A Series of Barbituric Acid Derivatives from Sulfa Drug: Synthesis and Antimicrobial Activity

This paper reports the synthesis and characterization of some new barbituric acid derivatives from sulfadiazine. A reaction of sulfadiazine with chloroacetyl chloride gave 2-chloro-N-(4-(N-pyrimidin2-ylsulfamoyl) phenyl) acetamide [A] which was reacted with thiourea and K2CO3 to give thiazole derivative [B]. Schiff base compounds [Sh1-Sh3] were prepared from condensation of thiazole derivative with different aromatic benzaldehydes. Then, addition reaction of acetyl chloride to Schiff bases afforded new tertiary amides compounds [D1-D3]. The latter compounds were allowed to react with 1, 3-bis (hydroxyl methyl) barbituric acids derivatives [E1-E2] via Williamson reaction to form new barbituric acid derivatives [F1-F3] and [G1-G3]. Thin layer chromatography, melting points, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (H-NMR) and carbon-13 nuclear magnetic resonance (C-NMR) techniques confirmed formation of the prepared compounds. Antimicrobial studies of the synthesized compounds were assayed against three different types of bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, and against two types of fungi Aspergillus flavus and Candida Albicans. Biological applications of the synthesized compounds showed a greater effect on antimicrobial activities than the standard.


Introduction
S u l f a d i a z i n e ( 4 -a m i n o -N -p y r i m i d i n -2 -y lbenzenesulfonamide) is a compound that contains two reactive locales, One of these aromatic amine and the other is sulfonamide [1].It is a drug that belongs to the sulfonamide group and has been utilized in veterinary and human therapy over sixty years [2], and in the treatment of urinary tract infections (UTI) [3].Thiazole is a main structure for an important class of nitrogen (N) and sulfur (S) containing heterocycles, in particular [4], The univalent radical is known as thiazolyl.The molecule of thiazole is planar, and the C-S bond length equals 171.3 pm, similar to that in thiophene compound [5].The applications of thiazoles were found in drug development for the treatment of hypertension, allergies, schizophrenia, inflammation, bacterial, HIV infections, and hypnotics and in treatment of pain [6].
Schiff bases are formed when any primary amine reacts with an aldehyde or a ketone in absolute alcohol with the presence of a few drops of glacial acetic acid or p-toluene sulfonic acid or concentrated hydrochloric acid [7].These compounds also have applications in food chemistry, agro chemical, dye industry and pharmaceuticals [8].Barbituric acid was prepared by Adolf Von Baeyer in 1864 from a fusion of the urea and malonic acid [9].It is an organic compound based on a pyrimidine heterocyclic skeleton [10].Barbituric acid is the parent compound of barbiturate drugs, although barbituric acid itself is not pharmacologically active [11], and the pharmacological properties of barbiturates mainly depend on the side groups attached to the C-5 atom of the pyrimidine ring [12].A number of 5-alkyl-and aryl barbiturates are used as hypnotic, sedative, anticonvulsant, and antihypertensive drugs [13].
This research involved the synthesis and characterization of some new barbituric acid derivatives from sulfadiazine and the study of their antimicrobial activities.

Experimental
All reagents and solvents were purchased from commercial sources and used without purification.Melting points were recorded using electro thermal melting point apparatus.Fourier transform infrared spectroscopy (FTIR) spectra were recorded using Shimadzu FT.IR-8400S infrared spectro-photometer by KBr disc, Kufa University.Proton nuclear magnetic resonance ( 1 H-NMR) and carbon-13 nuclear magnetic resonance ( 13 C-NMR) were recorded by Bruker spectrometer, operating at 300 and 400 MHz for 1 H-NMR and 75 MHz for 13 C-NMR with dimethyl sulfoxide-d6 (DMSO-d6), at Abu Ali Center Lab, Mashhad, Iran.Thin-layer chromatography (TLC) was performed on aluminum plates and coated with the layer of 0.25 mm silica gel; compounds were detected by iodine vapor.Autoclave was used to sterilize agar media, supplied by Prestige Medical-England.Incubator was used to maintain different temperatures required for the growth of organism, supplied by Memert-Germany.[14] To a stirred mixture of sulfadiazine (0.01 mol, 3 g) and trimethylamine (1.67 mL) in dimethylformamide (DMF) as solvent.Chloroacetyl chloride (0.01 mol, 0.95 mL) was added drop-wise by micropipette.After the addition was completed, the mixture was stirred for 3 hours without heating.Finally, the solvent was evaporated and the precipitate was filtered, dried and washed with distilled water and ether to generate chloro-N-(4-(N-pyrimidin-2-ylsulfamoyl) phenyl) acetamide [A].The crystalline precipitates were recrystallized with ethanol.Yield pale yellow = 87%, melting point (m.p.) = 220-222 °C, and retardation factor R f = 0.75.The TLC for the reaction was completed by using benzene : methanol at 4 : 1.

