Synthesis , Single Crystal X-Ray Structure , and Antimicrobial Activity of 6-( 1 , 3-Benzodioxol-5-ylmethyl )-5-ethyl-2-{ [ 2-( morpholin-4-yl ) ethyl ] sulfanyl } pyrimidin-4 ( 3 H )-one

1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 2 Pharmaceutical and Drug Industries Research Division, Medicinal and Pharmaceutical Chemistry Department, National Research Centre, Dokki, Giza 12622, Egypt 3 Department of Chemistry, Faculty of Science, Tanta University, Tanta 31527, Egypt 4Genetic Engineering and Biotechnology Division, Microbial Chemistry Department, National Research Centre, Dokki, Giza 12622, Egypt


Synthesis of 6-(1,3-Benzodioxol-5-ylmethyl)-5-ethyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (7).
A suspension of activated zinc dust [30] (7 g) in dry THF (30 mL) was heated to reflux.Few drops of ethyl 2-bromobutanoate (5) were added and the reaction mixture was refluxed for 10 min.The piperonylcarbonitrile (4) (2.42 g, 0.015 mol) was added in one portion while the rest of ester 5 (5.85 g, 0.03 mol) was added dropwise.After complete addition, the reaction mixture was refluxed for 30 min, then diluted with THF (100 mL), and quenched by addition of sat.aq.K 2 CO 3 solution (30 mL).The reaction mixture was stirred for 1 h at room temperature and then the THF layer was removed and the residue was washed with THF (3 × 20 mL).The combined THF fractions were stirred with 10% aq.HCl (20 mL) for 30 min.The solution was concentrated under reduced pressure and diluted with CH 2 Cl 2 (100 mL).The organic phase was washed with sat.aq.NaHCO 3 solution (2 × 50 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure to give the respective -ketoester 6 as a pale yellow viscous oil.The crude ketoester 6 (2.78 g, 0.01 mol) was added to a boiling solution of sodium ethoxide (prepared from 4.9 g of Na in 100 mL of absolute EtOH) containing thiourea (11.42 g, 0.15 mol).The reaction mixture was heated to reflux for 24 h.The solvent was evaporated to dryness under reduced pressure and the residue was dissolved in H 2 O (100 mL).The product was precipitated by addition of conc.HCl till pH = 4.The precipitate was filtered off, washed with H 2 O, and dried to give the corresponding thiouracil derivative 7. The crude 7 was purified by recrystallization (ethanol) to afford pure 7 in 46% yield as a white solid mp 183-185 ∘ C.

Crystal Structure Determination.
Slow evaporation of the pure compound 8 in dimethyl sulfoxide yielded its colourless single crystals.A colourless block-shaped single crystal of suitable size, 0.65 × 0.32 × 0.19 mm, was selected for X-ray diffraction analysis.Data were collected on a Bruker Venture D8 CMOS area diffractometer equipped with graphite monochromatic MoK \  radiation ( = 0.71073 Å) at 100 K. Cell refinement and data reduction were done by Bruker SAINT [34]; program used to solve structure and refine structure is SHELXTL [35].The final refinement was performed by full-matrix least-squares techniques with anisotropic thermal data for non hydrogen atoms on  2 .All the hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms.Multiscan absorption correction was applied by use of SADABS software [34].The crystallographic data and refinement information are summarized in Table 1.

Antimicrobial Activity by the Agar Disc-Diffusion Method.
Sterile nutrient, malt extract, and Czapek's dox agar media were inoculated, separately, with 100 L cell suspension of the chosen bacteria, Candida albicans, and Aspergillus niger, respectively, and poured into Petri dishes (20 cm diameter).The test compound 8 (200 g/10 mm diameter disc) was placed onto the surface of the agar Petri dishes.The antimicrobial potential was expressed as the diameter of the growth inhibition zone in mm [36].

Minimum Inhibitory Concentrations (MICs).
The minimum inhibitory concentration (MIC) of the test compound 8 was evaluated using serial dilutions technique [37].Different concentrations ranging from 50 to 200.0 g for the test compound 8 were dissolved in dimethyl sulfoxide (1 mL) and were placed on filter paper disc (1 cm diameter).The discs were deposited on the surface of inoculated agar plates and kept at low temperature before incubation which favours diffusion over microbial growth to detect the inhibition zone clearly.The plates were incubated at 30 ∘ C for 24 h for bacteria and Candida albicans and for 48 h for Aspergillus niger.

Results and Discussion
3.1.Chemistry.The commercially available piperonal (1) was elaborated to piperonylcarbonitrile (4) as portrayed in Scheme 1.Thus, piperonal (1) was reduced with sodium borohydride in methanol at room temperature to give the piperonyl alcohol (2) [31] in almost quantitative yield.The alcohol 2 was allowed to react with thionyl chloride to give the chloro derivative 3 [32].Subsequently, compound 3 was subjected to nucleophilic substitution using sodium cyanide and a catalytic amount of potassium iodide in dimethylformamide to yield the respective piperonylcarbonitrile (4) [33] in about 75% overall yield.

Crystal Structure of Compound 8.
Single crystal X-ray crystallography is a doubtlessly decisive analytical tool which can confirm the 3D structure of the target compound 8, particularly its S-alkylation.Fortunately, we have succeeded to get single crystals of compound 8 which were suitable for X-ray crystallography (Figure 1).
The asymmetric unit contains three molecules of the title compound 8.The molecular packing in the crystal structure is stabilized by the weak intermolecular interactions (Figure 2, Table 2), of which O3B, O2A, and O4B work as acceptor while N2B, C3A, and C19B work as donors.

In Vitro Antimicrobial Activity.
The in vitro antimicrobial activity of compound 8 was evaluated against Grampositive bacteria (S. aureus, B. subtilis, and B. cereus) and pathogenic fungi (C.albicans and Aspergillus niger).The obtained data, expressed as diameter of the inhibition zone (DIZ) and minimum inhibitory concentration (MIC) for the test compound 8 as well as for the reference antibacterial standard, ampicillin, and the reference antifungal standard, clotrimazole, are shown in Tables 4 and 5.
The preliminary antimicrobial potential of the test compound 8 was evaluated using DIZ assay and the results are given in    against Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa.Minimum inhibitory concentration assay was performed in order to get more insight into the antimicrobial activity of compound 8 as a new antimicrobial pyrimidinone derivative.Compound 8 displayed antimicrobial activity towards the tested bacteria and pathogenic fungi with MIC values in the range of 0.0619-0.1859mol/mL (Table 5).

Figure 1 :Figure 2 :
Figure 1: ORTEP diagram of the title compound 8 drawn at 50% ellipsoids for non hydrogen atoms.

Table 4 .
Compound 8 exhibited good antimicrobial activity with DIZ values in the range of 17-20 mm against the test microorganisms.Compound 8 showed no activity

Table 1 :
Crystallographic data and refinement information.

Table 4 :
Diameter of growth inhibition zone (mm) of the target compound 8, ampicillin, and clotrimazole towards the test microorganisms.The arithmetic mean of the inhibition zone diameters in mean ± standard deviation in mm. *

Table 5 :
The minimum inhibitory concentration (MIC, mol/mL) values for the target compound 8, ampicillin, and clotrimazole towards the test microorganisms.
*The lowest concentration of the test compound that inhibits the growth of microorganism (mol/mL).