti-cancer [26]- [28] activities.
It is well known that the anti-inflammatory activity is due to the ability to inhibit the cyclooxygenase (COX) activity of prostaglandin H synthase, an enzyme which mediates the production of prostanoids (including prostaglandins, prostacyclins and thromboxanes) from arachidonic acid.Prostaglandins act as mediators in the process of inflammation, thus the discovery of COX-2 specific inhibitors (Coxibs), which their pharmacological properties are correlated to their ability to decrease the COX-2 dependent prostanoid biosynthesis, providing a rational for the development of drug devoid of GIT disorders while retaining clinical efficacy as anti-inflammatory agent [29].The recent market withdrawal of some coxibs such as rofecoxibs (Vioxx ® ) and valdecoxib (Bextra ® ) due to their adverse cardiovascular side effects [30] clearly delineates the need to explore and evaluate alternative templates with COX-2 inhibitory activity.
Therefore, our aim was to design derivatives of existing clinically used NSAIDs, such as Tolmetin and Ketorolac [31] [32] which are well known pyrrole derivatives acting as anti-inflammatory drugs.In the light of these facts, this paper deals with the synthesis of novel pyrrole and condensed pyrrole derivatives and evaluates them for their anti-inflammatory activity.Furthermore, the extent of the pharmacological effects of pyrrole derivatives is reported to depend on the active groups which are attached to it, as several scientists have elucidated that in pyrrole system positions 2 and 3, it can be suitably modified by the introduction of groups [28], aromatic [19] [25] or heterocyclic moieties to show excellent pharmacological results [12].
Furthermore, compounds 4a,b were reacted with urea/thiourea in sodium ethoxide to give the target compounds 9a-d (Scheme 3), which were cyclized upon refluxing in pyridine to afford the pyrrolopyrimidine derivatives 10a-d which can also be obtained through refluxing of the o-aminonitrile derivatives 4a,b with urea/ thiourea in a mixture of glacial acetic acid and concentrated hydrochloric acid (3:1).Furthermore, the target phenylthioureaderivatives 11a,b was obtained via refluxing of compounds 4a,b with phenyl isothiocyanate in absolute ethanol.Also, refluxing of compounds 11a,b in pyridine afforded the cyclized compounds 4-iminopyrrolopyrimidine-2-thiones 12a,b which were also prepared directly via refluxing of compounds 4a,b with phenyl isothiocyanate in dry pyridine.
Moreover, chlorination of pyrrolopyrimidin-4-one derivatives 18a,b with excess phosphorus oxychloride followed by alkalinization to pH 10 using sodium bicarbonate furnished 4-chloropyrrolo [2,3-d]pyrimidines 21a,b (Scheme 6).However, the chlorinated compounds 21a,b were reacted with morpholine in ethanol under reflux in presence of a catalytic amount of triethylamine to furnish the 4-morpholino-pyrrolo [2,3-d]pyrimidines 22a,b.Furthermore, hydrazinolysis of chloro derivatives 21a,b was carried out by refluxing with hydrazine hydrate 98% in ethanol in presence of triethylamine as a catalyst to afford 4-hydrazinyl-7H-pyrrolo [2,3-d] Furthermore, stirring of compounds 13a,b with hydrazine hydrate 98% in absolute ethanol afforded 4-imino-4,7-dihydro-3H-pyrrolo [2,3-d]pyrimidines 24a,b (Scheme 7) which were used as good starting materials for preparation of several pyrrolo [3,2-e] [1,2,4]triazolo [1,5-c] This work was extended to shed more light on the activity and synthetic potential of the amino and carboxylate groups in compounds 4c,d (Scheme 9).Thus, compounds 4c,d reacted with hydrazine hydrate in absolute ethanol to afford the corresponding acid hydrazide derivatives 33a,b.However, acetylation of amino group in compounds 4c,d with acetic anhydride afforded the target acetamido derivatives 34a,b.However, the synthesis of different hydrazone derivatives through the condensation of hydrazine compounds with monosaccharide is well documented in the literature [33]- [35].Therefore, this information prompted us to explore the effect of glucose hydrazone derivatives 35a,b whichwere synthesized through the condensation of the hydrazide derivatives 33a,b with glucose in absolute ethanol.Additionally, the reaction of o-acetamidocarboxylate derivatives 34a,b were refluxed with excess hydrazine hydrate in ethanol to afford the target substituted pyrrolopyrimidinone derivatives 36a,b.The reaction mechanism is suggested to proceed first through formation of the hydrazone derivatives followed by tautomerism then intramolecular cyclization via elimination of an ethanol molecule.

