Synthesis , Characterization and Biological Activity of Symmetric Dinuclear Complexes Derived from a Novel Macrocyclic Compartmental Ligand

Um novo ligante macrocíclico binucleador, N,N-bis(2,6-diiminometil-1hidroxifenil)malonoildicarboxamida, foi preparado. Seus complexos metálicos foram preparados pelo método da moldagem por metal, através das reações de 2,6-diformil-4-metilfenol, malonoil diidrazida com os cloretos metálicos de Cu(II), Ni(II), Co(II), Cd(II), Zn(II) e Hg(II), em methanol, que levaram à formação de uma série de complexos dinucleares. Os complexos foram caracterizados por análises elementares, medidas de conductividade, dados de susceptibilidade magnética, IR, UVVis, EPR, RMN e dados de espectrometrica de massas-FAB. A natureza dinuclear dos complexos foi confirmada pelas análises elementares e dados de susceptibilidade magnética, EPR e de espectrometria de massas-FAB. O ligante e os complexos de Cu(II), Ni(II), Co(II) e Zn(II) tiveram suas atividades antibacteriana e antifúngica testadas contra Escherichia coli, Staphyloccocus aureus, Aspergillus niger e Fusarium oxysporum. As medidas de susceptibilidade magnética dos complexos de Cu(II), Ni(II) e Co(II) revelaram que estes complexos exibem acoplamento antiferromagnético devido à presença de dois íons metálicos próximos um do outro. O espectro de massas-FAB do complexo de Cu(II) forneceu evidencia clara da sua natureza dinuclear. O ligante e os complexos exibiram atividade muito fraca contra as bactérias testadas, entretanto, o ligante mostrou-se ativo contra o fungo Fusarium oxysporum.


Introduction
Efforts have been made in the last decades to the design and synthesis of macrocyclic or macroacyclic complexes and to study their physico-chemical properties. 1,2These investigations emphasized the great relevance of these systems in basic and applied chemistry.2][3][4] Many macrocyclic or macroacyclic systems containing an appropriate set of donor atoms and a suitable structural configuration are capable of producing new macromolecules which have been used in numerous chemical processes and technologies.Based on this concept many macrocyclic systems are available and are already applied in the fields of activation and catalysis, molecular materials, microelectronics, sensors, and so on. 4Bimetallic cores have been recognized at the active sites of many metalloenzymes. 5tudies on the geometry around the metal by electronic spectra and magnetic properties of the metal ions present in the active sites of the metalloenzymes are rather difficult owing to the fact that the metal ions in active sites are embedded in protein polymer back bone. 6Hence, design and synthesis of model compounds that mimic the physical and chemical properties of the active sites present in metalloenzymes is very essential and the studies on such compounds is becoming increasingly important in understanding biological functions of the bimetallic cores. 5inuclear complexes with two metal ions in close proximity show interesting magnetic, catalytic and electron transfer properties.Efforts to synthesize ligands capable of generating such dinuclear complexes have resulted in a large number of dinuclear ligands. 7One of such ligand systems was obtained from "2+2" condensation of 2,6-diformyl-4-methylphenol with 1,3diaminopropane. 8Macrocyclic ligands derived by template condensation of 2,6-diformyl-4-methylphenol with simple diamines such as 1,2-ethylenediamine, 1,3propanediamine, 1,4-butanediamine etc., generally produce 2:2 macrocyclic rings, encompassing two metal atoms, bridged by two phenoxide groups and each bound terminally by two imine nitrogen donors.Such complexes particularly in the case of copper are strongly antiferromagnetically coupled and exhibit well-defined one electron redox processes. 9A literature survey reveals that, for the synthesis of macrocyclic dinuclear complexes, the ligands employed were generally synthesized by the template condensation of 2,6-diformyl-4-methylphenol with various diamines.There are only a few references wherein the 2,6-diformyl-4-methylphenol has been condensed with aroyl hydrazones, 4,10 but there are no reports on the complexes of macrocycles obtained by the reaction of 2,6diformyl-4-methylphenol with dicarboxylic acid dihydrazides.In view of these observations, we report here the synthesis, characterization and biological activity of a novel macrocyclic ligand obtained by "2+2" condensation of 2,6-diformyl-4-methylphenol with malonoyl dihydrazide and its dinuclear complexes.

