SPECTROSCOPIC, STRUCTURAL, THERMAL AND ANTIMICROBIAL STUDIES OF 4,6-BIS (4-CHLOROPHENYL)-2-OXO-1,2-DIHYDROPYRIDINE-3- CARBONITRILE WITH SOME TRANSITION METALS

4,6-Bis(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (L) reacted with Mn(II), Fe(III), Co(II) and Ni(II) in methanol and acetone as a solvent at room temperature to form a new solid complexes. The isolated complexes were characterized by elemental analysis, magnetic properties, conductance measurements, mass, IR, UV-Vis and H NMR spectroscopic methods and thermal analyses. The thermogravimetric and infrared spectroscopic data confirmed the presence of water in the composition of the complexes and the ligand reacted as a bidentate. The metal – ligand binding in metal complexes is predicted using density functional theory. The ligand and their metal complexes were also evaluated for their antibacterial activity. The complexes showed high activities compared with free ligand.


INTRODUCTION
In recent years the chemistry of heterocyclic compounds are well known for their diverse therapeutic properties and exhibited antibacterial, anticancer, antiulcer, diuretics, anticonvulsant, antihypertensive, antitumor, antifungal, anti-AIDS and antiviral properties [1].
Enones are excellent starting materials for the synthesis of 4,6-bis(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (L) (Scheme 1) via the reaction of enone with ethyl cyanoacetate in the presence of ammonium acetate [2]. However, this procedure is time consuming and gives low yield. Jasinski et al. [2,3] had reported an easy one-step synthesis which gives a higher yield of ligand by heating a mixture of 4-chloroacetophenone, 4chlorobenzaldehyde, ethyl cyanoacetate and ammonium acetate in presence of glacial acetic acid as a catalyst.

Preparation of the metal complexes
The white solid complex [Mn(L) 2 4 .6H 2 O were prepared in similar manner described above by using acetone as a solvent and using Fe(NO 3 ) 3 .9H 2 O, CoCl 2 .6H 2 O and NiSO 4 .8H 2 O, respectively, in 1:2 molar ratio.
Elemental C, H and N analysis was carried out on a Perkin Elmer CHN 2400. The percentage of the metal ions were determined gravimetrically by transforming the solid products into metal oxide or sulphate and also determined by using atomic absorption method. Spectrometer model PYE-UNICAM SP 1900 fitted with the corresponding lamp was used for this purposed. IR spectra were recorded on FTIR 460 PLUS (KBr discs) in the range from 4000-400 cm -1 . TGA-DTG measurements were carried out under N 2 atmosphere within the temperature range from room temperature to 800  C using TGA-50H Shimadzu thermal analyzer. Electronic spectra were obtained using UV-3101PC Shimadzu spectrophotometer. The solid reflection spectra were recorded with KBr pellets. Mass spectra were recorded on GCMS-QP-2010 plus Shimadzu (ESI-70 eV).

