Synthesis , X-Ray Crystal Structures , Biological Evaluation , and Molecular Docking Studies of a Series of Barbiturate Derivatives

1Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia 2Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 21321, Egypt 3Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 4Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt 5H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan 6Department of Biochemistry, Abdul Wali Khan University, Mardan 23200, Pakistan

Because of the important biological activities, PYT is widely used as a synthon in the design of antitumor agents.It has a high efficacy to form hydrogen bonding with drug targets (6-9, Figure 1).Singh et al. have reported the activity of a series of new N-benzyl indole-pyrimidine-2,4,6-trione hybrids against a panel of 60 human tumor cell lines.Several of these analogs were also found to be inhibitors of DNA repair and replication stress response polymerases [18].
The discovery and development of effective antioxidants capable of supplementing body's antioxidant system is an important area of health care research.Similarly research efforts are focused on the identification of effective and safe inhibitors of -glucosidase as potential antidiabetic agents.
The focus in recent years is on the development of resource-efficient, environmentally benign, and sustainable chemistry practices.In continuation of our work in this field, we describe here the synthesis of a series of previously reported [17,19,20] and new PYT adducts.These compounds were evaluated for their antioxidant activity by using DDPH scavenging assay, as well as for in vitro enzyme inhibition activities against alpha-glucosidase, thymidine phosphorylase, and -glucuronidase enzymes.

Materials and Methods
The chemical reagents used for the preparation of the target compounds were commercially available and used without further purification. 1H-NMR and 13 C-NMR were measured using Bruker Avance AV-600 NMR spectrometer, MS were carried out with a Jeol, JMS-600 H instrument, and singlecrystal X-ray diffraction was collected using a Bruker SMART APEX II CCD diffractometer.

General Procedure for Aldol Condensation Michael Addition for the Synthesis of 4 and 5 (GP1).
A mixture of aldehyde 3 (1.5 mmol) and 1 and 2 (3 mmol) as well as Et 2 NH (1.5 mmol, 155 L) in 3 mL of degassed H 2 O (bubbling nitrogen through the water) was stirred at room temperature for 1-5 h until TLC showed complete disappearance of the reactants.The precipitate was removed by filtration and washed with ether (3 × 20 mL).The solid product was dried to afford pure products 4 and 5.
The full characterization of the synthesized compounds 4 and 5 and the X-ray crystal data of 5a, 5d, and 5f can be seen in the Supplementary Materials (see supplementary materials available online at http://dx.doi.org/10.1155/2016/8517243).

Biological Activity.
The samples of the synthesized compound were screened against DPPH radical, -glucuronidase

Results and Discussion
3.1.Chemistry.We have been focusing on the development of highly expedient methods for the synthesis of diverse heterocyclic compounds of biological importance via onepot multicomponent reactions (MCRs) and avoiding organic solvents in organic synthesis [17,19,20].This had led to the development of several efficient, environmentally benign, clean, and economical process technology (Green Chemistry Concepts).In current reaction strategy, equimolar amounts of barbituric acid (1) and dimedone (2) with aldehyde (3) in the presence of aqueous diethylamine medium at RT yielded salts of PYT adducts 4a-4y and 5a-5m were obtained in quantitative yields by simple filtration [17][18][19][20].

X-Ray Diffraction.
The molecular structures of compounds 5a, 5d, and 5f were unambiguously deduced by single-crystal X-ray diffraction technique (Figure 2).The crystals of the synthesized molecules 5a, 5d, and 5f were grown by slow diffusion of diethyl ether solution of pure samples 5a, 5d, and 5f in DCM/EtOH at room temperature followed by allowing standing for 24 h.The structures were resolved by direct methods by using the SHELXL97 program [21,22] in the SHELXTL-plus package and refined by a full-matrix least-squares procedure on  2 using SHELXS97.Diffraction data were collected on a Bruker APEX-II CCD diffractometer (Bruker AXS GmbH).The crystal structure and refinement data of compounds 5a, 5d, and 5f are listed in Table 1.The final atomic coordinates for all atoms and complete listing of bond distance and angles are presented Scheme 1: Synthesis of the target compounds.in supplementary information.ORTEP drawings of final Xray model of compounds 5a, 5d, and 5f with the atomic numbering scheme are presented in Figure 2, while crystal packing presentation of compounds 5a, 5d, and 5f is shown in Figure 3. Compound 5a crystallizes in orthorhombic crystal system in a space group of P2 1 2 1 2 1 ; on the other hand, compounds 5d and 5f crystalize in monoclinic crystal system in a space group of P2 1 /n.It was observed that the compound 5a has hydrogen bonding between the C 5 -O 2 ⋅ ⋅ ⋅ H⋅ ⋅ ⋅ O 4 -C 13 , while compounds 5d and 5f exist only in the enol form.There are two strong intramolecular hydrogen bonds on compound while compounds 5d and 5f have no intramolecular hydrogen bonding.The crystal structures of the tested compounds 5a, 5d, and 5f were formed by different hydrogen bonds between the molecules and diethyl amine and water solvent molecules in case of 5a to form network structures.Crystallographic data for the solved structures 5a, 5d, and 5f were deposited to the Cambridge Crystallographic Data Center as supplementary publication numbers CCDC-1444058, CCDC-1443783, and CCDC-1444050, respectively, which can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data request/cif.

