Molecular Modeling and Antimicrobial Screening Studies on Some 3-Aminopyridine Transition Metal Complexes

Seven transition metal complexes of Mn 2+ , Ni 2+ , Co 2+ , Cu 2+ and Zn 2+ with 3-aminopyridine (3-APy) as ligand have been synthesized, characterized by different techniques and their antibacterial activities were studied. Molecular modeling calculations were performed using DMOL 3 program in materials studio package which is designed for the realization of large scale density functional theory calculation (DFT). The quantum mechanical and chemical reactivity parameters such as chemical hardness, chemical potential, electronegativity, electrophilicity index and Homo-Lumo energy gap were obtained theoretically and were used to understand the biological activity of the prepared compounds. Some complexes were tested for their in-vitro cytotoxic activity in human lung cancer cell lines (A-549 cell line), and structure-activity relationships were established. In general, the coordination to Co 2+ increased the cytotoxicity while the Ni 2+ complexes show reduced cytotoxic activity compared to the metal-free 3-aminopyridine.


Introduction
Pyridine derivatives are very important chemicals with many biological applications. They are known to have moderate-excellent activities towards variety of biological targets varying from microbial diseases to viral organisms and cancerous cells by interacting with enzymes, proteins and DNA [1] [2] [3] [4].
Oxime and hydrazone derivatives of pyridine exhibit high activity against in-fluenza B-Mass virus, HIV and are also used as antidotes against poisoning by organophosphorus compounds [5] [6]. Many metal complexes with pyridine based ligands exhibit high cytotoxicity and have proved to be antitumor agents [7]- [11]. Molecular modeling (MM) is one of the fastest growing fields in science. It may vary from building and visualizing simple molecules in three dimensions (3D) to performing complex computer simulations on large proteins and nanostructures [12]. It is a collection of computer-based techniques for driving, representing and manipulating the structures and reactions of molecules depending on 3D structures. The techniques cover several issues among them computational chemistry, drug design, computational biology, nanostructures, and material science [13]. In recent years, density functional theory (DFT) has become an increasingly useful tool for theoretical studies. The success of DFT is due it is computationally less demanding than wave function based methods with inclusion of electron correlation [14]. In the present work seven Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ and Zn 2+ -3-aminopyridine (3-APy) complexes with variable stoichiometric ratios were prepared, characterized and their antibacterial activities were studied. Some complexes were tested for their in-vitro cytotoxic activity in human lung cancer cell lines (A-549 cell line), and structure-activity relationships were established. Also, the molecular structure of some selected metal complexes in view of chemical reactivity values such as chemical hardness, chemical potential electronegativity, electrophilicity index and HOMO-LOMO energy gap obtained theoretically were described.

Experimental
All reagents used were of the highest quality chemicals and were used without further purification. Aqueous solution of 1.00 mmol of metal salts was added with stirring to an ethanolic solution of 3-aminopyridine ligand with the desired molar ratio. The mixture was refluxed for ≈6 hours then cooled to room temperature, and the solid complexes so formed were filtered off and washed with distilled water followed by ethanol and dried under vacuum. Elemental analysis, spectroscopic studies and cytotoxic activity are as reported in our previous work [15].

Antimicrobial Screening
The antibacterial activity of some complexes toward some bacterial strains and fungi was evaluated by agar well diffusion method [16] [17]. The activity was evaluated by measuring the diameter of zone of inhibition in mm. A medium with DMF as solvent was used as a negative control whereas media with Ampicillin were screened separately for its standard antibacterial activity.

Molecular Modeling
The cluster calculations using DMOL 3 program [18] in materials studio package [19] which is designed for the realization of large scale density functional theory

Characterization of the Metal Complexes
The as-prepared complexes were characterized by different techniques and are proved to have the following structural formulae:

Thermal Analysis
Up to 800˚C, the prepared metal complexes degrade thermally through, more or less, three main steps. Representative thermogram is shown in Figure 1 and the thermal degradation patterns show that the physically adsorbed and coordinated water molecules are dehydrated from the coordination sphere in two successive steps within the temperature range 80˚C -220˚C followed by full thermal decomposition at temperature higher than 335˚C leading to metal oxides as final products.

Infrared Spectra
The IR spectra of the complexes M ← 3-aminopyridine were studied in terms of their molecular structure. The most important band frequencies (cm −1 ) are cited in Table 1 from which it is found that the spectra exhibit broad band within the range 3400 -3448 cm −1 and weak band within the range 2955 -3024 cm −1 due to the stretching vibrations of OH and NH 2 (V OH and V NH2 ) groups, respectively.
This remarkable broadness and shifting to lower frequencies indicates their contribution in coordination process. This is supported by the appearance of new broad bands within the range 3400 -3350 cm −1 and 574 -545 cm −