Biology
All the newly synthesized compounds 4a-40b were preliminarily evaluated for their anti-inflammatory and analgesic activities (using rat paw edema method and writhing test; respectively) as well as their gastric ulcerative effect (ulcerogenicity) an in-vitro antibacterial activity against Staphylococcus aureus (ATCC 25923) as a representative of Gram-positive bacteria; Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 8739) as representatives of Gram-negative bacteria.The compounds were also evaluated for their in-vitro antifungal activity against Candida albicans (ATCC 10231) (using the cup diffusion technique).
It is worth mentioning that, the highly potent compounds were those comprising 3-cyanopyrrole rings attached to different side chains in the 2 position, among these chains are the aryl imino function as in compounds 5b-d, thiourea group as in compounds 9c,d and ethoxymethyleneimino chain in compound 13b.Also, compounds containing pyrrole-3-carboxylate with the 2 position either unsubstituted as in compound 4d or substituted with thiourea side chain as compound 38b were highly potent.
Furthermore, among the highly potent compounds were thefused pyrrolopyrimidine compounds bearing 2-thioxo function with different substituents in the 4 position.Among these groups in the 4 position was the amino group as compound 10d, imino function in compounds 12a,b, 4-thioxo function as in compounds 31a,b and 4-oxo group as in compound 40a.Also, the 4-chloropyrrolopyrimidine derivative 21b as well as the 4-hydrazinopyrrolopyrimidine analogue 23b exhibited potent activity comparable to the reference drug Indomethacin (Indocin ® ).
As revealed from the results presented in Tables 1-3 that, compounds 4d, 5c, 5d, 6b, 10c, 10d, 17b, 31a and 31b exhibited the most potent analgesic activity with potency ranging from 1 -1.10 to the reference drug Indomethacin.It is to be noted that some functions are assumed to be responsible for the highly potent analgesic activity of these compounds.Among these functions are the ester function in 3-position as in compound 4d, the 2,4-dichlorobenzylidine imino function in pyrrole-2-position as in compounds 5c and 5d and pyrrolopyridine moiety in compound 6b.Also, the pyrrolopyrimidine thione function as in compounds 10c and 10d.
Furthermore, the carboxamide function as in compound 17b led to high analgesic activity.Additionally, pyrrolopyrimidine dithione function as in compounds 31a and 31b exerted the most potent analgesic activity.

Ulcerogenicity
Five compounds that exhibited the most potent anti-inflammatory activity; 12b, 23b, 31a, 31b and 38b were evaluated for their ulcerative effect on rats as revealed in Table 7.In general, all the tested compounds showed better results than the reference drug Indomethacin.Especially, compounds 12b and 31b which were devoid of any ulcerative effect compared to 94% of that of Indomethacin as illustrated in the previous table.

