Heterocyclic Synthesis via Enaminones : Synthesis and Molecular Docking Studies of Some Novel Heterocyclic Compounds Containing Sulfonamide Moiety

4-Acetyl-N, N-diethylbenzenesulfonamide (1) was condensed with dimethylformamide dimethylacetal (DMF-DMA) to yield the enaminone, which reacts readily with different reagents to give the corresponding pyrazoles, triazolopyrimidine, imidazopyridine, pyrimidopyrimidine, pyrazolopyrimidine, benzofuran, pyranone, pyridine, pyrimidine and pyrazolopyridazine derivatives. These compounds were designed to comply with the general features of sulfonamide pharmacophore which act as Cyclooxygenase (COX-2) inhibitors. Virtual screening using molecular docking studies of the synthesized compounds was performed by (MOE), the molecular docking results indicate that, some synthesized compounds suitable inhibitor against (COX-2) with further modification.


Introduction
Enaminones are polydentate reagents that have been utilized extensively in this decade as building blocks in organic synthesis [1]- [6].Also sulfonamides possess many types of biological activities and representatives of this class of pharmacological agents are widely used in clinic as antibacterial [7], antithyroid, diuretic, hypoglycaemic and anti-cancer [8]- [12].Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) are widely employed in musculoskeletal disease, as well as their anti -inflammatory properties [13].After widely evaluation, NSAIDs
To a mixture of enaminone 2 (0.01 mol) and thiourea or guanidine hydrochloride (0.01 mol) in ethanol (40 ml) was added a few drops of piperidine as catalyst.The reaction mixture was refluxed for 4 hr, then poured into cold water (50 ml) and neutralized with diluted hydrochloric acid.The precipitate that formed was filtered off, dried, and crystallized from the appropriate solvents to give:

General procedure for preparation of (24a,b).
A mixture of enaminone 2 (0.01 mol) and p-toluidine or p-phenitidine (0.01 mol) in a mixture of ethanol/acetic acid (50 ml) (1:1) was heated under reflux for 3 hr.During the reflux period, a crystalline solid was separated.The separated solid was filtered off, washed with ethanol and recrystallized from the appropriate solvents to give:

4-(3-Benzoyl-1-phenyl-1H-pyrazole-4-carbonyl)-N,N-diethylbenzenesulfonamide (30).
To a mixture of enaminone 2 (0.01 mol) and the hydrazonoyl bromide (85; 0.01 mol) in benzene (40 ml) an equivalent amount of triethylamine was added.The reaction mixture was heated under reflux for 2 hr. the solvent was distilled at reduced pressure and the residual viscous liquid was taken in ethanol then the resulting solid was collected by filtration, washed thoroughly with ethanol, dried and finally recrystallized to give the compound 30.

Preparation of Small Molecule
Molecular modeling of the target compounds were built using MOE, and minimized their energy with PM3 through MOPAC.Our compounds were introduced into the (COX) binding site accordance the published crystal structures of (1) bound to the kinase.

