Complexing Behaviour and Antifungal Activity of N-[ ( 1 E )-1-( 1 H-Benzimidazol-2-yl ) ethylidene ] morpholine-4-carbothiohydrazide and Related Ligand with Metal Ions

The coordination complexes of bivalent metal ions with N-[(1E)-1-(1H-Benzimidazol-2-yl)ethylidene]morpholine4-carbothiohydrazide (H 2 bmctz, H 2 L-1) and N-[(1E)-1-(1H-Benzimidazol-2-yl)(phenyl)methylidene] morpholine-4carbothiohydrazide (H 2 bpmctz, H 2 L-2) were prepared and their neutral, monoanionic, and dianionic forms of ligands of compositions [M(H 2 L)X 2 ] (M=CoII, NiII, CuII, ZnII, Cd, or Pd, X=Cl or Br, and H 2 L=H 2 L-1 or H 2 L-2), [M(HL) 2 ]nH 2 O where (M=CoII, CuII, MnII, NiII, ZnII, or Cd, H 2 L=H 2 L-1 or H 2 L-2, and n = 0 or 2), and [MLB]nB (M=CuII, NiII, ZnII, Cd, or Pd, B=H 2 O, Py, or Y-pic, n = 0 and n = 1 if B=H 2 O for Ni(II) and H 2 L=H 2 L-1 or H 2 L-2) have been characterised by magnetic susceptibility measurements, electrical conductance values and spectral properties. The magnetic moment value of [M(HL 2 )] (M=MnII, NiII, FeII or CuII) type complexes is consistent with high spin octahedral structure while those of [M(H 2 L)X 2 ] (M=CoII, NiII, CuII, ZnII, or Cd, X=Cl or Br) possess five coordinated trigonal bipyramidal geometry. The adduct complexes [MLB] ⋅ nB (M=NiII or CuII, B=H 2 O, Py, or Y-pic) are four coordinated planar and those of ZnII and Cd complexes [MLB], (H 2 L=H 2 L-1 or H 2 L-2, B=H 2 O, Py or Y-pic) are tetrahedral.These ligands have been suggested to coordinate as tridentate (N N S) donor molecule in complexes of type [M(H 2 L)X 2 ], [M(HL) 2 ], and [MLB]. The antifungal activity of ligands and some of their metal complexes were studied and it was observed that metal complexes show higher activity than free ligand.


Introduction
Preparation and structural aspects of various metal ions with a number of benzimidazole derivatives have been reported by one of us and a number of chemists [1][2][3][4][5][6][7][8][9][10].Medicinal properties of benzimidazole derivatives have established that benzimidazoles have privileged substructures for drug design [11,12].The most vital benzimidazole derivatives is Nribosyl-5,6-dimethylbenzimidazole which is axial ligand in vitamin B 12 possessing selective neuropeptide YY1 receptor antagonists [13], 5-lipo xyginase inhibitor [14] and poly (ADP-ribose) polymerase inhibitors [14].Benzimidazole derivatives are of immense interest because of their wide spectrum of biological activity such as anticancer [15], antiviral [16,17], antihistaminic [18], antifungal [19], anti-HBV [20], antibacterial [21], antitumoral [22], antiparasitic [22], antihelminitic [23], anti-inflammatory, local analgesic, hypotensive [24], antiulcer [25], and neuro leptic cardiotonic [26].Extensive biochemical and pharmacological studies have confirmed that various benzimidazole derivatives are effective against various strains of microorganisms [25].The wide ranging biological and industrial interest intrigued chemists to explore new benzimidazole derivatives and their metal complexes which might have novel mechanisms of action and drug activity [15][16][17][18][19][20][21][22][23][24][25].The metal ions selected for present studies Mn(II), Co(II), Cu(II), Fe(II), Ni(II), Zn(II), and Cd(II) are versatile essential micronutritents in biological systems [27][28][29][30][31][32].Copper is crucial for biological function of several enzymes and proteins, in energy metabolism, antioxidant, and mitochondrial respiration [27].Zinc(II) is essential for nucleic acid synthesis, cell growth division, and differentiation and its deficiency causes abnormalities in function of cell carboxypeptidase, amino peptidase, carbonic anhydrase, DNA and RNA polymerase.Zinc is also essential for life and it regulates the function of genes in the nuclei of thecell (Lipsocomb and Starter, 1996).Iron is the most vital element for life and iron-sulphur proteins formation (Richardson, 2002).Manganese is a component of nucleic acid and it accelerates the synthesis of cholesterol.Manganese enzymes are involved in metabolic pathway of DNA synthesis, sugar metabolism and protein modification.Cobalt is a component of vitamin B 12 and enzyme nutritional cofactor necessary for the formation of red blood cell [31].Nickel is remarkably useful metal in biological chemistry.It modifies antioxidant system and is associated with chromosomal aberrations and mutation [31].In continuation of our work on coordinating behaviour of benzimidazole and imidazole derivatives with various metal ions [2][3][4][5][6], and biological significance of above metal ions, we report in present communication, the preparation, characterization, and antifungal activity of complexes of these metals with of