Anti-Microbial Screening
For the tested compounds 4a-40b, the resulting inhibition zones were measured in mm diameter, Tables 8-10.
Among the tested compounds, compounds 5d, 9d, 11b, 12a, 13b, 15a, 15b, 17a, 22a, 27a, 32a, 33a, 39a and 39b were found to be the most active.Among the different functions attached to the free pyrrole ring which exerted potent antimicrobial activity against Gram negative bacteria are the 2,4-dichlorobenzylidineimino, thiourea, phenylurea, ethoxymethyleneimino as well as dimethylaminomethyleneimino groups as in compounds 5d, 9d, 11b, 13b and 15a, 15b; respectively, in addition to, the carboxamide and the acid hydrazide side chains attached to the pyrrole ring in compounds 17a and 33a; respectively.Furthermore, functions surmounted on the pyrrolopyrimidine skeleton as 4-imino-3-phenyl-2-thione and 4morpholino groups as in compounds 12a and 22a; respectively, also displayed significant antimicrobial activity.Besides to, the triazolopyrrolopyrimidine derivative 27a, pyrrolotriazine analogue 32a as well as the pyrroloxazine compounds 39a, 39b which exerted high antimicrobial activities against Gram negative bacteria.It is worth mentioning that, all compounds exerted weak activity against Gram positive bacteria except for the 3imidazolidine substituted pyrrole derivative 30b which exerted moderate antimicrobial activity.However, only the pyrrolopyrimidinone derivative 18a and the 1,2,6-thiadiazine-2,4-dione analogue 19a exerted moderate antifungal activity against C. albicans.

Docking on the Active Site of Cyclooxygenase-2 Enzyme (COX-2) 1) Diclofenac interactions with the active site of COX-2:
Diclofenac interacted as hydrogen bond acceptor via four hydrogen bonds via both the oxygen atoms of car-boxyl group with the amino acid residues Tyr 385 (2.73 A˚) and Ser 530 (2.65 A˚, 2.91 A˚ and 3.04 A˚) as shown in Figure 1.

Docking of Compound 4d into COX-2
Active site revealed that several molecular interactions were considered to be responsible for the observed affinity, as the N of pyridine moiety acted as a hydrogen bond acceptor with the side chain residue; His 90 (2.25 A˚) with a strength of 81.3%.In addition to a hydrogen bond interaction between the hydrogens of the amino group which acted as a hydrogen bond donor with the side chain residue Tyr 355 (2.61 A˚) with a strength of 5.3%.Besides to, hydrophobic interactions involving the pyridine C

General Procedure for Synthesis of Compounds 9a-d
The appropriate compound 4a,b (2 mmol.) was refluxed with equimolar amount of urea or thiourea (2 mmol.) in absolute ethanol (20 mL) containing sodium ethoxide [prepared by dissolving sodium metal (0.03 g, 2 mmol.) in absolute ethanol (20 mL)] for 10 h.The reaction was allowed to cool and the solid product was filtered and washed with ethanol.
Method 2: The selected urea or thiourea derivative 9a-d (2 mmol.) was refluxed in pyridine (10 mL) for 16 h.The solvent was evaporated under reduced pressure and the solid obtained was collected to yield the target compounds 10a-d in an average yield of 63%.
Method 2: The selected phenyl thiourea derivatives 11a,b was refluxed in pyridine (10 mL) for 18 h.The solvent was evaporated under reduced pressure to yield the target compounds 12a,b in an average yield of 48%.

General Procedure for Synthesis of Compounds 17a,b
The selected compound 4a,b (2 mmol.) was stirred at room temperature for 3 h.inconc.sulfuric acid (15 mL) then poured drop by drop on to crushed ice.The reaction mixture was neutralized with ammonium hydroxide and the obtained product was filtered, washed thoroughly with water, left to dry and recrystallized from ethanol.

General Procedure for Synthesis of Compounds 20a,b
The selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carboxamide 17a,b (2 mmol.) was refluxed in excess acetic anhydride (15 mL) for 10 h.The reaction mixture was concentrated to the minimum.The solid product was collected, washed with ethanol then recrystallized from dioxane.     ) and morpholine (0.17 g, 0.2 mL, 2 mmol.) was refluxed in absolute ethanol for 9 h (30 mL) in presence of a catalytic amount of TEA (3 -5 drops).The reaction mixture was allowed to cool then treated with 10% acetic acid.The obtained product was filtered, washed with ethanol then recrystallized from ethanol.