MOE Stepwise
The crystal structure of the (COX) with a Diclofenac (1) as inhibitor molecule, was used in the receptor molecule, water and inhibitor molecules were removed, and hydrogen atoms were added.The parameters and charges were assigned to the MMFF94x force field.After alpha-site spheres were generated using the SITE FINDER module of MOE.The optimized 3D structures of molecules were subjected to generate different poses of ligands using triangular matcher placement method, which generating poses by aligning ligand triplets of atoms on triplets of alpha spheres representing in the receptor site points, a random triplet of alpha sphere centers is used to determine the pose during each iteration.The pose generated was rescored using London dG scoring function.The poses generated were refined with MMFF94x forcefield, also, the solvation effects were treated.The Born solvation model (GB/VI) was used to calculate the final energy, and the finally assigned poses were assigned a score based on the free energy in kJ/mol 3. Results and Discussion.(Scheme 1).The structure of the enaminone 2 was confirmed on the basis of elemental analysis and spectral data.Thus, the IR spectrum of compound 2 revealed absorption bands at (v cm −1 ): 2910 (CH-aliph.)and 1646 (C = O), while its 1 H-NMR (δ ppm) spectrum (DMSO) indicated signals at: 2.90 and 3.13 (2s, 6H, N(CH 3 ) 2 ), 5.79 and 7.72 (dd, 2H, olefinic CH = CH; J = 12.25 Hz), which support that the structure in (E-form), not (Z-form).The reactivity of compound 2 towards some nitrogen nucleophiles was investigated.Thus, enaminone 2 was treated with hydrazine hydrate in refluxing ethanol/acetic, to produce intermediates 3 followed by acetylation to give 4-(1-acetyl-1H-pyrazol-3-yl)-N,N-diethylbenzenesulfonamide (4).In the same manner, the enaminone 2 reacted with 2-cyanoacetic acid hydrazide under the same experimental reaction conditions to afford 4-(1-(2cyanoacetyl)-1H-pyrazol-3-yl)-N,N-diethylbenzenesulfonamide (5) (Scheme 1).
In the same manner, the enaminone 2 reacted with another heterocyclic amine like: 5-amino-3-methyl thiopyrazole-4-carbonitrile [29] [30] and 5-Phenyl-2H-pyrazol -3-yl amine under the same experimental reaction conditions to afford pyrazolo [1,5-a]pyrimidine derivatives 13a,b.The formation of 13a,b was assumed to takes place via the addition of exoamino group of aminopyrazole to α,β-unsaturated moiety of enaminone 2 to yield the corresponding acyclic non-isolable intermediate 12a,b which undergoes intramolecular cyclization by the elimination of the dimethylamine and water molecules to afford the final product 13a,b not 11a,b (Scheme 3).
Furthermore, Compound 2 reacted with 1,4-benzoquin-one in glacial acetic acid at room temperature to yield a product which formulated as N,N-diethyl-4-(5-hydroxy-benzofuran-3-carbonyl)benzenesulfonamide (17).Elucidation of structure 17 and refusing of structure 15 was based on 1 H-NMR spectrum which indicates the disappearance of aldehydic signal and showed singlet signal at 9.52 ppm for H-2 benzofuran moiety.It's believed that electron rich (C-2) in the enaminone 2 initially adds to the activated double bond in the quinone yielding acyclic intermediate 16 which then cyclizes into 17 via dimethylamine elimination, and not afforded 15 (Scheme 4).
Enaminone 2 reacted with hippuric acid in acetic anhydride to give a product that was identified as N-(6-(4-(N,N-diethylsulfamoyl)phenyl)-2-oxo-2H-pyran-3-yl)benzamide (19), which confirmed on the basis of elemental analysis and spectral data, (Scheme 5).The formation of compound 19 was assumed to proceed via initial cyclization of hippuric acid into oxazolone derivative which then added to the activated double bond system of enaminone yielding 18 followed by intramolecular cyclization and rearrangement to give the final structure 19 (Scheme 5).
Enaminone 2 reacted with guanidine hydrochloride and thiourea in refluxing ethanol in the presence of pipridine to give 23a,b.A plausible mechanism for the formation of compounds 23a,b is outlined in (Scheme 7).Which then undergo intramolecular cyclization and subsequent aromatization via the elimination of dimethylamine and water molecules under the reaction conditions to give 23a,b as depicted in (Scheme 7).On the other  Hydrazonyl halides [31]- [33] has been reported to add to α,β-unsaturated carbonyl compounds to yield a mixture of isomeric pyrazolines [34] [35].In the present work the reaction of enaminone 2 with nitrileimine 26 (gen-erated in situ from the treatment of the hydrazonoyl bromide 25 with triethylamine in refluxing m-xylene) gave only one isolable product (TLC).From which two proposed structures 28 or 30 seemed possible (Scheme 8).The other possible regioisomer 28 was excluded on the basis of the spectral data of the isolated products.For example, in the pyrazole ring system, C-4 is the most electron-rich carbon, thus H-4 is expected to appear in the 1 H-NMR spectra at higher field, typically near 6.0 ppm.On the other hand, H-5 is linked to the carbon attached to a nitrogen atom and thus it is deshielded to appear near 8.0 ppm. 1 H-NMR spectrum of 30 exhibits a singlet at 8.41 ppm, which indicates the presence of the pyrazole H-5 rather than H-4 [36].Pyrazole derivative 30 was assumed to be formed via initial 1, 3-dipolar cycloaddition of nitrileimine 26 to the activated double bond in compound 2 forming non isolable intermediate 29 followed by the loss of dimethylamine (Scheme 8).Interaction of pyrazole derivative 30 with hydrazine hydrate in refluxing ethanol absolute afforded pyrazolo [3,4-d] pyridazine derivative (31), which confirmed by elemental analysis and spectral data (Scheme 8).