Material and Methods
The metal salts used were usually BDH Anal-R chemicals or E. Merk Extra pure reagent.Palladium(II) chloride was obtained from Johnson Matthey London.Solvents and organic reagent, hydrazine, morpholine, carbon disulphide, Chloroacetic acid were obtained from E. Merk, Nice, Sd.Fine and Ranbaxy.The metal contents of complexes were estimated by standard method sulphur content was determined as sulphate, and nitrogen content was estimated by Nitrometer (Duma , s method) in the laboratory.The infrared spectra of complexes were recorded on Shimadzu-FTIR spectrometer at IIT patna.The results of C, H, and N were obtained from CDRI Lucknow.The results of mass spectrum and U-V absorption spectrum of ligand and some complexes were obtained from IIT patna.The 1 HNMR spectrums were recorded at CDRI Lucknow.The magnetic susceptibilities of complexes were determined by Gouy method at room temperature.The electrical conductance values of complexes were determined in DMF on Systonic conductivity Meter Bridge at 30-31 ∘ C. The results of measurements are given in Tables 1 and 2.
Ligand molecules (H 2 bmctz, H 2 L-1 or H 2 bpmctz, H 2 L-2) were prepared by condensing 2-acetylbenzimidazole or 2-benzoylbenzimidazole [33] with morpholine-Nthiohydrazide [34] in aqueous ethanol containing a little amount of acetic acid.2-Acetylbenzimidazole or 2benzoylbenzimidazole in situ was prepared by oxidation of 1-(1H-benzimidazol-2-yl)ethanol and 1-(1H-benzimidazol-2-yl)phenylmethanol [35] with chromic acid [33].The 0.1 mole of 2-acetylbenzimidazole in 100 mL aqueous ethanol containing 2 mL acetic acetic acid was refluxed with 0.12 mole of aqueous ethanolic solution of morpholine-N-thiohydrazide(morpholine-4-carbothiohydrazide) on a steam bath for two hours.The ligand separated as cream of yellow product. ).About 20 millimoles of ligand was dissolved in 80-90 mL hot methanol and treated slowly with aqueous methanolic solution of 10 millimoles of appropriate metal acetate {chloride in case of Pd(II) and sulphate in case of Fe(II)} and refluxed on steam bath for half an hour.In some cases a little aqueous solution of sodium acetate was added to adjust the pH 6-8.In most of the cases complexes were obtained immediately on diluting them with water.The products were digested on a steam bath and collected on a filter, washed with aqueous methanol, and dried over CaCl 2 .Yield is 95-98%.

Preparation of [MLB]⋅nB (M=Cu
. About 5 or 10 millimole of metal chloride was dissolved in 30-40 mL aqueous methanol and treated with 5-6 mL pyridine or pic.The resulting solution was treated with 5 or 10 millimole of ligand dissolved in hot methanol (20-25 mL).The pH of the solution was raised by adding dilute ammonia when appropriate complexes separated.The aqua complexes were obtained on refluxing [MLPy] in hot water in presence of a little ammonia.The products were collected on a filter, washed with cold aqueous methanol, and dried over CaCl 2 .The results of elemental analysis and physical data of complexes are presented in Tables 1 and 2.