General Procedure for Synthesis of Compounds 28a,b
An equimolar mixture of the selected compound 24a,b (2 mmol.) and phenyl isothiocyanate (0.27 g, 0.24 mL, 2 mmol.) was refluxed for 7 h in absolute ethanol (30 mL) containing few drops TEA (2 -4 drops).The reaction mixture was left to cool and the obtained product was filtered off, washed with ethanol then recrystallized from ethanol.

Anti-Inflammatory Screening 1) Animals
The screening for anti-inflammatory activity for all the newly synthesized compounds 4a-40b was carried out by using adult albino rats of both sexes weighing 120 -150 g which were obtained from animal house laboratory of Nile company, Cairo, Egypt.Rats were divided into eighty four groups; each group consists of five rats per cage in the Department of Pharmacology, Faculty of Medicine, Al-Azhar University.
The rats were kept under constant temperature 30˚C and 12 hours light/dark cycle.All animals were acclimatized in the animal facility for at least two weeks prior the experiments.
The animals were kept fastened for 24 hours prior to the experiment, but they were allowed free access to water [37].The animal experiments described below comply with the ethical principles and guidelines for the care and use of laboratory animals adopted by the National Egyptian Community.
2) Anti-inflammatory activity: Rat paw edema assay was carried out according to Winter et al. [38].Prepared compounds (equimolar to the referencedrug) were dissolved in DMSO and administrated subcutaneously.
One hour after drug administration acute inflammation was induced by injection of 0.05 mL of 1% of carrageenan sodium (Sigma-Aldrich, St. Louis, USA) subcutaneously into the sub planter region of the right hind paw.
The thickness of the injected paw was measured (from dorsal to ventral surfaces) immediately after carrageenan injection and after (1, 2, 3, 4, 5 and 6 hours) by using a micrometer.The size of edema was expressed as the increase in the thickness in mm after carrageenan injection.
The percentage inhibition of edema thickness at each time interval was calculated from the mean effect in control and treated animals according to the equation [39] [40].

( )
% Inhibition of edema thickness Tc Tt Tc 100 where, Tc and Tt are the mean increase in thickness of the carrageenan injected paw of the control group and drug treated groups; respectively.Control group: received the excipients (water mixed with few drops of tween 80) followed by carrageenan after 1 hour.Indomethacin (Indocin ® ) (5 mg/kg) was used as the referencedrug [41].Then the potencies of compounds were calculated after 6 hours of carrageenan injection where the % edema inhibition reached maximum.
3) Statistical analysis Results are expressed as (mean ± standard deviation) statistically analyzed using two way analysis of variance (ANOVA) followed by Bonferroni test [42].

Ulcerogenicity
All animals subjected to this experimental test were sacrificed immediately after the last measurement (6 hour), by diethyl ether and stomachs were separated.An opening at the great curvature was made and the stomachs were washed with distilled water and cleaned gently by dipping in normal saline.The mucosal damage was inspected with a 3× magnifying lens for any evidence of hyperemia, hemorrhage or ulcer.For each stomach the mucosal damage was assessed [43].
The percentage ulceration for each group was calculated as follows: Number of animals bearing ulcerin a group % Ulceration 100 Total number of animals in the same group = ×