Materials
All chemicals used were of reagent grade.Solvents were distilled and dried before use according to standard procedures. 112,6-Diformyl-4-methylphenol 12 and malonoyl dihydrazide 13 were prepared by the literature methods.The metal salts used were generally in their hydrated form.

Methods
Elemental analyses for C, H, and N were obtained from Heraeus CHN-O rapid analyzer, IR spectra were recorded on Perkin-Elmer 1000 IR spectrometer using KBr pellets.Conductance measurements were carried out in DMF solution (1x10 -3 mol dm -3 ) using an Elico-CM 82 conductivity bridge.Electronic spectral measurements were carried out using Elico SL 159 spectrophotometer in the range 200-1000 nm. 1 H NMR spectra were recorded on Bruker AMX 400 MHz spectrometer, ESR spectrum of Cu(II) complex was recorded on X-band ESR spectrometer (Varian Associates).Magnetic measurements were made on a Cahn 2000 electro balance by Faraday Method using Hg[Co(SCN) 4 ] as calibrant.FAB mass spectrum of Cu(II) complex was recorded on a JEOL SX 102/DA-6000 mass spectrometer using Argon/Xenon as the FAB gas.

Biological activity
The in vitro biological screening of the ligand H 2 L and its Cu(II), Ni(II), Co(II) and Zn(II) complexes was undertaken against the bacteria Escherichia coli and Staphyloccocus aureus by the cup-plate method using nutrient agar as medium.In a typical procedure, molten nutrient agar kept at 45 °C was then poured into Petri dishes and allowed to solidify.Then holes of 6 mm diameter were punched carefully using a sterile cork borer and these were completely filled with test solutions (1000 µg cm -3 in DMF).The plates were incubated for 24 h at 37 °C.The diameter of the zones of inhibition for all the test compounds was measured and the results were compared with the standard drug Ciprofloxacin of the same concentration as that of the test compound under identical conditions.
The antifungal activity of the test compounds was evaluated against the Aspergillus niger and Fusarium oxysporum by cup-plate method cultured on potato-dextrose agar medium adapting similar procedure described above.The plates were incubated at 37 °C for 48 h.The diameters of the zone of inhibition for all the test compounds were measured and the results were compared with the standard drug Griseofulvin of the same concentration as that of the test compound under identical conditions.
Since all test compounds and standard drugs were prepared in freshly distilled DMF, its zone of inhibition was found to be very negligible and taken as zero mm.

Results and Discussion
All the complexes were sparingly soluble in common organic solvents but soluble in DMF, DMSO, and acetonitrile.The analytical data (Table 1) indicate that the complexes are dinuclear in nature.The molar conductance (Table 1) values measured in DMF solution (1x10 -3 mol dm -3 ) fall in the range 50-62 ohm -1 cm 2 mol -1 .These observed values of the molar conductance are well within the expected range for non-electrolytes. 14