General synthesis of tertiary amide derivatives [D 1 -D 3 ] [17]
In this step, acetyl chloride solution (0.01 mol) in 10 mL dry benzene was added dropwise to 0.01 mol Schiff bases [Sh 1 -Sh 3 ] dissolved in 20 mL dry benzene.The reaction mixture was stirred for 3 hours, without heating.The product obtained after the evaporation of the solvent was filtered, washed with solution of sodium carbonate of 2% and re-crystallized from hot ethanol (Table 2).Scheme 3 shows the general synthesis of tertiary amide derivatives [D 1 -D3].

Test of biological activity
The synthesized compounds and [G 3 ] were tested for their in-vitro antibacterial activity against three types of bacteria, Staphylococcus aureus as a Gram-positive bacterium, Escherichia coli and Pseudomonas aeruginosa as Gram-negative bacteria taking standard gentamicin and penicillin.The antibacterial activity was performed by filter paper disc plate method at concentrations of 0.03 and 0.06 μg/ mL using Muller Hinton agar medium, and dimethyl sulfoxide (DMSO) was used as a solvent control.While the antifungal activity for the same compounds was tested against Candida albicans and A. flavus by using filter paper disc plate method and potato dextrose agar (PDA), fluconazole, voriconazole and nystatin were used as standards for the antifungal activity

Results and Discussion
All the compounds were insoluble in water but soluble in organic solvents as DMF and DMSO.Some synthesized compounds were colored and stable by resonance and having high melting points relatively, which was another evidence on the extent stability.In this study, a series of new barbituric acid derivatives containing thiazole moiety were synthesized from sulfa drug, Sulfadiazine was the starting material for this research, It was converted to the 2-chloro-N- coli and Pseudomonas aeruginosa as Gram-negative bacteria by using Muller Hinton agar medium.The prepared compounds were measured for zone of inhibition around each disc.Solvent control (DMSO) did not inhibit bacteria or fungi.The test results are presented in Table 5 and Fig. 19.
Table 5 shows that [B] and [G 1 ] were highly active against S. aureous, P. aeruginosa and E.coli at 0.03 and 0.06 mg/mL.Compound [F 2 ] was highly active against P. aeruginosa and E. coli at 0.06 mg/mL, and it was moderately active against them at 0.03 mg/ mL, while it was inactive against S. aureous for both concentrations.
Compound [G 2 ] was highly active against S. aureous and E. coli at at 0.06 mg/mL, and it was moderately active against them at 0.03 mg/mL, while it was inactive against P. aeruginosa.
Antifungal activity of the synthesized compounds was tested against two types of fungi, Candida albicans and Aspergillus flavus by using potato dextrose agar (PDA) medium.The test results are presented in Table 6 and Fig. 2. Compounds [B], [G 1 ] and [G 2 ] were highly active against Candida albicans at 0.03 and 0.06 mg/mL, while compound [F 1 ] and [F 2 ] were inactive against the same fungus.
In Aspergillus flavus, compounds [B] and [G 2 ] were inactive at 0.03 and 0.06 mg/mL, while [F 1 ] and [G 1 ] were moderately active at 0.03 and 0.06 mg/mL.Compound [F 2 ] was moderately active at 0.03 mg/mL and highly active at 0.06 mg/mL.

Conclusions
In this study, the derivative compounds of barbituric acid from sulfa drugs were stable by resonance with high melting points relatively.They had good antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus.

Table 1
Physical properties of Schiff bases Note: M.p. = Melting point

Table 5
Antibacterial activity of some synthesized compounds

Table 6
Antifungal activity of some synthesized compounds