Analgesic Screening 1) Animals:
The screening for analgesic activity for all synthesized compounds 4a-40b was carried out by using mices of both sexes weighing 25 -30 g which were obtained from animal house laboratory, Nile company, Cairo, Egypt.
Mices were divided into eighty four groups; each group consists of four mices per cage in the animal facility of Faculty of Medicine, Al-Azhar University.The mices were kept under constant temperature 25˚C and 12 hours light/dark cycle.All animals were acclimatized in the animal facility for at least two weeks prior the experiments.The animals were kept fastened for 24 hours prior to the experiment, but they were allowed free access to water.
2) Assessment of analgesic screening: The analgesic activity was evaluated according to writhing test reported by Koster et al. [44].The newly synthesized compounds (equimolar to the reference drug) were dissolved in DMSO and administrated to the groups orally (using intragastric tube) followed by injection of 0.6% acetic acid solution (10 mL/kg) after 1 hour [45].Indomethacin (Indocin ® ) (2.5 mg/kg) was used as the reference drug.
Stretching movements (arching of the back, developments of the tension in the abdominal muscles, elongation of the body and extension of the forelimbs) were counted as a writhing response.The number of writhes was counted for 15 minutes immediately after the acetic acid injection.The percentage of inhibition of writhes number was calculated as follows: Nc Nt % of Inhibition 100 Nc
pyrimidine derivatives.Thus, compounds 24a,b were reacted with ethyl cyanoacetate in absolute ethanol containing few drops of glacial acetic acid to yield the cyanomethyl triazolopyrimidine derivatives 25a,b.While, refluxing of compounds 24a,b with carbon disulphide in absolute ethanol containing potassium hydroxide yielded the corresponding triazolopyrimidine-2-thione derivatives 26a,b.Moreover, the reaction of the precursors 24a,b with triethyl orthoformate in absolute ethanol gave the unsubstituted triazolopyrimidine compounds 27a,b.While, upon refluxing of 24a,b with phenyl isothiocyanate in absolute ethanol in presence of a catalytic amount of triethylamine afforded the phenyl amine substituted triazolopyrimidine derivatives 28a,b.Heating of the o-aminonitrile derivatives 4a,b with sodium azide in presence of ammonium chloride in dimethylformamide as a solvent to furnish 2-aminopyrrol-3-tetrazole derivatives 29a,b (Scheme 8).However, heating compounds 4a,b with ethylene diamine in the presence of carbon disulphide afforded the target compounds 2-aminopyrrol-3-imidazole derivatives 30a,b.Also, the o-aminonitrile derivatives 4a,b were refluxed with carbon disulphide in dry pyridine to yield the pyrrolopyrimidine-2,4-dithiones 31a,b.Besides, pyrrolo[2,3d][1,2,3]triazin-4-ones 32a,b were obtained via stirring of compounds 4a,b with sodium nitrite solution in a mixture of glacial acetic acid and concentrated hydrochloric acid at 0˚C -5˚C.The reaction mechanism is suggested to proceed first through partial hydrolysis of the cyano function to afford the corresponding o-aminocarboxamide derivatives that undergo subsequent diazotization then coupling with amino function of the carboxamide to yield the target 4-oxotriazine derivatives 32a & 32b.

Figure 1 .
Figure 1.Docking of Diclofenac into the active site of COX-2.

Figure 2 .
Figure 2. Docking of compound 4d into the active site of COX-2.

Figure 3 .
Figure 3. Docking of compound 5b in the active site of COX-2.

Figure 4 .
Figure 4. Docking of compound 5c in the active site of COX-2.

Figure 5 .
Figure 5. Docking of compound 5d in the active site of COX-2.

Figure 6 .
Figure 6.Docking of compound 6a in the active site of COX-2.

Figure 7 .
Figure 7. Docking of compound 6b in the active site of COX-2.

Figure 8 .
Figure 8. Docking of compound 9c in the active site of COX-2.

Figure 9 .
Figure 9. Docking of compound 9d in the active site of COX-2.

Figure 10 .
Figure 10.Docking of compound 10d in the active site of COX-2.

Figure 11 .
Figure 11.Docking of compound 12a in the active site of COX-2.

Figure 12 .
Figure 12.Docking of compound 12b in the active site of COX-2.

Figure 13 .
Figure 13.Docking of compound 13b in the active site of COX-2.

Figure 14 .
Figure 14.Docking of compound 19a in the active site of COX-2.

