Synthesis, Structure Delineation and Antibacterial Activity Study of Metal (II) Complexes of Schiff Base Derived from Kanamycin and Methyl Ester of Amoxicillin

A novel Schiff base ligand was synthesized from Kanamycin and methyl ester of Amoxicillin in stoichiometric ratio. Three metal chlorides viz. CuCl2.2H2O, CoCl2.6H2O and ZnCl2 were used for the synthesis of respective metal complexes from synthesized novel ligand. The proposed structure of the complexes have been established by various microanalytical and spectral techniques like elemental analysis, TG/DTA studies, conductivity measurement, IR, H NMR, Electronic and mass spectrometry. Various shifting in the band positions in IR spectra of the complexes suggested coordination of metal ions through azomethine N and O-atoms of the novel ligand. Spectral studies concluded pentadentate nature of ligand. The correct stereochemistry of the complexes was optimized by MM2 calculations programmed in CsChem3D Ultra-11 software. The crystal Original Research Article Chaudhary and Mishra; IRJPAC, 7(4): 165-180, 2015; Article no.IRJPAC.2015.065 166 structures of the complexes have been determined by X-ray powder diffraction techniques. The particle size calculation by Scherrer's formula suggested nanocrystalline nature of the complexes. The Coats-Redfern equation was used to calculate the thermal decomposition kinetic parameters of the metal complexes. Non-spontaneous decomposition of the complexes has been suggested by positive ΔG and negative ΔS values. The pharmacological potentiality of the Schiff base ligand and its metal complexes were assayed in vitro by Kirby Bauer paper disc diffusion method against four bacterial pathogens viz. S. aureus, E. coli, B. subtilis and K. pneumoniae. The results of these studies revealed moderate to better antibacterial potency of the complexed Schiff base against uncomplexed.


INTRODUCTION
Aminoglycosides and β-lactam antibiotics are considered the new compounds for the formation of Schiff base which is the condensation product of active carbonyl group and amine. Azomethine linkage (-CH=N-) along with many potential donor atoms in it provides uniqueness in coordination chemistry. Moreover, the multifunctional applications of transition metal complexes of Schiff base viz. antibacterial, [1,2] antitumor, [3][4][5] antifungal, [6,7] catalytic [8][9][10] & luminescence [11][12][13] properties etc. have spurred the interest of coordination chemists to develop new type of compounds with superior biological functions and drew the researcher's intelligence in the field of chemical and medical sciences.
Recently, Schiff bases have considered as a privileged ligand, as most of the bio-molecules in the living system are structurally similar to this class of compound, so more attentions have to be paid in its structural design and synthesis [14]. The abundance of potential anchoring sites in this ligand, due to presence of various donor atoms viz. N, O and S etc., make easy in the coordination process with metal ions of various types at different oxidation states, which form stable chelate complexes [15]. πelectrons sharing in conjugation with metal ions in the ring of metal complexes authenticate stability of the complexes [16,17].
Kanamycin, in complex form has three component units: Kanamycin A [major component], B and C. The disulphate of Kanamycin A, at its low concentration is a broad spectrum antibiotic, very actively used to treat the infections caused by many gram-positive bacteria. In unmodified form, Kanamycin has shown severe toxic effects like nephrotoxicity and ototoxicity which results in kidney failure and irreversible loss of hearing respectively [18,19]. The four amine groups in Kanamycin A, in combination with seven hydroxyl groups serve it as a better substrate to obtain semi synthetic and improved antibiotics. Amine groups in sugar rings of Kanamycin A, not only provide anchoring sites for the metal ions but also serve as the reactive site for the formation of Schiff base by the condensation with active carbonyl groups. Besides four amine groups of Kanamycin, amine-1 is best-fit search for ligand formation that has been supported by energy optimization calculation through MM2 software and minimum steric structural effect. Amoxicillin is a class of βlactam antibiotic, widely used in clinical therapy for the treatment of severe infections caused by various gram-negative bacteria. The biofunctional activity of amoxicillin is related to lactam ring that inhibits bacterial growth by proteolysis mechanism. Many of this class of drugs are under bacterial resistance because of anti-chemical transformation by forming βlactamase enzyme, which are responsible for failures of antimicrobial therapy by hydrolyzing βlactam ring [20]. Several research papers revealed that classic drugs used to treat bacterial pathogens have become outdated.
So, in order to address their therapeutic failures and serious limitations, their structural design in derived form has considered as a topic of research [21]. In the present investigation, we report here the synthesis, spectral characterizations coordination behavior and antibacterial activity of M(II) complexes of novel Schiff base ligand derived from Kanamycin and esterifies Amoxicillin, in order to study their combined antibacterial activities against different species of bacteria. The chemical structures of these antibiotics are given in the Figs

Physical Measurements
The elemental microanalysis (C, H and N)  . The XRD powder pattern was recorded on a vertical type Philips PW 1130/00 x-ray diffractometer, operated at 40kV and 50 Ma generator using the monochromitised Cu Kα line at wavelength 1.54056 Å as the radiation source and measurements were taken over the range of 2θ =10 -80º. Crystallographic data were analyzed by CRYSFIRE and CHECKCELL software programme. The molecular structure of the complexes was optimized by CsChem 3D Utra-11 programme.