Docking Studies
In brief, two isoforms of COX protein are known: COX-1 is responsible for the physiological production of prostaglandins, which is expressed in most tissues; and COX-2, is responsible for the increasing production of prostaglandins during process of inflammation, which is induced by endotoxins, cytokines and mitogens in inflammatory cells [37].Recently, from analysis of X-ray cocrystal of arachidonic acid with COX-2 showed that, carboxylate coordinated with Tyr-385 and Ser-530 [38], as well as the action of NSAIDs, through the interaction carboxylate group with Tyr-385 and Ser-530, which stabilize the negative charge of the tetrahedral intermediate [39], and demonstrated that, Tyr-385 and Ser-530 have a structural and functional evidence for the importance of them in the chelating of the ligands [40].Molecular docking of the synthesized compounds into the active site of COX was performed, in order to obtain biological data on a structural basis, through rationalized ligand-protein interaction behavior.All calculations for docking experiment were performed with MOE 2008.10 [41].The tested compounds were evaluated in silico (docking), using X-ray crystal structures of COX-2 (ID: 4COX).The tested compounds were docked into active sites of both enzymes COX-2.The active site of the enzyme was defined to include residues within a 10.0 Å radius to any of the inhibitor atoms.The scoring function of the most stable docking model for testing compounds was applied to evaluate the binding affinities of the inhibitors complexes with (COX) active site (Table 1).The complexes were energy-minimized with an MMFF94 force field [42] till the gradient convergence 0.05 kcal/mol was reached.The active compounds docked successfully into the COX-2 active site.The compounds 2.10 and 17 in COX-2 active site exhibited, binding scores (−98.09,−118.71 and −125.06)Kcal/mol, respectively.Structure activity relationships: In order to get a deeper insight into the nature and type of interactions of docked compounds, the complexes between each compound and COX-2 receptor were visualized, and depicted in (Figures 1-3).Since, the H bonds play an important role in the structure and function of biological molecules, the current ligand-receptor interactions were analyzed on the basis of H bonding.In order to reduce the complexity, hydrophobic and π-cation interactions (>6 Å) are not shown in the Figures 1-3.
On COX-2 binding site (Figure 3); i-Compound 2 arranged in the binding pocket by adjusting phenyl ring   perpendicular with N-ethyl and formed an electrostatic bond with Ser-530 and Tyr-385; ii-Compound 10 and 17, were interacted with different modes with binding pocket, by forming two strong hydrogen bond interaction with important residue Arg-120, which explain increasing of the binding energy of its compounds.The results obtained clearly reveals that, the amino acid residues close to the reference molecules are mostly the same as those observed in the most active compounds complexes with protein (Figures 2 and 3).These results indicate that, the compounds 2, 10 and 17 act as selective inhibitors against COX-2.ADMET factors profiling: Oral bioavailability was considered to play an important role in the development of bioactive molecules as  therapeutic agents.Many potential therapeutic agents fail to reach the clinic, because of their ADMET (absorption, distribution, metabolism, elimination and toxic) Factors.Therefore, a computational study for prediction of ADMET properties of the molecules was performed for most active compounds, by the determination of topological polar surface area (TPSA), a calculated percent absorption (%ABS) which was estimated by Zhao et al. equation [29], and "rule of five" formulated by Lipinski [30], which established that, chemical compound could be an orally active drug in humans, if no more than one violation of the following rule: 1) ClogP (partition coefficient between water and octanol) < 5.
In addition, the total polar surface area (TPSA) is another key property linked to drug bioavailability, the passively absorbed molecules with (TPSA > 140) have low oral bioavailability [43].All calculated descriptors were performed using MOE Package, and their results were disclosed in Table 1.Our results revealed that, the CLogP (factor of the lipophilicity) [44] was less than 5.0, hydrogen bond acceptors between (5 and 7), hydrogen bond donors (1), this data show these compounds fulfill Lipinski's rule.Also, the absorption percent is ranged between (~66% -90%).
The HOMO and LUMO of a molecule play important roles in intermolecular interactions [33], through the interaction between the HOMO of the drug with the LUMO of the receptor and vice versa.The interactions were stabilized inversely with energy gap between the interacting orbitals.Increasing HOMO energy and decreasing LUMO energy in the drug molecule lead to enhancement stabilizing interactions, and hence, binding to the receptor.Furthermore, the global and local chemical reactivity descriptors for molecules have been defined (Table 1), like softness (measures stability of molecules and chemical reactivity), hardness (reciprocal of softness), chemical potential, electronegativity (strength atom for attracting electrons to itself), electrophilicity index (measuring lowering energy due to maximal flowing electron between donor and acceptor) [45]- [51].The results were shown in Table 1 and may explain the less toxicity and high affinity of its compounds against COX.