Figure 15 .
Figure 15.Docking of compound 19b in the active site of COX-2.

Figure 16 .
Figure 16.Docking of compound 21b in the active site of COX-2.

Figure 17 .
Figure 17.Docking of compound 23b in the active site of COX-2.

Figure 18 .
Figure 18.Docking of compound 31a in the active site of COX-2.

Figure 19 .
Figure 19.Docking of compound 31b in the active site of COX-2.

Figure 20 .
Figure 20.Docking of compound 38b in the active site of COX-2.

Figure 21 .
Figure 21.Docking of compound 40b in the active site of COX-2 enzyme.

Table 1 .
Anti-inflammatory and analgesic results for compounds of Scheme 1 and Scheme 2.

Table 2 .
Anti-inflammatory and analgesic results for compounds of Scheme 3.

Table 3 .
Anti-inflammatory and analgesic results for compounds of

Table 4 .
Anti-inflammatory and analgesic results for compounds of

Table 5 .
Anti-inflammatory and analgesic results for compounds of

Table 6 .
Anti-inflammatory and analgesic results for compounds of

Table 7 .
Ulcerogenic effects of the tested compounds.

Table 10 .
Inhibition zones (IZ) in mm diameter for compounds of Scheme 9 & Scheme 10.

5. 2.3.6. Docking of Compound 6a into COX-2
6 and p-chlorophenyl C 3 carbon as well the CH 3 group of ester function and the following amino acid residues: His 90, Met 113, Val 116, Leu 117, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Leu 384, Tyr 385, Trp 387, Phe 518, Met 522, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure2.Active site revealed the presence of arene cation interaction between the pyrrole ring and the amino acid residue Arg 120.In addition to, hydrophobic interactions involving pyridine C 4 and C 6 carbons and the following amino Active site revealed the presence of four hydrogen bonds in which the cyano nitrogen acted as a hydrogen bond acceptor for three hydrogen bonds with the amino acid residues His 90, Arg 513 and Tyr 355 (2.82 A˚, 3.44 A˚ and 2.91 A˚; respectively) with a strength of 20.4%, 7% and 7.3%; respectively.While, the amino group acted as a hydrogen bond acceptor with the amino acid residue Tyr 355 residue (3.24 A˚) with a strength 5.5%.In addition to, hydrophobic interactions involving the carbonyl oxygen with the following amino acid residues: His 90, Met 113, Val 116, Leu 117, Arg 120, Ser 530, Val 349, Leu 352, Ser 353, Tyr 355, Leu 384, Try 385, Ala 527 and Leu 531 as shown in Figure 6.Pro 86, Val 89, His 90, Arg 120, Val 349, Leu 352, Tyr 355, Arg 513, Ala 516, Phe 518, Val 523, Glu 524, Gly 526, Ala 527 and Ser 530 as shown in Figure acid residues: His 90, Met 113, Val 116, Leu 117, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Trp 387, Arg 513, Phe 518, Met 522, Val 523, Ala 527, Ser 530 and Leu 531 as shown in Figure 3.2.3.4.Docking of Compound 5c into COX-2Active site illustrated the presence of several interactions of the cyano group with different amino acid residues as it acted as a hydrogen bond acceptor with the side chain residues; His 90, Tyr 355 and Arg 513(3.35A˚,2.43A˚and 3.16 A˚; respectively) at a strength of 2.1%, 90.6% and 13.4%; respectively.This beside hydrophobic interactions among the cyano function and the following amino acid residues: His 90, Val 116, Leu 117, Arg 120, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Trp 387, Arg 513, Ala 516, Ile 517, Phe 518, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 4.2.3.5.Docking of Compound 5d into COX-2Active site revealed the presence of hydrogen bond interaction between the cyano group, as it acted as a hydrogen bond acceptor with the amino acid residue Ser 530 residue (3.32 A˚) with a strength of 1.8%.In addition to, arene cation interactions involving the p-chlorophenyl ring with the amino acid residue Phe 518 and the 2,4-dichlorophenyl ring with the amino acid residue Arg 120.There are also hydrophobic interactions involving the pyridine C 2 , C 5 , C 6 as well as the pyridine nitrogen atom with the following amino acid residues: His 90, Val 116, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Leu 384, Phe 518, Met 522, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure While the cyano nitrogen atom acted as a hydrogen bond acceptor with the amino acid residue His 90 (3.41 A˚) with strength of 2.2%.In addition to, two arene cation interactions among the p-chlorophenyl moiety and the amino acid residue Arg 120 and Arg 513.Besides to hydrophobic interactions involving the pyridine ring, pyrrole C 2 carbons as well as the chlorine atom and the p-chlorophenyl C 3 carbon with the following amino acid re-sidues:

7. 2.3.8. Docking of Compound 9c into COX-2
Active site revealed the presence of two hydrogen bond interactions between the cyano nitrogen, as it acted as a hydrogen bond acceptor with the amino acid residue side Arg 120 and Tyr 355 (3.21 A˚ and 1.60 A˚; respectively) with a strength of 12.3% and 95.9%; respectively.In addition to, hydrophobic interactions involving the cyano nitrogen and chlorine atom with many amino acid residues: His 90, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Phe 381, Tyr 385, Trp 387, Arg 513, Ala 516, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 8. Active site revealed the presence of one hydrogen bond between the pyridyl nitrogen atom as it acted as a hydrogen bond acceptor with the amino acid residue His 90 (2.82 A˚) with a strength of 4.8%.In addition to, hydrophobic interactions concerning 4-chlorophenyl C 2 carbon, pyridine C 2 and C 4 carbons, the thiourea amino group and sulphur atom with the following amino acid residues: His 90, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Phe 381, Leu 384, Tyr 385, Trp 387, Met 522, Val 523, Gly 526 and Ala 527 as shown in Figure 9.
2.3.10.Docking of Compound 10d into COX-2Active site revealed hydrogen bond interaction between the N atom of pyridine moiety as it acted as a hydrogen bond acceptor with the side chain residues His 90 and Arg 513 (2.74 A˚ and 3.54 A˚; respectively) with a strength of 21.7% and 1.2%; respectively.Besides to, arene cation interaction between the p-chlorophenyl ring and the amino acid residue Arg 120.In addition to, hydrophobic interactions among the p-chloro phenylC 2 carbon, chlorine atom as well as the pyrimidine thioxo function, N 3 atom and C 4 carbon with the following amino acid residues: His 90, Val 116, Arg 120, Val 349, Leu 352, Ser 353, Leu 359, Trp 387, Arg 513, Ala 516, Ile 517, Phe 518, Met 522, Val 523 and Ala 527 as shown in Figure 10.2.3.11.Docking of Compound 12a into COX-2 Active site revealed the presence of only hydrophobic interactions involving the phenyl C 2 carbon and the p-chlorophenyl C 3 and C 6 carbons as well as the chlorine atom with the following amino acid residues: His 90, Met 113, Val 116, Arg 120, Ile 345, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Arg 513, Ala 516, Phe 518, Met 522, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure

14. 2.3.15. Docking of Compound 19b into COX-2
Active site revealed the presence of hydrogen bond interactions between the oxygen atoms of pyrimidine C 2 and C 4 oxo functions as they acted as hydrogen bond acceptor with the side chain residue Arg 513 (3.27 A˚, 2.3%) and Arg 120 (2.28 A˚, 9.5%); respectively.In addition to, another hydrogen bond between the pyridyl nitrogen atom as it acted as hydrogen bond acceptor with the amino acid residue Arg 513 (3.45 A˚, 1.7%).In addition to, hydrophobic interactions concerning the carbon atoms of p-chlorophenyl moiety and the following amino acid residues: Pro 86, His 90, Arg 120, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Arg 513, Ala 516, Val 523, Glu 524, Ala 527, Ser 530 and Leu 531 as shown in Figure