RESULTS AND DISCUSSION
4,6-Bis(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (L) complexes of Mn(II), Fe(III), Co(II) and Ni(II) were obtained in moderate good yields (68-78%) as solids of a color characteristics of the metal ion. Single crystals suitable for X-ray crystallographic measurements were not obtained. Table 1 summarizes the carbon, hydrogen and nitrogen elemental analysis as well as melting points and magnetic properties of the isolated solid complexes. The results obtained indicated that all of the isolated complexes are formed from the reaction of the metal salts with L in 1:2 molar ratios. All of the complexes reported here in are hydrates with various degrees of hydration and air stable solids at room temperature. Elemental analyses of C, H, N, S and M (M = Mn, Fe, Co or Ni) are in accordance with the proposed compositions of the complexes. The molar conductivities at room temperature in DMF solvent at 1.0×10 -3 M of free ligand (L) and their metal complexes were in the range 15.9 to 230.6 S cm 2 mol -1 which indicated that the complexes were electrolyte in the solvent with three categories [10]. Conductivity measurements have frequently been used to predicts the structure of metal chelates within the limits of their solubility. They provide a method of testing the degree of ionization of the complexes. It is clear from the conductivity data that the complexes are electrolytes. The obtained results were strongly matched with experimental data which indicated that sulfate, chloride and nitrate groups are found as counter ions (outside the complexes sphere). The magnetic moments (as B.M.) of the complexes were measured at room temperature. The Mn(II), Fe(III), Co(II) and Ni(II) complexes are found in paramagnetism character and octahedral geometry (high spin) around the metal ion with measured magnetic moment values at 5.81, 5.62, 3.72 and 2.73 B.M., respectively.  Figure 1). The proposed structure for all complexes is represented by Scheme 2 The four donor atoms of the two ligands (L) coordinated to central metal ions equatorially in a plane forming tetragon with the two oxygen atoms of the two coordinated water molecules axial. According to the proposed structure for the complexes under investigation, the complexes possess a two-fold axis and two planes of symmetry and hence they are C 2ν symmetry. The C 2ν complexes, [M(L) 2 (H 2 O) 2 ] n+ are expected to display 213 vibrational fundamentals which all are monodegenerate and distributed between A 1 , A 2 , B 1 and B 2 motions; all are IR and Raman active, except A 2 modes which are only Raman active.
The infrared spectrum of free ligand (L) shows two bands at 2215 and 1628 cm -1 ( Table 2) which are attributed to the stretching vibration of ν(CN) for carbonitrile group and ν(C=O) keto group [11,12]. Upon comparison of the IR spectra of the complexes with that of free ligand the change in intensity of ν(CN) from medium to strong (Table 2) confirming that the L molecule coordinated to metal ion through carbonitrile nitrogen. Also, the shift of ν(C=O) to higher frequency value (1636 cm -1 ) in the spectra of the complexes may indicate an increase of the C=O bond strength upon coordination. The possibility that the electron density on the oxygen atom is decrease upon coordination to metals means decrease in the electron repulsion between the oxygen lone pair and the double bond electrons leading to a stronger C=O bond and then a higher bond frequency. The increase of ν(C=O) frequency upon coordination was reported by the other workers in the spectra of other complexes [13,14]. These changes of the IR spectra suggest that the free ligand is coordinated to the metal ions via oxygen of keto group and nitrogen of carbonitrile group.
The bands in the range 3533-3378 cm -1 in the spectra of the complexes can be attributed to the ν(O-H) vibration of the water molecules [15]. The ν(N-H) vibration appears in the region of 3289-3267 cm -1 and the stretching vibrations ν(C-H) of phenyl groups are assigned as a number of bands in the region 3136-3017 cm -1 [16].

UV-Visible solid reflection spectra
The formation of the ligand and its metal complexes was also confirmed by the electronic solid reflection spectra in the range 200-800 nm ( Figure 2). The free ligand reflects at 208, 279, 322 and 344 nm, respectively ( Table 3) that may be attributed to π-π * and n-π * transitions which occur in unsaturated hydrocarbons containing ketonic groups [18]. On complexation, The 208 nm is absent besides a bathochromic shift of the 279, 322 nm bands and the appearance of new reflections. The complexes have bands in the range from 518 to 522 nm which may be assigned to ligand to metal charge-transfer [19]. Also, the presence of new bands in the range of 569 to 682 nm in all complexes may be attributed to d-d transition.  The formation of the metal complexes was also confirmed by 1 H NMR spectra ( Figure 3). The 1 H NMR spectra for complexes exhibit new peaks in the range 3.17-3.39 ppm, (Table 4) due to the presence of water molecules in the complexes [1]. Also, the compounds showed peacks in the range δ: 12.85-12.93 for NH amide and at 6.66-8.11 ppm for ‫ــ‬CH aromatic. On comparing main peaks of free ligand with its complexes, it is observed that all the peaks of the free ligand are present in the spectra of the complexes with chemical shift upon binding of free ligand to the metal ion [20].

Mass spectra
Mass spectrum of the free ligand (L) is in a good agreement with the suggested structure. The ligand (L) showed molecular ion peak (M .+ ) at m/z = 340 (24%) and M +2 at m/z = 342 (8%) (Figure 4)