Scheme 7 .
Scheme 7. Synthesis of pyrimidine derivatives.hand,when enaminone (2) was treated with primary aromatic amines namely (p-toluidine and p-phenitidine) in a mixture of ethanol/acetic acid at reflux temperature afforded 4-(3-((p-substituted amino)acryloyl)-N,N-diethylbenzene sulfonamides (24a,b) (Scheme 7).The 1 H-NMR spectrum of compounds 24a,b support this structures is (Z-form) not (E-form), where the coupling constant of the doublet signals for olefinic protons equal to 7.6, 7.8 Hz, respectively.Stabilization of (Z-form) is achieved by intramolecular hydrogen bonding (Scheme 7).Hydrazonyl halides[31]-[33] has been reported to add to α,β-unsaturated carbonyl compounds to yield a mixture of isomeric pyrazolines[34] [35].In the present work the reaction of enaminone 2 with nitrileimine 26 (gen-erated in situ from the treatment of the hydrazonoyl bromide 25 with triethylamine in refluxing m-xylene) gave only one isolable product (TLC).From which two proposed structures 28 or 30 seemed possible (Scheme 8).The other possible regioisomer 28 was excluded on the basis of the spectral data of the isolated products.For example, in the pyrazole ring system, C-4 is the most electron-rich carbon, thus H-4 is expected to appear in the 1 H-NMR spectra at higher field, typically near 6.0 ppm.On the other hand, H-5 is linked to the carbon attached

Figure 1 .
Figure 1.Docking compound 2 into the active site COX-2 with highest docking score H-bonds are in pink.

Figure 2 .
Figure 2. Docking compound 10 with a highest docking score into the active site COX-2.

Figure 3 .
Figure 3. Docking compound 17 with a highest docking score into the active site COX-2.

General Procedure for the Reaction of 2 with Active Methylene Compounds to Form ( 20a,b) and (21).
The reaction mixture was refluxed for 3 h, then poured into cooled water (50 ml) and neutrallized with diluted hydrochloric acid.The precipitate that formed was filtered off, dried, and crystallized from the appropriate solvents to give:

Table 1 .
Pharmacokinetic parameters important for good oral bioavailability of most active compounds.