Binding Interactions of 𝛼-Glucosidase
Enzyme.To define inhibitors interactions and their specificity, the docking study of the most active compound 4p against -glucosidase  was carried out [23][24][25].Ten different conformations of protein-ligand interactions were generated and the top ranked pose was selected to explore the binding interactions.In our docking study, we have observed two interactions with the active site residues of -glucosidase.The polar imidazole ring of His 239 showed hydrogen bond through its H atom with the lone pair of oxygen of the pyrimidinedione moiety of the compound, whereas Arg 312 formed hydrogen bond with the OH group of the same moiety of compound (Figure 4).The presence of polar and electron rich center in the form of pyrimidinedione and chlorobenzene groups of the compound was observed here as key factors for the interaction between compound and protein.A number of hydrogen bond donor and acceptor pairs with their suitable orientations were observed in the interaction pose.Furthermore, several hydrophobic interactions between the nonpolar active site residues and the compound 4p were also observed.

Binding Interactions of Thymidine Phosphorylase
Enzyme.To explore the binding modes of these newly synthesized compounds in the active site of thymidine phosphorylase, the most active compound 4v was docked against this enzyme.The docking results showed that compound 4v well accommodated in the active site of thymidine phosphorylase (Figure 5).The active site residue Ala 206 established a hydrogen bond through its lone pair of oxygen with the active hydrogen of hydroxyl moiety of the pyrimidinedione group of the compound.However, the substituted phenolic group and the other electron rich centers of the pyrimidinedione showed poor behavior regarding interaction which may depend on the nature and orientation of the surrounding active site residues.Moreover, several weak hydrophobic interactions between the active site residues and the compound were also observed.

Binding Interaction of 𝛽-Glucuronidase
Enzyme.The predicted binding mode of most active compound 4w against -glucuronidase showed that a hydrogen bond is formed between the lone pair of oxygen of cyclohexanedione component of the compound and imidazole hydrogen of the important active site residue, His 509.An arene-arene and arenecation interaction was also observed between the pi electronic cloud of dichlorobenzene ring of compound and imidazole ring of His 509 (Figure 6).Besides hydrogen bonding and arene-arene interactions, several hydrophobic interactions between compound 4w and the active site residues were also observed.The pyrimidinedione moiety in this case was observed to be passive regarding interactions.

Conclusions
In conclusion, barbiturate salts were synthesized by one-pot multicomponent reactions via tandem Aldol/Michael addition reactions between barbituric acid and dimedone with aldehydes, mediated by aqueous diethylamine.The molecular structures of 5a, 5d, and 5f were deduced by singlecrystal X-ray diffraction techniques.All of the synthesized compounds were evaluated for their antioxidant potential in vitro by using DPPH radical scavenging assay as well as enzyme inhibition activities against -glucosidase, thymidine phosphorylase, and -glucuronidase enzymes.Compounds 4a-4g (IC 50 = 101.8± 0.8-124.4± 4.4 M) were found to be the potent antioxidants as compared to the standards, BHT (IC 50 = 128.8± 2.1 M), and N-acetylcysteine (IC 50 = 107.6 ± 2.8 M).Compound 4p (IC 50 = 186.8± 8.1 M) was found to be a potent -glucosidase inhibitor.Compound 4v (IC 50 = 8.7 ± 1.2 M) was found to be a potent thymidine phosphorylase inhibitor.Promising results indicate that these compounds need to be investigated as antioxidant agents to treat various associated oxidative disorders.Similarly their enzyme inhibitory potential can be reported on drug discovery program.

Figure 3 :
Figure 3: Molecular packing of 5a, 5d, and 5f viewed approximately three-dimensional network, along  axis and three-dimensional network, respectively.Intermolecular interaction is drawn as dashed lines.

a
SEM: standard error of mean, b PC3: human prostate cancer cell line, c HeLa: Henrietta Lacks cervical cancer cell line, d MCF-7: Michigan Cancer Foundation-7 breast cancer cell lines, and e BHT: butylated hydroxytoluene.

Table 2 :
Result of in vitro biological evaluation of the synthesized compounds.