Thermal analysis
The 4,6-bis (4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (L) of Mn(II), Fe(III), Co(II) and Ni(II) complexes are stable at room temperature and can be stored for several months without any changes. To establish the proposed formulae for the new complexes of Mn(II), Fe(III), Co(II) and Ni(II) under investigation, thermogravimetric (TGA) and differential thermogravimetric (DTG) analyses were carried out for the solid complexes under N 2 flow from ambient temperature to 800  C ( Figure 5). Table 5 give the maximum temperature values, T max /  C together with the corresponding weight loss for each step of the decomposition reactions of the above compounds. The obtained data strongly support the proposed chemical formulae of the compounds and indicated that the ligand (L) is thermally stable at room temperature. Decomposition of the L started at 350  C and finished at 600  C with one stage at maximum 411  C and is accompanied by a weight loss of 82.50% corresponding to loss of five molecules of acetylene, one molecule of carbon monooxide and two molecule of cyanogen chloride [21].
The thermal decomposition of Mn(II), Fe(III) and Co(II) complexes proceed approximately with one main degradation step at 200 and 360  C for Mn(II), at 320  C for Fe(III) and at 502  C for Co(II) leaving MnSO 4 , Fe 2 O 3 and CoO as a final products ( Table 5).
The TGA curve of [Ni(L)(H 2 O) 2 ]SO 4 .6H 2 O shows two stages of decomposition. The first stage occurs at maximum temperature 80  C corresponds to the loss of six water molecules with mass loss of 11.56% (calc. 11.01%). The relatively low value of temperature of this step may indicated that these water molecules undergoes less H-bonding. The second step of decomposition occurs at two maxima at 337 and 451  C, is accompanied by a weight loss of 91.51% which leaving NiO+3C, as resdue [22,23].    i.e., the inhibition zones exceed the plate diameter. Statistical significance P NS P not significant, P >0.05; P +1 P significant, P < 0.05; P +2 P highly significant, P < 0.01; P +3 P very highly significant, P < 0.001; student's-test (paired).

Antimicrobial activity
The efficiencies of L and their metal complexes have been investigated against two Gramnegative, Escherichia coli and Pseudomonas aeruginosa and two Gram-positive, Staphylococcus aureus and Bacillus subtilis also antifungal screening was studied against two species Aspergillus fumigatas and Candida albicans microorganisms. The results of the antibacterial study of L and their complexes ( Table 6) have inhibitory action against all four types of bacteria and antifungal activity ( Figure 6). The complexes of Mn(II) shows a highly significant against Escherichia coli and Pseudomonas aeruginosa and moderate activity against Bacillus subtilis, Staphylococcus aureus than free ligand. Fe(III) shows very highly significant against Pseudomonas aeruginosa, a highly significant against Escherichia coli and moderate significant against Gram-positive. The Co(II) shows very high significant against Pseudomonas aeruginosa and highly significant against Escherichia coli, Staphylococcus aureus and Bacillus subtilis and moderate significant for antifungal species. For Ni(II) complex shows very highly significant against Pseudomonas aeruginosa but, for Escherichia coli, Bacillus subtilis and Aspergillus fumigates are highly significant and moderate significant against Staphylococcus aureus and Candida albicans. The nature of the metal ion coordinated to a drug may have a significant role to this diversity. In general for metal complexes showing antimicrobial activity, the following five principal factors [13,14] should be considered: i) the chelate effect; ii) the nature of the ligands; iii) the total charge of the complex; iv) the nature of the ion neutralizing the ionic complex; and v) the nuclearity of the metal center in the complex. Figure 6. Statistical representation for biological activity of (L 2 ) and its metal complexes.

Computational method
The geometric parameters and energies were computed by DFT at the B3LYP/CEP-31G level, using the GAUSSIAN 98W package of the programs, on geometries that were optimized at CEP-31G basis set. The high basis set was chosen to detect the energies at a highly accurate level. The atomic charges were computed using the natural atomic orbital populations. The B3LYP is the keyword for the hybrid functional, [24], which is a linear combination of the gradient functional proposed by Becke [25] and Lee et al. [26] together with the Hartree-Fock local exchange function [27].
Structural parameters and models (4,6)bis-4-chlorophenyle-2-oxo-1,2-dihydropyridine-3-carbonitrile (L) The biological activity of pyridinones L is mainly determined by its fine structure, the L has many characteristic structural features. The molecule is not highly sterically-hindered, the two benzene rings in the same plane of the pyridinone ring. This observation is supported by the values of calculated dihedral angles of C3C4C5C8 and C6C5C8C9 are 180.00˚ and 0.00˚ (Table  7) while the dihedral angles of C11C17C21C22 and C11C17C18C19 are 180.00˚and 180.00˚. Scheme 5 shows the optimized geometrical structure of L molecule, the dihedral angles of N16C15C9C13 and N16C15C9C8 are 0.00˚ and 180.00˚ which confirms -CN group in the same plane of molecule.
The bond distance of C13-O14 is 1.289Å and the bond angle of C9C15N16 is 164.29˚ reflects on sp hybridization of C15 and the bond length of C15N16 is 1.208Å. The values of bond distances are compared nicely with that obtained from experimental data [28].
There is a significant built up of charge density on oxygen atom and two nitrogen atoms of pyridinone and cyano group, so L is bi-dentate ligand (O14 and N16 atoms) and the molecule is not highly dipole µ = 7.37 because the planarity of L molecule. The charge accumulated on Cl7and Cl23 are -0.287 and -0.277, respectively, also, the charge on N12 and N16 are -0.218 and -0.258, respectively. The total energy of the optimized geometry of L molecule is -191.782 au. The metal ligand binding, dipole moment, total energy and optimized geometrical structure of all complexes were studied and the DFT of Mn(II) complex is used as example in my article.