Results and Discussion
The   [39].In case of Mn(II) no distinct d-d transitions could be located definitely, since all d-d transitions of Mn(II) complexes are of spin forbidden type.Iron (II) complexes display a shoulder near 440 and 450 nm attributable to 5 T 2g → 5 E g transitions in octahedral field [37][38][39] (See Figures 1 and  2).The IR spectra of ligands and their complexes were recorded as KBr optics in range 4000-400 cm −1 .The diagnostic I.R. bands of ligands and some of their complexes are recorded in Table 3.The ligand H 2 L-1 displays medium I.R. bands at 3350, 3240, 3120, 3040, 2949, 2860, and 2840 cm −1 attributed from ](NH), phenyl (C-H) stretches, and morpholine ring ](CH 2 ) vibrations and most of them are retained in complexes.The ](C=N), ](N-N) of imidazole ring and azomethene part of ligand were observed at 1653, 1610 and 1585 cm −1 .The (NH) of ligand was located at 1515 and 1485 cm −1 .The IR spectrum of Cu(H 2 L-1)Cl 2 displays azomethene ](C=N) at 1622 cm −1 and imidazole ring ](C=N) at 1590 which is lower than free ligand molecule that indicated of types [M(HL-2) 2 ] and [M(L-2)B] supporting bonding through deprotonated thiol sulphur of thioamide group [40,41].The I.R. spectral band of both H 2 L-1 and H 2 L-2 shows similar trend of bonding of donor atoms.Infar I.R. range, the I.R. spectra of complexes were recorded above 400 cm −1 , therefore ](M-S) and ](M-N) stretches could not be located definitely.However, the new I.R. bands in complexes at 420-435 cm −1 are tentatively suggested to ](M-N) and ](M-S) vibrations (Table 3).In Finger print region H 2 L-1, H 2 L-2, and their complexes display a number of I.R. bands due to phenyl ring skeletal vibrations and (CH 2 ), (CH 3 ), ](N-N), ](C-N), and ](C-C) vibrations as well as ring deformation vibrations [40,41] C-H) proton signal at  = 6.93-7.28 as multiplet (9H).The benzimidazole ring (NH) and hydrazide (N-H) proton signals were located at  = 7.415 and 8.215 ppm.The mass spectrum of H 2 L-1 gave  + / at 303(3%), 288(3.8%),217(40%), 117(100%), and 86(30%).The calculated value of mass was 303.The mass spectral results of H 2 L-2 indicated  + / values 365(5%), 288(3%), 279(8%), 86(30%), and 117(100%) as base peak.The mass calculated for H 2 L-2 was 365.The 1 HNMR spectra of complexes could not be recorded due to their poor solubility (See Figure 3).method [43,44].Czapek agar medium was prepared by dissolving 20 g starch, 20 g agar agar and 20 g glucose in one litre distilled water.The resulting medium was added requisite amount of test compound to get 100 and 200 ppm of solution.The resulting medium was then poured into Petri plates and the spores of fungi were placed on medium with the help of inoculum needle.The petri plates were wrapped in polythene bags containing two to three drops of ethanol and then placed in incubator at 30 ± 0.5 ∘ C. The linear growth of fungus was evaluated by measuring the fungal zone diameter after five days.The percentage inhibition was calculated from the relation 100 × ( − /) where  and  are the diameter of the fungus colony and control test plate, respectively.The fungi used in present investigation for screening against the fungi, Aspergillus niger, Aspergillus flavus, F. oxysporum, Rhizoctonia bataticola, and R. phaseoli.The control solution was mycostatin.The result of activity is shown in Table 4.It has been found that the ligand containing phenyl substituent is more active than methyl substituent.The Cu(II) and Zn(II) complexes in general show larger activity than Mn(II) and Fe(II) complexes.The bromo complexes show larger activity than that of chloro or pydridine or -picoline adduct.The chemical Mancozeb fungicides were used as standard for this process.In general the antifungal activities observed for Cu(II) and Zn(II) halo complexes were much larger than other metal complexes and ligand.The antifungal activity observed for R. phaseoli is larger compared to other fungi, indicating selectivity of complexes for particular fungi.The enlarged activity of complexes may be attributed to increased delocalisation of -electrons of ligand over the whole chelate ring which enhances the lipophilicity of the complexes.The increase lipophilicity enhances the penetration of complexes into fungi membrane and blocking the active binding sites in the microorganism.In addition the complexes disturb the respiration process of the fungi cell thus blocking the synthesis of proteins essential for fungi growth and restrict the further growth of the organism.The activity of complexes of phenyl substituted ligand is larger than methyl derivatives due to larger -delocalised electron in phenyl substituted ligand than that of methyl derivatives and free ligand as well.
In addition to above facts, the chelate formation reduces the polarity of metal ions, increases the lipophilic character of the chelate, and increases the interaction between metal ion and fungi cell membrane.Consequently it blocks the cell growth of microorganism.According to overtone's concept the lipophilicity of complexes is major factor which favours International Journal of Inorganic Chemistry  antimicrobial activity [45,46].The hydrogen bond formation between fungi and complex molecule through azomethene (C=N) and heteroatoms S and O and halogens of complexes interacts with cell constituents resulting in interference with the normal cell process.These factors are tentatively suggested for enhanced antimicrobial activities of metal chelates complexes of N and S donor.
From the results of I.R. and physical data, the probable structures of complexes are shown in Figures 4, 5, 6, and 7.

