Antimicrobial activity of synthesized, novel hydroxamic acid of pyrimidine-5-carboxylic acid and its complexes with Cu(II), Ni(II), Co(II) and Zn(II) metal ions

Bhawani Shankar, Rashmi Tomar, Madhu Godhara, Vijay Kumar Sharma

ABSTRACT

Four 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 Dilution Method. Metal complexes were found more active against both bacteria as well as fungi in antimicrobial screening test.

Keywords

Hydroxamic acids, antimicrobial activity, metal complexes

INTRODUCTION

Hydroxamic acids show a wide spectrum of biological activities and generally have low 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 treatment 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-methoxy-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 fields 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 iron 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 type 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.

EXPERIMENTAL

Reagents and methods

All 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, cobalt acetate tetrahydrate and zinc acetate dihydrate were purchased from E-Merck. Triethyl amine and ethyl chloroformate were purchased from Spectrochem. Hydroxylamine hydrochloride potassium 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 using 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 using 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 acid

2,4,6-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 follows:

Step 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-tolylbarbituric 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 stirred 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 yield 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 described by Griffith et al 32. The mixture of hydroxylamine hydrochloride 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 water to yield pure compound 3.

Synthesis of metal complexes

Synthesis 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 vacuo. Yellowish brown precipitate 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 taking nickle acetate tetrahydrate, cobalt acetate tetrahydrate and zinc acetate dihydrate respectively.

Infrared Spectra

In the IR spectra (Table 1), carbonyl stretching vibrations of hydoxamic acid exhibit a medium sharp intensity band 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 suggesting the coordination of ligand to the metal ion through oxygen of the N-O moiety 34. The presence 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 appearance 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 data of hydroxamic acid 3 and its metal complexes 4a-d.

H¹-NMR Spectra

The hydroxamic acid 3 shows a one proton singlet at 1.14 due to –NH-O proton, probably due to magnetic 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 disappeared in the spectra indicating the possibility of –OH proton. Six protons multiplet for two Ar–CH₃ group protons of hydroxamic acid 3 appear at 2.01 – 2.09. The hydroxamic acids 3 show a one proton singlet due to –C₅–H 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. Spectra

Cu(II) complex

In the electronic spectra of Cu(II) complex, 4a, three absorption bands in the region. 13157, 16949 and 23809 cm^-1 have been observed, which correspond to the transitions ²B_1g → ²A₁g, ²B_1g → ²B_2g and ²B₁g → ²E₁g suggesting distorted octahedral geometry 38-40.

Ni(II) complex

The electronic spectra of Ni(II) complex, 4b, exhibit three bonds in the region 13333, 16129 and 20833 corresponding to the transitions ³A_2g → ²T_2g(F), ³A_2g → ³T_1g(F), ³A_2g → ³T_1g(P) respectively which show an octahedral geometry for these complexes 41-42.

Co(II) complex

In the electronic 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 ⁴T_1g→ ⁴T_2g(F), ⁴T_1g­ → ⁴A_2g (F) and ⁴T_1g → ⁴T_1g(P), respectively, indicating an octahedral geometry 43-44.

Zn (II) Complex

No significant 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 activity

Synthesized ligand 3 and metal chelates 4a-d were tested for their antimicrobial activity against two bacteria Staphylococcus 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 beef 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 (2000µg/ml) of seeded broth (obtained by 1:100 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 37^oC for 24 hours for bacteria and at 28^oC for 96 hours for fungi. The Minimum Inhibitory Concentration (MIC) values 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 (1500µg/ml) of seeded broth against all microorganisms.

RESULTS AND DISCUSSION

In 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 data (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.

Antimicrobial Activity

The 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 cell 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 (µg/ml) MIC values of hydroxamic acid 3 and their metal complexes 4a-d.

CONCLUSION

Four 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. Among the metal complexes, Cu (II) complex 4a was found most active against both bacteria and fungi.

ACKNOWLEDGEMENT

One of the authors Ms. Rashmi Tomar is grateful to UGC, Bahadur Shah Zafar Marg, New Delhi, for providing fellowship.

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