Synthesis of Methyl Ester of Amoxicillin (MEA)
The compound MEA was prepared by mixing amoxicillin trihydrate (

Synthesis of Novel Schiff Base Ligand [H 2 L]
To 4 mmol (2.33 g) of Kanamycin sulphate in 30 ml warm and homogenously stirred aqueous methanol (1:1), was added a well stirred and hot solution of 4 mmol (1.5 gm) methyl ester of Amoxicillin (MEA) dissolved in 30 ml distilled methanol, as reported in the previous literatures [22]. The pH 7 of the solution was adjusted by adding few drops of NaOH solution. The mixture was stirred under heat on magnetic stirrer and was refluxed for 15 hrs at 35ºC. The light yellow solid was obtained by reducing its volume by placing over the hot plate. It was further recrystallized and dried under vacuum over anhydrous CaCl 2, Yield 70% and m. p. 180ºC.

Synthesis of Metal Complexes of Schiff Base [ML]
To the hot and magnetically stirred 50 ml (1:1) aqueous methanolic solution of novel Schiff base ligand (2 mmol, 1.662 gm), was added 5 ml (2 mmol) methanolic solution of metal chloride salts. The mixture was then stirred and refluxed alternately at 45ºC for another 15 hrs. On cooling, the precipitates of metal complexes of different color were obtained, filtered and washed with methanol. The complexes were purified by re-crystallization from DMSO/H 2 O mixture. Yield 55-65%, m. p. > 275ºC. The synthesis of the ligand and its metal complexes is given in Scheme 1.

Antibacterial Study
In vitro antibacterial potency of newly synthesized novel Schiff base and metal complexes were assayed by Kirby Bauer paper disc diffusion method, [23,24] using Mueller-Hinton's nutrient agar media. The pH was maintained at 7.4 for better bacterial growth. Several well isolated colonies of the fresh cultures of standard bacteria viz. E. coli, B. subtilis, S. aureus and Klebsiella pneumoniae were collected from microbiology laboratory of Suraksha Prashuti Hospital, Biratnagar, Nepal and inoculated in 5 ml of tryptone soya broth. The broth was incubated for 5 hrs at 37ºC until there was visible growth shown by opaqueness. The broth was spread over nutrient agar media, prepared as usual in Petri plates using stick swab and the well sterilized Paper discs of 6 mm diameter (Whatman no. 1) impregnated with test compounds at the concentration of 10 μg/μl in DMSO were stuck on the previously seeded bacterial culture [25,26]. Gentamicin 30 μg per disc of 6 mm size (HIMEDIA co.) was used as standard (+ve control). DMSO, which exhibited no antimicrobial activity against the test bacterial pathogens, was used as negative control. Afterwards, the Petri plates were incubated at 37ºC and the diameter of zone of inhibition around each disc was measured by using antibiogram zone measuring scale after 48 hrs of incubation.

RESULTS AND DISCUSSION
Presence of -COOH group in amoxicillin might interfere in Schiff base formation that can interact with amine group of Kanamycin, so this reaction of amide formation was inhibited by the formation of MEA. Esterifies amoxicillin now safely interact with amine group of Kanamycin. The ligand was soluble in hot methanol and polar solvents like DMSO and DMF. The melting points of ligand (180ºC) and metal complexes (> 275ºC) suggest that the complexes are much more stable than the ligand and all of them are air and moisture stable. Different coloured metal complexes were obtained by synthetic routes proposed in Scheme-1. IR spectral measurements are completely in consistent with the proposed formulation of the ligand and complexes. The metals are bound to the ligand through the azomethine nitrogen, N-atom of amine-1 and cyclic O-atom of Kanamycin moiety and amide N and O-atoms of Amoxicillin moiety, as evidenced by negative shift in band positions of such groups due to sharing of electrons towards the metal centers. IR spectral data are presented in the