Conclusion
The physicochemical data suggested octahedral structure for [M(HL) 2 ] type of complexes of Ni(II), Co(II), and Cu(II) while four coordinated planar to Pd(II) complexes.The monoligated Ni(II) and Pd(II) complexes [M(L-1)/(l-2)B] show characteristics of planar geometry.The complexes of [M(H 2 L)Cl 2 ], (M=Co II , Ni II , Cu II , Zn II or Cd II ) have been suggested to possess trigonal bipyramidal structures.The ligand H 2 L-1 and H 2 L-2 coordinate as neutral, monoanionics or dianionic tridentate (N, N, S) donor molecules.The biological activity of phenyl substituted ligand and its complexes are larger than those of methyl substituted products probably due

Table 1 :
Analytical results and physical data of complexes.
The elemental analysis of complexes of Mn II , FeII, Ni II , Co II , Cu II , Zn II , Cd II , and Pd II corresponds to compositions [M(H 2 L)X 2 ] (M=Cu II , Ni II , Zn II or Cd II and X=Cl − or Br − ) [M(HL) 2 ]H 2 O (M=Mn II , Fe II , Co II , Ni II , Cu II , Zn II , Cd II or Pd II , H 2 L=H 2 L-1 or H 2 L-2,  = 0 but 2 for Co II and Ni II ) and [MLB]B (M=Ni II , Cu II , Pd II , Zn II or Cd II and H 2 L=H 2 L-1 or H 2 L-2, B=Py, -pic or H 2 O and  = 0 or 1).These complexes are quite stable at room temperature and do not lose texture in desiccator.On heating the complexes [M(HL) 2 ⋅ H 2 O (M=Co II , or Ni II , and HL=HL-1 or HL-2) in air oven between 60 and 80 ∘ C for one hour, is loss H 2 O and loss in weight incurred in complexes correspond to loss of required for 2H 2 O, and no change in colour of the complexes that was noticed which indicated that H [37,38]oordinating molecule capable of forming five membered strong chelates with The elemental analysis of complexes of Ni II , Co II , Cu II , Zn II , Cd II and halide formed in dry ethanol corresponds to compositions [M(H 2 L)X 2 ], and (H 2 L=H 2 L-1 or H 2 L-2, X=Cl or Br) metal ions.Depending on pH of medium, these ligands coordinate as neutral, monoanionic, and dianionic donor molecules.2Omolecules are not coordinated to metal atom rather they are held up in crystal lattices.The weight loss incurred in complexes Ni(L-1)2H 2 O and Ni(L-2)2H 2 O at 120 ∘ C corresponds to loss of only one H 2 O, indicating that one H 2 O is coordinated to nickel (II) in complex [NiL(H 2 O)]H 2 O and the second H 2 O is held up in crystal lattices.The dihalo complexes are formed in dry ethanol by interacting solutions of ligand with metal halides.The bis ligated complexes [M(HL) 2 ]H 2 O were formed in neutral or weakly basic medium (pH 6-8) by interacting aqueous methanolic or ethanolic solutions of metal acetate or metal chloride and appropriate ligand in 1 : 2 molar proportion.The ligands coordinate as dianionic tridentate molecule in much basic medium (pH 9-12) and form complexes of stoichiometry, [MLB]⋅nB (M=Cu II , Ni II , Zn II , Cd II or Pd II and H 2 L=H 2 L-1 or H 2 L-2, B=H 2 O, py or -pic and  = 0 or 1).The complexes are all insoluble in water but aqueous ethanolic suspension of [M(H 2 L)X 2 ] gradually dissociates into M(HL) 2 and MX 2 ] show magnetic moment value (4.54-4.71B.M) similar to five coordinated TBP structure[37,38].As expected zinc(II) or Cd(II) complexes M(HL) 2 and [M(H 2 L)X 2 ] (X=Cl − or Br − ) are diamagnetic.The electronic absorption spectra of ligand (H 2 L-1 or H 2 L-2) and their complexes were determined in ethanol or ethanol