IR spectra
The important bands in the IR spectra of the ligand as well as complexes together with their assignments are listed in Table 2.The free ligand showed a broad band at 3432 cm -1 due to ν OH in case of ligand has disappeared in all the complexes indicating its involvement in the coordination with the metal ions via deprotonation. 15The free ligand showed a medium intensity band at 3210 cm -1 assigned to ν NH vibrations, 16 which has been observed in the 3203-3207 cm -1 region in the case of the complexes.It can be observed that there is no considerable shift in the ν NH vibrations in the case of the complexes compared to the ligand, indicates non-involvement of amide NH function in the coordination.A strong intensity band observed at 1675 cm -1 and a medium intensity band at 1630 cm -1 in the case of the ligand are assigned to ν C=O and ν C=N functions respectively.In the case of complexes the band due to ν C=O was observed in the 1670-1676 cm -1 region, indicating its non-involvement in the complexation. 17The band due to ν C=N observed at 1630 cm -1 in the case of the ligand has shown a negative shift of 14-3 cm -1 in the complexes which suggests the involvement of azomethine group (C=N) in the coordination with the metal ions and bonded through the nitrogen atom. 18The band due to phenolic C-O observed at 1240 cm -1 in the case of the ligand has shifted towards higher wave number and this positive shift of the band suggests the coordination of the phenolate anions with the metal ions via deprotonation. 15urther, the appearance of new bands in the 1536-1555 cm -1 region in all the complexes suggest phenoxide bridging with the metal ions. 19The presence of broad stretching vibrations in the 3444-3401 cm -1 region can be attributed to coordinated or lattice water molecules in all the complexes. 15The low frequency skeletal vibrations due to M-O and M-N stretching provide direct evidence for the complexation.In the present investigation, the bands in the 579-515 cm -1 region have been assigned to ν M-O and the bands observed in the 479-417 cm -1 region for ν M-N vibrations respectively. 20,21The bands due to ν M-Cl were observed in the 339-313 cm -1 region and are characteristic of chloro-bridged binuclear complexes. 22

Magnetic susceptibility
The room temperature magnetic susceptibility values are recorded in Table 1.Cu(II) complex of ligand H 2 L showed a magnetic moment of 1.42 B.M. which is considerably lower than the spin only value for Cu(II) complexes.The low value of the magnetic moment is attributed for the antiferromagnetic coupling interaction between two metal ions.This fact suggests the dinuclear nature of the Cu(II) complex. 23On the other hand, Ni(II) and Co(II) complexes have shown magnetic moment values 2.77 and 4.21 B.M. respectively which are lower than the respective spin only values and indicate weak antiferromagnetic coupling interaction 24 between the metal ions which further confirm the dinuclear nature of the complexes.

H NMR spectra
The 1 H NMR spectrum of ligand H 2 L recorded in DMSOd 6 showed a peak at δ 2.09 (s, 6, 2×ArCH 3 ) due to methyl group at 4-position on the phenol rings.Four methylene protons have resonated at δ 3.61 (s, 4, 2×CH 2 ) as a singlet and the four azomethine protons have resonated as a singlet at δ 8.51 (s, 4, 4×CH=N-) and protons due to the amide function have appeared as a singlet at δ 11.70 (s, 4, 4×NH).Two protons due to phenolic -OH have appeared at δ 12.06 (s, 1H) as a singlet whereas the four aromatic protons have resonated at δ 7.23 (s, 4, 2×ArH 2 ) as a singlet.The 1 H NMR spectrum of Cd(II) complex of ligand H 2 L scanned in DMSO-d 6 showed a peak at δ 2.24 (s, 6, 2×ArCH 3 ) due to six methyl protons at 4-position of phenyl rings.Four methylene protons have resonated at δ 3.61 (s, 4, 2×CH 2 ) and four aromatic protons of the phenyl ring have resonated as a singlet at δ 7.58 (s, 4, 2×ArH 2 ) respectively.Four azomethine protons have appeared at δ 8.47 (s, 4, CH=N-) as a singlet.The signal observed at δ 12.06 due to the protons of phenolic -OH group, in case of ligand has disappeared indicating coordination of the phenoxide ions to the metal ions via deprotonation. 15Four -NH protons of amide function have resonated at δ 11.65 (s, 4, 4xNH) as a singlet.These observations reveal that the involvement of phenolic -OH groups in the complexation with the metal ions via deprotonation indicated by the disappearance of the signals due to -OH groups in the Cd(II) complex.Further, the changes in the chemical shifts of azomethine function and aromatic protons in the Cd(II) complex suggest coordination of the azomethine nitrogen with metal ions.