Electron Spin Resonance (ESR) Spectra
The X-Band ESR spectra of the powder CuCl 2 ← 3-APy (1:2) and Cu(Ac) 2 ← 3-APy (1:2) complexes were recorded at room temperature using DPPH as reference standard (c.f. Figure 2). The ESR exhibits anisotropic spectra with g⊥ > g II > g e (g e = 2.0036 free ion value) characteristic for compressed axial symmetry of the coordination sphere (one coordination axis (z) is significantly shorter than the other two (x, y) with d x2-y2 ground state. One unpaired electron in Cu (II) complex with 2 B 1g as ground state lies in d z2 orbital. Analysis of spectra gave the g II and g┴ values as cited in Table 2.  Magnetic moments and electronic spectral data give information about the oxidation state and stereochemistry of the central metal ion in coordination complexes. The data are presented in Table 3.
The magnetic moment of Mn(II) complex is 5.85 BM indicating d 5 high spin (t 2g 3 e g 2 ) configuration,(spin only value (5.92 BM) while that for Co(II) complex is 5.17 BM indicating d 7 high spin (t 2 g 5 eg 2 ) configuration (spin only value 3.88) which agree with octahedral range 4.3 -5.2 BM. Ni(II) complexes showed the magnetic moment values of 3.20 BM consistent with their d 8 (t 2 g 6 eg 2 ) octahedral environment. Cu(II) complexes showed magnetic moments of 1.77 BM, slightly higher than the spin-only value (1.73 BM) expected for one unpaired electron d 9 (t 2 g 6 eg 3 ), offering the possibility of octahedral geometry [21]. These geometries are further confirmed by electronic transitions. The Zn (II) complex is observed to be diamagnetic as expected from its electronic configuration. The electronic spectral data (measured using Nujol mull technique) are used for assigning the stereochemistry of metal ions in the complexes based on the d-d transitions observed (c.f. Table 3).

Biological Activity
The antimicrobial and antifungal activities of selected complexes were tested against two bacterial; Staph. aureus, E. coli and two fungi; Asper. ocheratious, Asper. niger species. Standard drug; Ampicillin and DMF solvent control were screened separately for their antibacterial activity. The results and percent activity (c.f.   Inspection of the data cited above shows that, as a general trend, the activity of the complexes is higher than that of the free ligand. The increase in activity is simply due to the fact that chelation process reduces the polarity of the ligand due to partial sharing of its negative charge with metal ion favoring transportation of the complexes across the lipid layer of the cell membrane [22] [23]. This is in accordance with the calculated quantum chemical analysis data, gathered from molecular modeling studies (as will be shown later in Section 3.

Antitumor Activity
The cytotoxic activities of some selected complexes were tested against Lung carcinoma cells A-549 cell line and compared to that of Vinblastine as a standard drug. The 50% inhibitory concentration (IC 50 ) was estimated from graphic plots of the relation between surviving cells and complex concentration. The lethal concentrations (IC 50 ) values with a brief comment on the data are listed in Table 5. From the data cited in the Table, it is clear that Co(II) and Cu(II) complexes have high activity toward lung carcinoma cell with a low IC 50 value (81.6 ± 2.4 and 108. ± 2.7 µg/ml, respectively) while Zn(II) complex showed moderate activity. On the other hand, Ni(II) complex showed weak activity against the tested cell line (IC 50 value 354 ± 8.7 µg/ml).

Molecular Modeling
The molecular modeling, the total density function, deformation density function and frontier orbital energy (the HOMOs and LUMOs) for the ligand 3-aminopyridine and its Ni 2+ , Cu 2+ and Zn 2+ complexes were determined using DFT method. The quantum chemical parameters of the studied complexes were calculated as given in Table 6.
The HOMOs and LUMOs are known as Frontier molecular orbitals (FMOs), which play an important role for evaluating molecular chemical stability, and hardness-softness of the molecule [24]. The energy gap, ΔE (E HOMO − E LOMO ), represents the chemical reactivity of compounds, (system of lower value of ΔE is more reactive). As depicted in Table 6, the free ligand (3-APy) has the largest energy gap (2.889 eV) which decreases in case of complex species indicating their stability. Another global reactivity descriptor electrophilicity index (ω) which describes the electron accepting ability of the systems quite similar to hardness and chemical potential. High values of electrophilicity index increases electron accepting abilities of the molecules. The latter values increase in case of complex species indicating the increase in their electron accepting abilities. The direction of the charge transfer is completely determined by the electronic chemical potential (μ) of the molecule because an electrophile is a chemical species capable of accepting electrons from the environment and its energy must decrease upon accepting electronic charge. Electronegativity (χ), chemical potential (µ), global hardness (η), global softness (σ), additional electronic charge (ΔN max ) and global electrophilicity index (ω) [25] [26] were calculated and listed in Table 6:  Table 7.

Conclusion
Seven Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ and Zn 2+ -3-aminopyridine (3-APy) complexes with variable stoichiometric ratios were prepared, characterized then, their antibacterial and cytotoxic activities toward some bacteria, fungi and human lung cancer were studied. The molecular structure of some selected metal complexes in view of chemical reactivity values such as chemical hardness, chemical potential electronegativity, electrophilicity index and HOMO-LUMO energy gap obtained theoretically were described. The latter data are used to understand their biolog-