Scheme 1. Proposed route for the synthesis of Schiff base ligand & metal complex
One H 2 O molecule coordinated with metal center in the inner sphere of the crystal system satisfies the proposed crystal structure of the complexes.
XRD measurements revealed the crystalline nature of complexes with particle size in nanometer range, so called nano-structured molecules. Thermograms of colored complexes revealed complete decomposition above 600 0 C. Higher dehydration temperature of the complexes suggested that the water molecule is coordinated to the metal ion, which is also supported by broad IR absorption band above 3100 cm

Elemental Analysis
The data for the elemental analysis and other physical measurements of the Schiff base and its metal complexes are computed in the

Infrared Spectra
The IR spectra of the Schiff base and its metal complexes as presented in the Table 2 suggest that, the ligand binds to metal centre through five anchoring sites, behaving as a pentadentate ligand that use N atom of azomethine and amide group and also O-atom of amide carbonyl group and ring O-atom. The ligand behaves as a dinegative ion that coordinates to metal centre by forming covalent bond using deprotonation of amine-2 and C-NH of lactam ring. The lactam ν(C=O) band appears at 1774 cm -1 in the spectrum of MEA. The IR spectrum of ligand shows no band due to lactam ν(C=O) vibrational mode coming from MEA. The real coordination mode of the ligand towards metal ion has been deduced by IR spectral comparison of the free ligand and coordinated ligand [27]. The significant differences in the absorption frequencies were observed in the region between 1775-1200 cm -1 and at lower frequencies below 600 cm -1 . Some characteristic and recognizable spectral changes have been noticed in the IR spectra of metal complexes compared to free ligand that provide additional support of the bonding mode. The absence of lactam ν(C=O) band in Schiff base ligand and appearance of new band at 1644 cm -1 has been attributed to ν(C=N) of Schiff base ligand. The IR spectra of metal complexes display absorption bands in the range of 1635-1641 cm -1 range which can be assigned to (C=N) stretching frequencies of coordinated ligand. In fact, in all the complexes, the absorption band for ν(C=N) were shifted to lower wave numbers, indicating their participation in the coordination with metal ions. The IR absorption band at 1675 cm and a medium intensity band in the region of 500-530 cm -1 assignable for ν M-N and ν M-O stretching vibrations, which were absent in free ligand [28]. These overall data suggest that there is coordination of Schiff base ligand with metal ions through azomethine-N, amide-N, amine-2 N of Kanamycin moiety, heterocyclic-O and amide-O of the ligand.

1 H NMR Spectra
1 H NMR spectral comparison of novel Schiff base ligand and its metal complexes was made to confirm the binding nature of ligand with metal ions viz. Co(II), Cu(II) and Zn(II). The integral intensities of each signal in the 1 H NMR spectra of ligand and metal ion complexes are found to agree with the number of different types of protons present. In the 1 H NMR spectrum of Schiff base ligand, single peak attributed to CH 3 , COOCH 3 and Ar-OH groups appeared at 1.79, 3.65 and 12.5 ppm respectively. These peaks were still present in the metal complexes, indicating no participation of these groups in coordination process. Multiple peaks in the range of 6.6-7.8 ppm due to Ar-H were also present. Doublet peaks at 4.75 and 5.45 ppm correspond to N-CH and N=C-CH protons on the β-lactam ring and at 9.05 ppm due to NH proton lying between amide carbonyl and β-lactam ring were observed. Disappearance of NH proton peak (9.05 ppm) in the metal complexes is the indication of deprotonation which involve in coordination process. Side chain methylene proton of kanamycin moiety linked to azomethine nitrogen showed doublet peak at 2.09 ppm. No appreciable shift in 1 H NMR peak positions have shown in the spectra of metal complexes that were due to kanamycin moiety except decrease in intensity of NH 2 peak. Ring protons of kanamycin showed resonance as triplet at 3.5-3.8 ppm. The intense single peak at 3.2 and 4.07 ppm may be attributed to NH 2 and OH protons of Kanamycin. The decrease in peak intensity of NH 2 protons in metal complexes is also an indicative of its participation in coordination. The 1 H NMR spectrum of Zn-complex is shown in the Fig. 3.

Mass Spectra
The mass spectra of ligand and its metal complexes were recorded and are used to compare their stoichiometric compositions. The mass spectrum of Schiff base ligand shows a molecular ion peak at m/z 845. The molecular ion (MH) .+ peaks for metal complexes appear at m/z 921, 925 & 927 for Co, Cu and Zn complexes respectively and confirmed the stoichiometry of metal chelates as ML.H 2 O type. It was in good agreement with the microanalytical data.