ESR spectrum of Cu(II) complex
The X-band ESR spectrum of the powder Cu(II) complex (Figure 2) was recorded at room temperature using DPPH as a reference standard.The spectrum appeared to be slightly broadened with g ⊥ =2.08 and g ⎟⎥ =2.17.Generally, chlorobridged binuclear Cu(II) complexes give broad ESR signals. 22he observed ESR spectrum is characteristic of square pyramidal geometry.'g' values averaged to overall directions and 'G' which is a measure of extent of exchange interaction between the metal ions have been calculated using the relations g 2 av =1/3(2 g 2 ⊥ + g 2 ⎟⎥ ) and G= (g ⎟⎥ -2)/(g ⊥ -2).By observing the g-values it is clear that g e < g ⊥ < g ⎟⎥ (2.0023 < 2.0844 < 2.1719)) which suggest that d x2-y2 orbital is in the ground state and the d 9 configuration is (e g ) 4 (a 1g ) 2 (b 2g ) 2 (b 1g ) 1 .The 'g' values are related to the axial symmetry 22 and g ⎟⎥ > g ⊥ suggests square pyramidal geometry for Cu(II) complex. 27In the present case the value of 'G' was found to be 2.03.According to Hathaway, 28 if G value is greater than 4, the exchange interaction is negligible whereas G value less than 4 indicates considerable exchange interaction between metal ions in the solid complex.In the present case G=2.03 indicate considerable exchange interaction between the Cu(II) ions in the complex which further supports the dinuclear nature of the complexes.

FAB mass spectrum of Cu(II)-complex of ligand H 2 L
FAB mass spectrum of Cu(II)-complex of ligand H 2 L has been depicted in Figure 3.The spectrum showed a molecular ion peak M + at m/z 770, which is equivalent to its molecular weight.The molecular ion by the loss of four hydrogen radicals gave a fragment ion peak A 1 at m/z 766.The fragment ion A 1 by the loss of three water molecules, two chloride and two methyl radicals simultaneously gave  a fragment ion A 2 , at m/z 612.This fragment ion A 2 by the expulsion of C 5 H 4 N 4 O 2 species gave another fragment ion A 3 at m/z 460.Further, the fragment ion A 3 by the loss of C 3 O 2 species gave a fragment ion A 4 at m/z 391.Finally, the fragment ion A 4 by the loss of C 7 H 4 NO species gave a fragment ion A 5 at m/z 273.The fragmentation pattern is given in Scheme 1.It clearly indicates dinuclear nature of the complex and two Cu(II) ions are held in the macrocyclic compartment of the ligand H 2 L. The metal ions are bonded to two phenoxo bridges which endogenously coordinate to the metal ions and the other coordinating sites in the ligand are the azomethine nitrogen atoms.Both phenoxide and azomethine groups surround the two metal ions which are in close proximity within the ligand molecule to form a square base.A chloride ion coordinates to each metal ion from opposite sides to give square pyramidal configuration to the metal ions.The FAB mass spectrum confirms the dinuclear nature of the metal complex.

Biological activity
The ligand H 2 L and its Cu(II), Ni(II), Co(II) and Zn(II) complexes were tested for their antibacterial and antifungal activity.The zones of inhibitions of the antimicrobial activity have been presented in the Table 4.The results of the antibacterial activity of the ligand H 2 L and its Cu(II), Ni(II), Co(II) and Zn(II) complexes revealed that the ligand as well as its Cu(II), Ni(II), Co(II) and Zn(II) complexes displayed very weak activity against both the bacteria E. coli and S. aureus with 12-14 mm inhibition compared to the standard drug ciprofloxacin which showed 24 mm and 22 mm inhibition against E.coli and S. aureus respectively.
The antifungal activity results of the ligand H 2 L and its complexes revealed that the ligand showed less activity against A.Niger and good activity against F.oxysporum with 14 mm and 18 mm inhibition respectively, compared to the standard drug griseofulvin with 24 mm and 23 mm inhibition.Cu(II), Ni(II), Co(II) and Zn(II) complexes were found to be less active with 10-15 mm inhibition against

Figure 3 .
Figure 3. FAB mass spectrum of the Cu(II) complex.

Table 2 .
IR spectral assignments of the ligand H 2 L and its complexes Figure 1.Structures of the ligand H 2 L and its complexes.

Table 4 .
Antibacterial and antifungal activities of the ligand H 2 L and its complexes