Antimicrobial Activity of Pyrimidine-5-carboxylic Acid

Antimicrobial Activity of Pyrimidine-5-carboxylic AcidAntimicrobial activity of synthesized, novel hydroxamic blistery of pyrimidine-5-carboxylic acerb and its difficultes with Cu(II), Ni(II), Co(II) and Zn(II) admixture ionsBhawani Shankar, Rashmi Tomar, Madhu Godhara, Vijay Kumar SharmaABSTRACTFour metal complexes of new hydroxamic acid, 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3) with Cu(II), Ni(II), Co(II) and Zn(II) metal ions have been synthesized. The hydroxamic acid and its metal complexes were characterized by simple analytical techniques such as repeated melting point (M.P.) determination, elemental analysis, running their thin layer chromatography for single spot, and spectroscopic techniques such as I.R., H1-NMR and UV-Vis. (only for metal chelates) spectroscopy. Antimicrobial activity of the hydroxamic acid and their metal complexes were screened against two species of bacteria and two species of fungi by serial publi cation Dilution Method. Metal complexes were found more than active against both bacteria as intimately as fungi in germicide screening test.KeywordsHydroxamic acids, antimicrobial activity, metal complexesINTRODUCTIONHydroxamic acids show a wide spectrum of biological activities and generally have suffering toxicities 1-2. Hydroxamic acids are very well known for their antibacterial 3-5, antifungal 6-7, antitumor 8-9, anticancer 10, antituberculosis 11 and antimalerial 12 properties. Hydroxamic acids are inhibitors of enzymes such as prostaglandin H2 synthatase 13, peroxidase 14, urease 15 and matrix metalloproteinase 16. Cinnamohydroxamic acids are used for manipulation of the symptoms of asthma and other obstructive airway diseases which inhibit 5-lipoxygenase 17. A number of hydroxamic acid analogues have been shown to inhibit DNA (dinucleic acid) synthesis by inactivating the enzyme ribonucleotide reductase (RNR) 18. Naturally occurringhydroxamic acid, 2,4-dihydroxy-7-meth oxy-1,4-benzoxazin-3-one (DIMBOA) is a powerfulantibiotic present inmaize 19. Antiradical and antioxidant properties of hydroxamic acids have also been observed 20. Hydroxamic acids play important role in many chemical, biochemical, pharmaceutical, analytical, and industrial handle 21-25. These diverse biological activities of hydroxamic acids are due to their complexing properties towards transition metal ions 26-27. Siderophores are Fe(III) complexes of naturally occurring hydroxamic acids, involved in the processes of squeeze transport from the environment to the living organisms 28-29. Hydroxamic acids after deprotonation acts as bidentate ligands and octahedral complexes are formed through the co-ordination of two oxygen atom of the CONHO- group. This case of co-ordination have been studied with Cr(III), Fe(III), Ni(II), Co(II) and Zn(II) ions in solid state as well as in solutions, indicating the formation of octahedral complexes 30.We report herein the synthesis, structural features and antimicrobial activity of new hydroxamic acid, 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3) as well as their metal complexes 4a-d with Cu(II), Ni(II), Co(II) and Zn(II) metal salts.EXPERIMENTALReagents and methodsAll chemical used in the present investigation were of analytical reagent grade. 1,3- Di-p-tolylbarbituric acid was synthesized by previously known method in the laboratory. Copper acetate monohydrate, nickle acetate tetrahydrate, atomic number 27 acetate tetrahydrate and zinc acetate dihydrate were purchased from E-Merck. Tri ethyl group aminoalkane and ethyl chloroformate were purchased from Spectrochem. Hydroxylamine hydrochloride cat valium hydroxide and diethyl ether were obtained from S.D. fine chemicals limited, India. All the synthesized compounds were analysed for C, H and N by elemental analyser, model 1108 (EL-III). H1-NMR spectra (400MHz) were recorded on JNM ECX- 400P (Jeol, USA) spectrometer app ly TMS as an internal standard. IR absorption spectra were recorded in the 400-4000 cm-1 range on a Perkin-Elmer FT-IR spectrometer model 2000 employ KBr pallets. UV-Vis. spectra of metal complexes were recorded in DMSO solvent at room temperature on Simadzu Spectro Photometer model no. 1601. Melting points were determined using Buchi M-560 and are uncorrected. These reactions were monitored by thin layer chromatography (TLC), on aluminium plates coated with silica gel 60 F254 (Merck). UV radiation and iodine were used as the visualizing agents.Synthesis of the hydroxamic acid2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3)Synthesis of ligand 3 was carried out in two steps as followsStep 1 Synthesis of ethyl 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylate (2).Ethyl 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylate (2) was synthesized by the reported method of Kuhne et al 31. 1,3- Di-p-tol ylbarbituric acid 5g, 0.016 mol. and triethyl amine 2.30ml, 0.0168 mol. and dimethyl aminopyridine (DMAP) 0.10g were dissolved in 20 ml of dichloromethane (DCM) and the solution was cooled to 00 C. Then ethyl chloroformate 1.60ml, 0.0165 mol. was added drop-wise over half an hour. The mixture was subsequently aflame for 12 hours at 00C, then, allowed to warm to the room temperature for 7 hours. The product is extracted in chloroform and dried over Na2SO4. Further, chloroform was evaporated to dryness and crude product was recrystallised from ethyl alcohol to outlet pure 2.Step 2 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3) from ethyl 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylate (2).Synthesis of 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3) was carried out by adopting a method similar to that exposit by Griffith et al 32. The mixture of hydroxylamine hydroc hloride 1.87g, 0.026 mol. and aqueous potassium hydroxide 2.19g, 0.039 mol. was added drop-wise to a methanolic solution of ethyl 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylate (2) 5g, 0.013 mol. . The solution was stirred at room temperature for 72 hours and then acidified to pH 5.5 using 5% HCl solution. After filtration the solvent was removed in vacuo to yield a solid. The crude product was recrystallised from hot pissing to yield pure compound 3.Synthesis of metal complexesSynthesis of Cu(II), Ni(II), Co(II) and Zn(II) complexes of 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3).Copper acetate monohydrate 0.136g, 0.00068 mol. in cold water was added with stirring to 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3) 0.50 g, 0.00136 mol. in EtOH (20 ml) in a round bottom flask. The contents were stirred for about 6 hours and then reduce to half volume under vacu o. Yellowish brown hasty of 4a was appeared after adding petroleum ether. The precipitate was filtered, washed with small amounts of Et2O and dried over CaCl2 in a vacuum desiccator.Similarly, complexes 4b of Ni(II) , 4c of Co(II) and 4d of Zn(II) with 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3) were synthesized by pickings nickle acetate tetrahydrate, cobalt acetate tetrahydrate and zinc acetate dihydrate respectively.Infrared SpectraIn the IR spectra (Table 1), carbonyl stretching vibrations of hydoxamic acid exhibit a medium sharp intensity pile in the region 1660 cm-1 33. This band has shifted towards negative region 1626-1609 cm-1 in the metal complexes indicating the coordination of the ligand with the metal ion through oxygen of the carbonyl group. The symmetric N-O stretching vibrations, obtained in the region 1120 cm-1 in the IR spectra of ligands, have shifted to lower side in the IR spectra of their metal complexes sugge sting the coordination of ligand to the metal ion through oxygen of the N-O moiety 34. The strawman of water molecules within coordination sphere of all chelates were supported by broad bands in the region 3450-3280 cm-1 and 850-800 cm-1 due to stretching and deformation modes of coordinated water molecules, respectively. The port of new band in the IR spectra of metal chelates in the region 551-519 cm-1 is probable due to formation of M-O bonds 35.Table 1. IR spectral info of hydroxamic acid 3 and its metal complexes 4a-d.Compound(C=O)cm-1(C-N) cm-1(N-O) cm-1(M-O) cm-131660134911204a1609132710365514b1624135510235194c1626138410235404d162913501025541H1-NMR SpectraThe hydroxamic acid 3 shows a one proton singlet at 1.14 due to NH-O proton, probably due to magnetised anisotropy of the neighboring carbonyl group, electronegativity of nitrogen and H- bonding 36. One proton singlet in hydroxamic acid 3 appear at 2.49 due to N-OH proton 37. Due to proton exchange in D2O this signal dis appeared in the spectra indicating the possibility of OH proton. Six protons multiplet for two ArCH3 group protons of hydroxamic acid 3 appear at 2.01 2.09. The hydroxamic acids 3 show a one proton singlet due to C5H proton at 5.26. A multiplet due to eight protons of aromatic rings, Ar-H was observed at 7.17 7.20. H1-NMR of metal complexes 4a-d was not taken due to very less solubility in suitable organic solvents.UV- vis. SpectraCu(II) complexIn the electronic spectra of Cu(II) complex, 4a, three absorption bands in the region. 13157, 16949 and 23809 cm-1 have been observed, which play off to the transitions 2B1g 2A1g, 2B1g 2B2g and 2B1g 2E1g suggesting distorted octahedral geometry 38-40.Ni(II) complexThe electronic spectra of Ni(II) complex, 4b, exhibit three bonds in the region 13333, 16129 and 20833 corresponding to the transitions 3A2g 2T2g(F), 3A2g 3T1g(F), 3A2g 3T1g(P) respectively which show an octahedral geometry for these complexes 41-42.Co(II) complexIn the el ectronic spectra of Co(II) complex, 4c three absorption bands in the region 12903, 14925 and 20200 cm-1 were seen, which may correspond to the transition 4T1g 4T2g(F), 4T1g 4A2g (F) and 4T1g 4T1g(P), respectively, indicating an octahedral geometry 43-44.Zn (II) ComplexNo probative absorption was noticed in Zn(II) complex, 4d, above 400nm probably due to diamagnetic nature and completely filled d- orbitals. In the Zn(II) complex only transitions due to * and n* were seen.Antimicrobial activitySynthesized ligand 3 and metal chelates 4a-d were tested for their antimicrobial activity against two bacteria staph aureus and Escherichia coli and two fungi Aspregillus flavus and Aspergillus niger by adopting Serial Dilution Method 45-46.. The micro-organisms were cultured in nutrient agar medium 46 which was prepared by taking 6.0 gm peptone, 1.50 gm boot extract, 1.0 gm dextrose, 3.0 g yeast extract, 1.50 g agar (for slant) in 1 liter distilled water for bacteria and 10.0g peptone, 20. 0g dextrose, 20.50g agar (for slant) in 1 liter distilled water for fungi. Measured quantities of the test compounds were dissolved in propylene glycol. First set was prepared for primary screening by taking 1ml (2000g/ml) of seeded caudex (obtained by 1100 dilution of the incubated micro-organism broth culture) in 10 well cleaned sterilized test tubes and gradual dilution process was continued for all the ten tubes using a fresh pipette each time. All the above sets of tubes were incubated at 37oC for 24 hours for bacteria and at 28oC for 96 hours for fungi. The Minimum Inhibitory Concentration (MIC) determine were determined at the end of incubation period. Active synthesized compounds, found in the primary screening were further tested for secondary screening by taking 1ml (1500g/ml) of seeded broth against all microorganisms.RESULTS AND DISCUSSIONIn this present work synthesis of 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylic acid hydroxamide (3) was carried out by adding an aqueous solution of hydroxylamine hydrochloride and potassium hydroxide drop-wise to a methanolic solution of ethyl 2,4,6-trioxo-1,3-di-p-tolyl-1,2,3,4,5,6-hexahydropyrimidine-5-carboxylate (2). The solution was continuously stirred for 72 hours at room temperature, which on acidification give crude solid. (Scheme I). Compound 3 on stirring with different metal salts, gave corresponding metal complexes 4a-d (Scheme II).All the metal complexes obtained were solid and stable at room temperature and insoluble in most of the common organic solvents. The spectroscopic and analytical info (Table 2) are in good agreement with theoretical values for the ligand and metal complexes.Table 2. Analytical data and physical properties of the hydroxamic acid 3 and metal complexes 4a-d.S.No.CompdMolecular FormulaColorPercentage Elemental AnalysisCalc./ (Found)M.P. /D.T. (oC)Yield (%)CHN13C19H17N3O5Dark Pink62.12(61.90)4.63(4.52)11.44(11.28)1560C85%24aCu(C19H16N3O5)2.2H20Yel lowish Brown54.87(53.27)4.33(4.30)10.10(9.90)2480C71%34bNi(C19H16N3O5)2.2H20Light Pink55.22(54.70)4.39(4.25)10.13(10.10)2700C70%44cCo(C19H16N3O5)2.2H20Pink55.20(54.70)4.35(4.25)10.16(10.20)3220C75%54dZn(C19H16N3O5)2.2H20Brown54.67(53.80)4.32(4.30)10.07(9.89)3100C70%Antimicrobial ActivityThe newly synthesized hydroxamic acid 3 and its metal chelates 4a-d were tested for their antimicrobial activity against two bacteria Staphylococcus aureus and Escherichia Coli and two fungi Aspergillus niger and Aspergillus flavus. The experimental results of MIC values (Table 3) show moderate activity of all the compounds against both bacteria and fungi. Further, it has been found that the metal complexes were more active than hydroxamic acid. This increased antimicrobial activity of the complexes as compared to the hydroxamic acid is probably due to the fact that chelation increases the lipophilicity of the complexes, which subsequently enhances the penetration through the lipid layer of prison c ell membrane and restricts further multiplicity of the microorganism 46. Among the metal complexes, Cu (II) complex 4a was found most active against both bacteria and fungi. The higher antimicrobial activity of Cu (II) complex may be due to higher stability constant of copper complexes.Table 3. The minimum inhibitory concentration (g/ml) MIC values of hydroxamic acid 3 and their metal complexes 4a-d.S.No.CompoundBacteriaFungiStaphylococcus aureusEscherichia coliAspergillus nigerAspergillus flavus1332532525032524acxxv12525025034b32550050032544c50025025032554d500250250250CONCLUSIONFour new metal chelates, 4a-d with ligand 3 have been synthesized and characterized. Octahedral geometries were proposed for the prepared metal complexes. All the synthesized hydoxamic acids and their metal chelates were screened for antimicrobial activity. A comparative study of the MIC values of the ligand and its complexes show that complexes exhibit higher antimicrobial activity than free ligand. 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