Description of the structure of [Mn(L) 2 (SO 4 )]
The structure of complex with atomic numbering scheme is shown in Scheme 6. The complex is six-coordinate with distorted octahedral environment around the metal ion. The Mn(II) is coordinated through two oxygen and two nitrogen atoms of two ligand and two oxygen atoms of SO 4 -2 group. The Mn-O25 and Mn-O33 bond lengths are 2.026 Å and 2.025 Å, respectively [29,30]. The Mn-N27 and Mn-N29 are 1.795 Å and 1.796 Å, respectively, (Table 8) and the bond distance between Mn-O2 and Mn-O3 of sulfate group are 1.849 Å and 1.849 Å, respectively [31]. Also, the angles around the Mn (II) with surrounding four oxygen atoms and two nitrogen atoms vary from 73.46˚ to 172.31˚; these values differ legally from these expected for a regular octahedron.
The energy of this complex is -434.252 au while the dipole moment is lower than free ligand 4.425D also, the charge accumulated on O25 and N27 of the first molecule are -0.396 and -0.179, respectively. O33 and N29 of the second molecule are -0.386 and -0.193, respectively. The bond angle of O4SO5 of sulfate group is 130.76˚ [32].  The structure of complex with atomic numbering scheme is shown in Scheme 7. The complex consists of two units of L molecule and two water molecules with Mn(II). The complex is sixcoordinate with distorted octahedral environment around the metal ion. The Mn(II) is coordinated to one O keto atom and one N car atom of L ligand and two oxygen atoms for water. The total energy of this complex is obtained by using density functional theory combined with B3LYP/CEP-31G as basis set. The bond angle between O22MnO30 is 96.07 and also the angle between N24MnN26 is 164.18˚ (Table 9) so the two L molecules are not lying in the same plane they are perpendicular respect to each other with angle equal 90˚. The angle between O2MnO49 is 91.04˚ this value reflects that the two water molecules not in trans-form respect to each other but they are lying in cis-form and then they are perpendicular to each other. The bond length between Mn-O30 and Mn-O22 are 2.026 Å and 2.025 Å are longer than that Mn-N26 and Mn-N24 (1.799 Å and 1.800 Å) [29][30][31], while the bond distance between Mn-O H2O vary from 1.845 Å and 1.846 Å [32,33]. Also, the angles around Mn(II) with surrounding oxygen atoms vary from 73.44˚ to 166.15˚; these values agree with these expected for a distorted octahedron.
The bond distances between Mn(II) and surrounded oxygen atoms and nitrogen atoms of L in water complex are shorter than that in sulfate complex. Also, the charge accumulated on O keto (-0.396 and -0.386) and N car (-0.179 and -0.193), in sulfate complex while, O keto (-0.415 and -0.421) and N car (-0.207 and -0.205), in water complex. There is a strong interaction between Mn (II) which become has charge equal +0.546 in case of water complex while, in case of sulphate complex the charge accumulated on Mn(II) is 0.343. The energy of the water complex is more negative than sulfate complexes -474.816 au and relatively weak dipole 7.425D. For all these reasons the water complex is more stable than other complexes and Mn(II) favor coordinated with two molecules of water more than one molecule of sulfate ion to complete the octahedron structure.