Electronic Spectra, Magnetic Moment and Molar Conductivity
The electronic transition spectrum of free ligand showing absorption band higher than 430 nm have shifted to lower frequencies (bathochromic shift) due to coordination of the ligand with metal centers. The entire complexes showed electronic absorption band in the visible region of spectrum which may be attributed to charge transfer band. The electronic spectrum of Cu-complex showed absorption band at 660 nm which may be assigned to 2 A 1g (F) → 2 B 1g (P) transition and corresponds to square planar geometry of the complex. The magnetic moment value 1.81 BM of the complex further supports the observed geometry. The electronic transition at 235 nm may be due to π-π * transition and three closely spaced bands at 325, 322 and 350 nm may be due to n-π * transition [29,30]. The d-d transition band displayed at 670 nm is in good agreement with the square planar geometry of the Cucomplex. The electronic absorption spectrum of Co(II) complex display d-d transition band at 615 nm which corresponds to 4 T 1g → 4 T 1g (P) transition and revealed octahedral geometry of the complex. The observed magnetic moment value of 3.12 BM further supports octahedral geometry. The electronic spectrum of Zn(II) complex exhibited a sharp band of high intensity at 365 nm, which has been due to ligand metal charge transfer and assigned tetrahedral environment around Zn(II) ion. With a view to study the electrolytic nature of the metal complexes, their molar conductivities were recorded in DMF at 10 , which indicate their non-electrolytic nature. This further generates the idea of lack of any counter ions in the proposed structure of the mononucleate metal complexes.

X-ray Powder Diffraction Study
So far, single crystal growth of the complexes had been unsuccessful, X-ray powder diffraction technique was carried to get useful crystal information data to deduce accurate cell parameters, crystal system and the cell volume [31]. Powder diffraction patterns of ligand and the metal complexes were recorded over the 2θ = 10º-80º range and crystallographic data are listed in the Table 3. The indexing procedures were performed using Crysfire program package software. The diffraction pattern revealed well defined crystalline peaks indicating crystalline nature of the complexes and the data confirmed the triclinic crystal systems for ligand, Co and Cu complexes. Zn complex exhibited orthorhombic crystal system. The average particle size of the crystalline metal complexes was calculated using Scherrer's formula (d XRD = 0.9λ / βCosθ) by measuring the full width at half maximum of the XRD peaks [32][33][34]. The average particle size in the range of 45-66 nm suggests nanocrystalline nature of the compounds.

Thermal Analysis
Thermal stability of the complexes was extensively studied by TG/DTA analytical techniques with a heating rate of 10ºC / minute and their decomposition profiles were noticed at different stages of temperature ranges, resulting various thermally stable products, as explained according to the Scheme 2.
TG/DTA profile of the complexes revealed change in curve area corresponding to their decomposition at various temperature ranges. Nearly 2% weight loss due to coordinated water molecule in all the complexes occurred in the temperature range of 120-135ºC. Thermogram of Co-complex showed that nearly 50% of the total mass of complex reduced at 150-170ºC, followed by considerable decomposition above 550ºC that corresponds to the decomposition of ligand moiety leaving Co 2 O 3 as stable end residue. Similarly, thermogram of Cu and Zn-complexes showed 50% weight loss in between 350-375ºC and final decomposition above 450-600ºC resulting into final pyrolysis product ie. stable metal oxide (CuO and ZnO) residue [35,36].
Above this temperature, horizontal curve showed no further loss in weight. This inferred metal oxide as the final residue. The calculated metal content in the oxide residue was compared with that obtained from analytical determinations. Hence these decomposition patterns were in good agreement with suggested formulae of the complexes.

Thermal Decomposition Kinetics
Thermal decomposition profiles obtained by thermo gravimetric (TG) and differential thermo gravimetric (DTA) analysis were used to calculate thermodynamic and kinetic parameters for the non-isothermal decomposition of the complexes such as order of reaction (n),  ( 0 ) 10-80 10-80 10-80 10-80 Limiting indices  ) and Gibb's free energy change of activation (ΔG # ) [37]. By the analysis of the non-isothermal TG, using the integral method of Coats-Redfern relation, kinetic parameters of decomposition were calculated [38].
Where α is the mass loss up to the temperature T, R is the gas constant, E # is the activation energy in J mole -1 and φ is the linear heating rate. A straight line plot of left hand side of the equation against 1000/T gave a slop from which E # was calculated while its intercept value corresponds to frequency factor (A). The entropy of activation ΔS # in J K -1 mol -1 , enthalpy change (ΔH # ) and Gibb's free energy change (ΔG # ) [39] were calculated by using the equations: Where, k B is the Boltzmann constant, h the plank's constant and T is the DTG peak temperature. The Coats-Redfern linearization plots, confirmed the first order kinetics for the decomposition process. The calculated values of thermodynamic activation parameters for the decomposition steps of the metal complexes are reported in Table 4. The high value of activation energy of the complexes revealed their high thermal stability. The positive sign of ΔG # and negative sign of ΔS # suggested that the thermal decomposition steps are non-spontaneous process. Further negative entropies of activation of complexes indicated that the studied complexes are in more ordered state and the decomposition reactions proceed with a much slower rate than the normal. Among metal complexes, activation energy increases as Zncomplex < Co-complex < Cu-complex. Thus, thermal stability of metal complexes follows the order Co-complex > Zn-complex > Cu-complex.