15. 2.3.16. Docking of Compound 21b into COX-2
Active site revealed the presence of hydrogen bond interaction between the N 1 of the pyrimidine moiety, as it acted as a hydrogen bond acceptor with the side chain residue Arg 120 (2.35 A˚) with strength of 64.2%.In addition to, arene cation interaction between the pyridine moiety and the amino acid residue Arg 120.Besides to, hydrophobic interactions involving the pyrimidine C 2 carbon, pyrrole C 5 carbon and pyridine C 6 carbon with many amino acid residues: Arg 120, Val 349, Leu 352, Tyr 355, Tyr 385, Trp 387, Arg 513, Val 523, Glu 524, Gly 526, Ala 527 and Leu 531 as shown in Figure16.
2.3.17.Docking of Compound 23b into COX-2Active site revealed the presence of three hydrogen bond interactions in which the hydrazine NH 2 proton acted as a hydrogen bond donor with the amino acid residue Val 349 (3.21 A˚, 1.5%), while the nitrogen atom acted as a hydrogen bond acceptor with the amino acid residue Ser 353 (2.62 A˚, 2.5%).Also, the hydrazine NH proton acted as a hydrogen bond donor to the amino acid residue Val 349 (1.38 A˚, 93.7%).Besides to, hydrophobic interactions among the p-chlorophenyl moiety and the pyrimidine C 4 carbon with the following amino acid residues: His 90, Tyr 348, Val 349, Leu 352, Ser 353, Tyr 355, Phe 381, Leu 384, Tyr 385, Phe 518, Met 522, Val 523, Gly 526, Ala 527 and Ser 530 as shown in Figure 17.2.3.18.Docking of Compound 31a into COX-2 Active site showed arene cation interaction between the 3-triflouromethylphenyl ring and the amino acid residue Arg 513.In addition to, hydrophobic interactions involving the 3-trifluoromethylphenyl C 6 and fluorine atom as well as the two sulphur atoms of the two thioxo functions of pyrimidine ring with the following amino acid residues: His 90, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Leu 384, Tyr 385, Trp 387, Arg 513, Ala 516, Ile 517, Phe 518, Met 522, Val 523, Gly 526, Ala 527 and Ser 530 as shown in Figure

18. 2.3.19. Docking of Compound 31b into COX-2
Active site revealed the presence of hydrophobic interactions involving the pyridine C 6 carbon, chlorine atom, the sulphur atom of the 2-thioxo function of pyrimidine ring as well as the pyrimidine N 3 NH moiety with the following amino acid residues: His 90, Val 349, Leu 352, Ser 353, Tyr 355, Leu 384, Tyr 385, Trp 387, Arg 513, Phe 518, Met 522, Val 523, Gly 526, Ala 527 and Leu 531 as shown in Figure

19. 2.3.20. Docking of Compound 38b into COX-2
Active site showed one hydrogen bond between NH-phenyl proton which acted as a hydrogen bond donor with the side chain residue Tyr 355 (2.03 A˚, 0.8%).In addition to, two arene cation interactions between both pyridine and phenyl rings and the amino acid residue Arg 120.Besides to, hydrophobic interactions involving the phenyl C 4 carbon and the sulphur atom with many amino acid residues: Pro 86, Val 116, Arg 120, Tyr 348, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Trp 387, Arg 513, Val 523, Glu 524, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure

.22. General Procedure for Synthesis of Compounds 23a,b
A mixture of compounds 21a,b (2 mmol.) and hydrazine hydrate 98% (0.1 g, 0.1 mL, 2 mmol.) was refluxed in absolute ethanol (30 mL) in presence of TEA as a catalyst for 10 h.The reaction mixture was allowed to cool and poured on to crushed ice.The obtained solid was filtered, washed with water and recrystallized from dio-