The synthesis and antimicrobial activity of novel Zn(II) metal complexes derived from three novel heterocyclic Schiff base ligands 8-[(
The treatment of infectious diseases still remains an important and challenging problem because of various factors like emerging infectious diseases and the increasing number of multidrug resistant microbial pathogens. In recent years, bacterial resistance to antibiotics has been a matter of great concern. Antibiotic resistance is the ability of bacteria or other microbes to resist the effects of an antibiotic. Antibiotic resistance occurs when bacteria change in some way that reduces or eliminates the effectiveness of drugs designed to cure or prevent infections. The bacteria survive and continue to multiply causing more harm [
Due to increasing resistance of these bacterial strains, effective antibacterial medicines like Vancomycin, Ciprofloxacin, Methicillin, and so forth become less effective in treatment of diseases caused by such infections. Over the past several decades, the incidence of resistant Gram positive organisms has risen in the world. Methicillin, a resistant
To overcome such challenges in treating patients with infections of such antibacterial resistant strains, new antimicrobial agents, that is, new medicines, need to be researched and continuous efforts are necessary to explore small molecular structures as new medicines. A lower molecular weight cutoff of 500 Daltons (as part of Lipinski’s “rule of five”) [
It is well known that the cost of developing a new medicine, that is, new chemical entity, is enormous and takes many years to develop the same due to prolonged biological safety studies and human clinical trials. It also takes a lot of research and development efforts to develop multistep synthesis process and scale up of complex molecules. The number of chiral centres in a molecule also increases its cost to develop and time to market. Hence, new cost-effective, shorter routes of synthesis and relatively small molecules are a need of hour in new chemical entity research [
The antimicrobial properties of metals have been recognised for centuries and have represented some of the most fundamental breakthroughs in medicinal history [
In order to begin our efforts for such new medicines as effective anti-infective agents against bacteria and fungi, we thought of combining heterocyclic aniline scaffold with simple ortho hydroxy benzaldehydes like salicylaldehyde to get a Schiff base and its conversion to transition metal complex like Zn(II), Cu(II), Ni(II), and Co(II). In our initial efforts, to screen compounds derived from coumarin scaffold and aliphatic diamino compound like N,N-dimethyl ethylene diamine, we got encouraging results with respect to biological assays against Gram positive bacteria and fungi [
This diverted our focus to search for new molecular structures having less complex structure and few synthesis steps. We thought of heterocyclic aniline scaffolds and condensed with salicylaldehyde to get corresponding Schiff base and then complexation with zinc metal. Schiff bases were synthesised, isolated, and characterised. Zn(II) complexes were prepared by template method and characterised. Schiff bases and their corresponding Zn(II) metal complexes were evaluated for antibacterial and antifungal activities by MIC method.
All chemicals and solvents used in this work were of analytical grade. Salicylaldehyde was purchased from Merck Chemicals. Zinc chloride, DMSO, and oxalic acid were purchased from SD Fine chemicals.
7-hydroxy-4-methyl-2-oxo-2
Synthesis of Schiff base 7-hydroxy-4-methyl-8-[(
The Schiff base, that is, the ligand 8-[(
As the oily Schiff base was unstable in nature, it was difficult to characterize the compound. Therefore, its oxalate salt was prepared for spectral characterization.
4-Methyl-7-hydroxy 8-formyl coumarin (1.0 g, 0.0049 mole) was dissolved in 10 mL ethanol and N-methylpropane-1,3-diamine (0.431 g, 0.0049 mole) was added. A drop of concentrated hydrochloric acid was added and the reaction mixture was refluxed for an hour. Oxalic acid (0.555 g, 0.0041 mole, 0.9 eq.) was added and further refluxed for an hour. On cooling, the product was isolated as oxalate salt which was recrystallized from alcohol. The product was filtered and dried in oven till constant weight. Weight: 1.1 g, (yield: 70%). Colour: yellow, M.P. 205–207°C, elemental analysis observed (calculated): C 56.4% (56.02%), H 5.53% (5.69%), N 7.69% (7.22%), UV:
Synthesis of Zn(II) complex of Schiff base 7-hydroxy-4-methyl-8-[(
The preparation of the Zn(II) complex was carried out by taking 7-hydroxy-4-methyl-2-oxo-2
Synthesis scheme of Schiff base TMPIMP and complex [Zn(TMPIMP)2]·2H2O.
4-(1
4-(1
4-(1
Synthesis scheme of Schiff base ligand HBABO and complex [Zn (HBABO)2]·2H2O.
(4
(4
(4
IR (KBr):
13C NMR (DMSO-d6, 75 MHz) 163.04 (–C=N azomethine), 160.26 (oxazolidone –C=O), 158.60 (–C–O phenolic), 146.43, 135.48, 133.18, 132.53, 130.49, 121.36, 119.10, 116.55, 67.97, 52.42, 39.90.
All the metal complexes are stable at room temperature and are nonhygroscopic in nature. On heating, they decompose at high temperatures. The complexes are insoluble in water but are soluble in DMSO. The elemental analysis, physical properties, and analytical data of the ligand and complexes are summarized below.
Due to the diamagnetic nature of Zn(II) metal complexes, it was possible to scan 1H NMR spectrum in DMSO-d6. Diamagnetic zinc metal complexes do not interfere in magnetic field of NMR instrument; however, paramagnetic metal complexes interfere and it is not possible to lock NMR instrument for scanning samples.
It was observed that the azomethine proton in [Zn(NMAPIMMTC)2]·2H2O complex appeared at 8.83 ppm after complexation with zinc metal. It was shifted significantly downfield due to deshielding effect exerted by zinc metal atom. Apart from the downfield shift of azomethine, following other interesting observations were also made. Aromatic protons of coumarin ring were observed at 6.56 ppm and 7.56 ppm as doublets due to the electron withdrawing mesomeric effect exerted by central zinc metal atom. Olefinic proton of coumarin ring was also shifted downfield to 5.98 ppm due to electron withdrawing mesomeric effect operating through the conjugation across the aromatic ring over the
The azomethine proton in [Zn(TMPIMP)2]·2H2O complex was observed at 8.63 ppm and that of [Zn(HBABO)2]·2H2O was observed at 8.94 ppm.
Thus, the azomethine H–C=N protons which appeared at about 8.83 to 9.12 in free Schiff bases were shifted to downfield to 8.63 to 8.94 due to electron withdrawing effect of central metal atom (see Figure
Azomethine proton shift values before and after complexation with Zn(II) metal atom.
The azomethine carbon atom appeared most downfield as reported in literature values. In [Zn(NMAPIMMTC)2]·2H2O complex, it was observed at 162.73 ppm, in [Zn(HBABO)2]·2H2O complex, it was observed at 163.04 ppm, and, in [Zn(TMPIMP)2]·2H2O complex, it appeared at 163.70 ppm (Table
13C NMR assignments of metal complexes.
Atom number | Group | 13C ppm | |
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|
2, 2′ | C | 162.733 |
3, 3′ | CH | 107.248 | |
4, 4′ | C | 106.985 | |
4a, 4a′ | C | 106.322 | |
5, 5′ | CH | 130.107 | |
6, 6′ | CH | 120.355 | |
7, 7′ | C | 154.867 | |
8, 8′ | C | 156.861 | |
8a, 8a′ | C | 160.229 | |
9, 9′ | C | 173.636 | |
10, 10′ | CH2 | 58.48 | |
11, 11′ | CH2 | 33.582 | |
12, 12′ | CH2 | 47.342 | |
13, 13′ | CH3 | 27.064 | |
14, 14′ | CH3 | 18.892 | |
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14, 14′ | HC=N | 163.04 |
5, 5′ | C=O | 160.26 | |
20, 20′ | C–O–Zn | 158.60 | |
10, 10′ | C | 146.43 | |
7, 7′ | C | 135.48 | |
16, 16′ | CH | 133.18 | |
18, 18′ | CH | 132.53 | |
8, 8′, 12, 12′ | CH | 130.49 | |
9, 9′, 11, 11′ | CH | 121.36 | |
15, 15′ | C | 119.27 | |
19, 19′ | CH | 119.10 | |
17, 17′ | CH | 116.55 | |
2, 2′ | CH2 | 67.97 | |
3, 3′ | CH | 52.42 | |
6, 6′ | CH2 | 39.90 | |
|
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|
14, 14′ | HC=N | 163.70 |
20, 20′ | C–O–Zn | 160.23 | |
2, 2′ | CH | 151.44 | |
10, 10′ | C | 147.82 | |
5, 5′ | CH | 144.20 | |
7, 7′ | C | 134.69 | |
16, 16′ | CH | 133.37 | |
18, 18′ | CH | 132.52 | |
8, 8′, 12, 12′ | CH | 129.12 | |
9, 9′, 11, 11′ | CH | 121.60 | |
15, 15′ | C | 119.24 | |
17, 17′ | CH | 119.16 | |
19, 19′ | CH | 116.58 | |
6, 6′ | CH2 | 51.77 |
Normally carbon attached to phenolic –OH group appears at about 155 ppm, but, in these complexes, it was observed at 155–160 ppm which may be due to electron deshielding effect of zinc metal atom.
In [Zn(NMAPIMMTC)2]·2H2O, lactonyl carbon appeared at 173.63 ppm, and, in [Zn(HBABO)2]·2H2O complex, the oxazolidinone carbonyl carbon appeared at 160.26 ppm.
The formation of Schiff bases is confirmed by the presence of intense molecular ion peak in the mass spectra of Schiff base metal complexes such as [Zn(NMAPIMMTC)2]·2H2O, [Zn(HBABO)2]·2H2O, and [Zn(TMPIMP)2]·2H2O. Other prominent peaks may be due to the elimination of CH3NH, –CH2–CH2–CH2– units of propyl side chain in case of [Zn(NMAPIMMTC)2]·2H2O. In other complexes, prominent peaks may be due to the fragmentation of heterocyclic rings in the molecules.
Some other peaks may be due to loss of tropylium ion and so forth from the parent ion and subsequent fragmentation. The mass spectra of the Zn(II) complexes showed molecular ion peaks corresponding to [M(L)2] stoichiometry. Peaks corresponding to L+ and fragments of L+ are also present in the spectra. Detection of [M+] and [M+1]+ peaks in mass spectra indicated and confirmed Zn : (L)2 stoichiometry of the complexes.
The interpretation of IR spectra provides valuable information regarding the nature of functional group attached to the metal atom and helped in confirmation of bond formation. In order to study the bonding mode of Schiff base ligand to the central metal atom, the IR spectra of the free ligands were compared with the spectra of the complexes. The main IR bands and their assignments are listed in Table
FT-IR bands for metal complexes and their assignments.
Complex | Lattice water |
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3122 | 1727 (lactone) | 1631 | 1371 | 543 | 453 |
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3282 | 1750 (Oxazolidinone) | 1625 | 1446 | 530 | 449 |
|
3312 | NA | 1619 | 1452 | 522 | 447 |
The Schiff base HBABO has an oxazolidinone –N–C=O functional group and it has been observed as carbonyl stretching band at 1737 cm−1 in [Cu(HBABO)2]·2H2O complex and at 1750 cm−1 in [Zn(HBABO)2]·2H2O complex. All the above metal complexes have also shown absorption bands in the region 3400–3500 cm−1 due to coordinated water molecules Table The phenolic –OH band does not appear in metal complexes spectra. However new bands have appeared at 1621 cm−1 to 1639 cm−1 due to new –C=N, that is, azomethine double bond, which is characteristic of Schiff base and confirms the formation of Schiff bases and further complexation with central metal atom. The IR spectra of all the metal complexes show prominent band at about 1240–1280 cm−1 due to There are no prominent bands appearing in the 1600–1800 cm−1 region of the spectra indicating participation of the azomethine nitrogen and phenolic oxygen atom in coordination with the metal atom [ The broad signals in the region of 2500 cm−1 to 3500 cm−1 of the Schiff base ligands disappeared in the spectra of all the metal complexes indicating complexation with central metal cation. However, the spectra of the metal complexes in this region show a number of signals arising from The low frequency region of the spectra indicated the presence of two new medium intensity bands at about 450 cm−1 to 470 cm−1 due to
Thermogravimetric analysis showed a loss of about 5.5% in weight corresponding to weight of two water molecules from the compound. This is water coordinated to central metal atom. Further heating resulted in continuous loss in weight with rise in temperature indicating decomposition of samples above 250°C.
It is clear from the data presented above that the experimental values of each compound are in good agreement with the theoretical values calculated for 1 : 2 ratio of metal : ligand stoichiometry. This is confirmed by M+ and [M+1] peaks in high resolution mass spectra.
From the discussion of the results of various spectroscopic details presented above, it may be concluded that the proposed geometry for the transition metal complexes with general formula ML2·2H2O is octahedral for Zn(II) complexes. The probable structures are shown in Table
Proposed structure of
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Antimicrobial activity of the Schiff base ligand and its metal complexes was screened against two Gram negative bacteria:
The following ATCC strains were procured from Institute of Microbial Technology, Chandigarh, India:
Inoculum used was matched with 0.5 Mac Farland standard, that is, equal to 3 × 105 CFU/mL.
DMSO was used as solvent control. The solvent DMSO had no antimicrobial effect at the concentrations employed. DMSO used was commercially available. 10 mg of the test compound was dissolved in 1 mL of DMSO and this solution was used as stock solution for the test.
Nine dilutions of each drug were done with brain heart infusion (BHI) for MIC. In the initial tube, 20
Ciprofloxacin and Fluconazole were used as standards. Microbroth dilution method was used for the standard drugs.
Antifungal activity was carried out in a biosafety cabinet to avoid the contamination.
A comparative study of MIC values of Schiff base and its complexes indicated that metal complexes exhibit higher antimicrobial activity than the free Schiff base ligands and the same is indicated from the results given in Table
Showing comparative antibacterial and antifungal screening results by MIC method.
Test compounds | Test organism and sample concentration in |
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NMAPIMHMC.oxalate | 50 | 50 | 50 | 50 | 0.8 |
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50 | 50 | 12.5 | 3.12 | 0.8 |
TMPIMP | 100 | 50 | 12.5 | 50 | 1.6 |
|
100 | 100 | 6.25 | 3.12 | 3.12 |
HBABO | 100 | 100 | 6.25 | 12.5 | 6.25 |
|
100 | 100 | 12.5 | 25 | 3.12 |
Standard Ciprofloxacin | 2 | <4 | 2 | — | — |
Standard Fluconazole | — | — | — | 16 | 8 |
There was no promising antibacterial activity observed against Gram negative bacteria, that is,
The sensitivity of the test organisms to the test compounds may also be associated with cell wall structure. The major role of action involves highly specific coordination of metal ion to thiol groups on proteins containing L-cysteine [
However, in case of
In case of antifungal activity against
However, in case of antifungal activity against
In almost all the comparative studies done, metal complexes showed enhanced activity compared with Schiff base ligand. These observations are due to heterocyclic rings of coumarin moiety, triazole heterocyclic ring, and oxathiazolidinone heterocyclic ring incorporated in the molecular structure of the metal complexes. These structural scaffolds might interfere in the mechanism of cell multiplication as discussed above and hence stop further growth of fungus.
It is known that chelation tends to make the ligand act as more powerful and potent bacterial agent. This may be probably due to the greater lipophilic nature of the complexes. Such increased activity of the metal chelates can be explained on the basis of chelation theory [
According to yet another plausible mechanism, these complexes might be inhibiting DNA gyrase enzyme, which is responsible for DNA multiplication phases. Since DNA gyrase is inhibited by metal complexes, multiplication of bacterial cells is stopped, ultimately resulting in antibacterial activity [
Three novel Schiff bases 8-[(
The physical and spectral analytical data show that the metal ligand stoichiometry in all these complexes is 1 : 2. The spectral data show that the ligand is bidentate which coordinates through the azomethine nitrogen of Schiff base ligand and oxygen atom of salicylaldehyde fragment. Based on analytical and spectral data, all these complexes are assigned to be in octahedral geometry.
Some of the Zn(II) metal complexes have shown significant antifungal activities compared to its Schiff base ligand and moderate antibacterial activity. Schiff base Zn(II) metal coordination complexes can be used not only as an approach to enhance their activity but also to overcome the drug resistance.
In conclusion, the “
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank Dr. Kishore Bhat of Governmental Dental College, Belgaum, for facilitating antimicrobial assays and providing the procedure for the same. They also thank Dr. Ranjan Das and Ajay Patil of TIFR, Mumbai, Dr. Moneesha Fernandes of NCL, Pune, and Dr. Avinash Kumbhar of Department of Chemistry, University of Pune, for supporting with 1H and 13C NMR spectra; without these supports, this work would not have been complete. They also thank the management of Patkar-Varde College, Goregaon (W), Mumbai, for constant encouragement throughout this work.