Coordination Sites
The novel Schiff base ligand obtained from Kanamycin and amoxicillin has many potential donor atoms at various positions which can bind to metal center forming multinucleate chelate. In the present investigation, spectral studies revealed penta-dentate nature of ligand, encapsulating metal through azomethine nitrogen (C=N), carbonyl oxygen of amide group, cyclic oxygen of ring A, nitrogen of amine 2 of kanamycin moiety and nitrogen of CH-N of lactam ring. Besides these, there is coordination with one water molecules and form octahedral geometry of Co-complex and square planar geometry of Cu-complex.

Antibacterial Activity
The novel Schiff base ligand and its metal complexes were screened in vitro against four bacterial pathogens viz, E. coli, B. subtilis, S. aureus and K. pneumoniae to assess their antibacterial potency. The results are quite promising. The prescription of the antimicrobial results showed graphically (Fig. 4) reveals that they exhibited moderate to better antibacterial activity [40,41]. Further, metal complexes were found to have higher biological activity than the parent Schiff base. Such increased activity of metal complexes may be considered due to insertion of metal ions in chelation process with Schiff base that cause to increase lipophilic nature of these complexes due to delocalization of π-electrons over the whole chelate ring. This increased lipophilicity enhances the penetration of complexes into the lipid membranes and blocks the metal binding sites in enzymes of microorganisms [42]. These complexes also disturb the respiration process of the cell by deactivating enzymes responsible for this and thus block the synthesis of proteins, which restricts further growth of the organism. The potential activity of the complexes against bacterial pathogens may be related to cell wall structure of the bacteria which possibly occurs due to inhibition of synthesis step of peptidoglycan layer of bacterial cell wall. The data revealed that the activity of the ligand enhanced on complexation comparable to the standard used. Overall comparison of observed data gave information that metal complexes are more active than free ligand against all bacteria. Specifically, Zn-complex showed better antibacterial potency against S. aureus bacteria.

MOLECULAR MODELLING
3D molecular modeling of the proposed structure of the metal complexes were studied by CsChem 3D Ultra program package and optimized structures revealed octahedral geometry for Co, square planar geometry for Cu and tetrahedral geometry for Zn-complexes [43]. This investigated geometry of the complexes is also supported by several spectral techniques. The correct stereochemistry was assured through, manipulation and modification of the molecular coordinates to obtain reasonable and low energy molecular geometries. Energy minimization was repeated several times to find the minimum [44,45]. The energy minimization values for the metal complexes suggested their maximum stability. The change in bond length values of metal-nitrogen and metal-oxygen in the complexes compared with ligand further suggested their coordination. The details of the bond lengths and bond angles as per 3D structure of the metal complexes optimized by MM2 calculations are given in the Table 5

CONCLUSION
In the present study, we have successfully synthesized referenced metal(II)-complexes of novel Schiff base ligand from Kanamycin and methyl ester of Amoxicillin. Instrumental protocols like spectral, thermal and other analytical data measurements revealed the real correlation with the proposed and suggested structure. The investigated Schiff base ligand act as neutral pentadentate ligand. The electronic transition band observed at lower wavenumbers in metal complexes may be ascribed to d-d electronic transition within the metal ions which were absent in the spectrum of free ligand. Molar conductivity measurement recommended nonelectrolytic nature of the complexes. Further molecular modeling and electronic spectral data measurement strongly recommend square planar geometry for Cu-complex, octahedral geometry for Co-complex and tetrahedral geometry for Zncomplex. Negative entropy values obtained by thermal decomposition calculation by using Coats-Redfern equation indicated slow pyrolytic decomposition of metal complexes. Two antibiotics in conjugation with metals lead to better antibiotic activities. The antibacterial screening of the synthesized ligand and its metal complexes against various pathogenic bacteria suggested remarkable antibacterial activities. Comparable antibacterial sensitivity test of the ligand and metal complexes showed that they bear strong activity against S. aureus. Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal