Synthesis, Spectral Analysis and Biological Potency of Hydrazoneoxime Ligands Incorporating Pyrazolone Moiety and Their Metal Complexes

A modest attempt has been made for the synthesis of hydrazoneoxime ligands bearing pyrazolone group (1-4) and their successive metal complexes such as: 1(a-c), 2(a-c), 3(a-c) and 4(a-c). The precursor (1Z,2E)-2-(hydroxyimino) ethanehydroximohydrazide (GH2) was obtained through coupling of (1Z,2E)-N-hydroxy-2-(hydroxyimino) ethanimidoyl chloride and hydrazinium hydroxideto generate hydrazonoxime compounds bearing the pyrazolone group. The ligands (1-4) were reacted with MX2.nH2O, where M = Co(II), Ni(II) and Cu(II) to obtain the successive metal coordinated compounds into good yields. The ligands and their metal complexes were investigated by using 1H NMR, 13C NMR, FT-IR, elemental analysis and magnetic susceptibility measurements. Tautomerism in the ligands is investigated spectroscopically and biological activities are evaluated as well. Finally, the findings of present study were found within good egreement with other workers.


INTROdUCTION
Pyrazolone is considered as an important component or structural unit which is present in various active compounds. Owing to its convenient synthesis as well asversatile biological applications particularly its comprehensive antiseptics, antitumor, antibacterial action [1][2][3][4] , pyrazolone and its complexes have acquired a significant consideration in coordination as well as in medicinal chemistry.
Pyrazolone derived product sare paramount kind of heterocyclic compounds that occurs in various drugs as well as in synthetic products 5,6 . These compounds show extraordinary analgesic 7 , antitubercular 8 , antifungal, antibacterial 9 , antiinflammatory 10 , antioxidant and antitumor activities 11 . Because of their facile synthesis and character is ticbiological activity, pyrazolone arrangement performs a significant function and reflects an effective example for combinatorial and pharmaceutical chemistry. Furthermore, pyrazole derived products showed outstanding biological asset, for example, anti-microbial 12 , analgesic 13 , anti-inflammatory 14 and anticancer activities 15 . This provided anenormous boost to explore for potentially active compounds having pyrazole substituents.
Here in, the derivatization of hydrazone oxime ligands bearing pyrazolone group (1-4) and their metal complexes1(a-c), 2(a-c), 3(a-c), 4(a-c) was reported. The ligands and its complexes were characterized by 1 H NMR, 13 C NMR, FT-IR spectroscopy, elemental analysis and magnetic susceptibility techniques. The proposed general structure of the ligand is given in scheme.1 ethanehydroximohydrazide (GH 2 ) was prepared by the action of the (1Z,2E)-N-hydroxy-2-(hydroxyimino) ethanimidoyl chloride 16 and hydrazinium hydroxide. Since the parent compound, GH 2 is unstable at normal temperature, so it was utilized without further purification or as it was received. Hydrazone oxime ligands bearing pyrazolone group (1-4) have beenprepared with the 5-pyrazolones as mentioned in the literature 17 The preparation procedures were followed as given in theliterature [18][19][20] . For the synthesis of (1-4) the common route follows as given in the Scheme 2.

General procedure of synthesis Synthesis of hydrazoneoxime ligands bearing pyrazolone group(1-4)
Aniline derivatives 10 mmol (0.69 g, aniline; 1.38 g, 4-nitroaniline; 1.23 g, p-anisidine; or 1.04 g, p-toluidine respectively) were taken and dissolved in a mixture of 1:1 ratio of glacial acetic acid and concentrated hydrochloric acid (20 mL) and cooled the solution upto 0-5 o C. Sodium nitrite (0.69 g or 0.01 mol) was taken and dissolved in 10 mL water and then added dropwise to the above-prepared solution mixture with vigorous stirring for nearly one hour to maintain the solution temperature between 0-5 o C. The obtained diazonium solution has been mixed in aliquots for 30 min to thesolution of 2-[(1Z,2Z)-N-hydrokis-2-(hydrokisimino)etanimidoil]-5-metil-2,4-dihidro-3Hpirazol-3-on (0.76 g or 0.01 mol) in 10 mL of ethyl alcohol and stirred vigorously, added the NaOH solution for maintaining the pH level between 7-8 . The mixture has bee stirred for 2 h and maintain the solution temperature between 0-5 o C. The obtained product was separated by means of diluting with water (50 mL), filtered, washed several times with distilled water and finally dried.

Instruments and reagents
Reagent sused in the experiment have been obtained from Sigma-Aldrich, Merck and Fluka and were handled as received. The melting point of the synthesised complexes and the ligands were varified and calculated by the Büchi SMP-20 apparatus using an open capillary method. For the calculation of FT-IR spectra, KBr discs on a Perkin Elmer Mattson 1000 spectrophotometer were used, 1 H and 13 C NMR spectra have been reported by Bruker-Spectrospin Avance DTX 400 Ultra-Shield in deuterated dimethyl sulphoxide (DMSO-d 6 ) and tetramethylsilane (TMS) used as an internal standard and chemical shifts considered in ppm. The Leco CHNS-932 analyzer used for the elemental analysis: and for the pH measurements, an Orion Expandable Ion Analyzer EA 940 was used. The melting point, colors, molecular weights, percentage of yield, molar conductance, magnetic susceptibilities (Sherwood Scientific) have been calculated and are given in the following sections.

Compound's Synthesis
For the synthesis of all three complexes, a general method was used 16 . The 2 mmol of the above ligands (1-4), were dissolved in 10 mL absolute ethyl alcohol at refluxing temperature. A solution of NiCl 2 .6H 2 O or CuCl 2 .2H 2 O or CoCl 2 .6H 2 O (1 mmol) with water (15 mL) is taken and then mixed dropwise over the ligand solution with vigorous stirring, a noticeable color change as well as a reduction in the pH value (~3.0-3.5) was observed. Sodium hydroxide (1%) in water (20 mL) has been mixed to adjust the pH ~5-5.5 and maintained the temperature of the reaction mixture nearly to room temperature. Mixtures have been stirred by the hour at 50 o C on a water bath so as to ensure complete precipitation of complexes. After one hour the precipitated solid has been filtered, washed by means of hot ethanol (3x5 mL) also dehydrated in vacuo, over anhydrous CaCl 2 . Proposed structures for the monomeric Co(II), Ni(II) Cu(II) as well as the ligands (1-4) are given in Scheme 3.

In vitro antimicrobic activity
The antimicrobic activity of the test compounds was evaluated by way of agar well diffusion method [21][22] . 0.1mL of the diluted inoculums (106 CFU/mL) of test organisms are taken and spread on NA/SDA (Nutrient agar/Sabouraud dextrose agar) plates. Wells of 6 mm diameter have been punctured into the agar medium and filled one byone with 150 mL of compound (150 µg/L) solvent blank and anantibiotic (chloramphenicol, 100 µg/L) to which the test the sensitivity of bacteria. Fluconazole at the concentration of 100 µg/L has been applied for the monitor versus Candida albicans, Candida tropicalis and Candida glabrata. The plates were incubated for at 37°C 24 hours. Antimicrobial activity was has been assessed in order to the zone of inhibition versus the test organism.

Spectral studies: structure of ligands and complexes
Synthesis of new hydrazoneoxime ligands bearing pyrazolone group (1-4) that were prepared by reaction of (1Z,2E)-N-hydroxy-2-(hydroxyimino) ethanimidoyl chloride [16][17] with hydrazinium hydroxide and their metal complexes1(a-c), 2(a-c), 3(a-c),  4(a-c)were reported (Scheme 2 and 3). The ligands used in this work were prepared using the literature mentioned elsewhere [18][19][20] . The atomic arrangement of the synthesized ligands(1-4) including their metal complexes were established on the basis of their elemental analysis, FI-TIR, 1 H NMR, 13 C NMR, along with the calculation of their magnetic susceptibility as well. The analytical and physical properties of the prepared ligands including their metal complexes be summarised in Tables 1,3,5, along with 7.
The FT-IR spectrum related to the synthesized ligands(1-4) as well as metal complexes1(a-c) ,  2(a-c), 3(a-c) and 4(a-c). 1-4 and 1(a-c), 2(a-c),  3(a-c) and 4(a-c) exhibited a carbonyl (keto) band at 1655-1667 cm -1 and NH band (hydrazo) at 3168-3271 cm -1 as shown in the IR spectral data of the representative ligand (1) (Fig. 1).The IR frequencies of the representative ligand and its complexes are shown in Table 1. The IR spectral collected statistics of the synthesized metal complexes has to begiven into Tables 2,4,6 and 8. Such said values indicated that each complexes are exist in keto-hydrazo form (T 2 ) as well as in solid-state. [23][24][25] The 1 H NMR spectra of all ligands (1-4) in DMSO-d 6 showed a single peak lying between 2.16-2.20 ppm corresponding to methyl protons (pyrazolone-CH 3  The coordination compounds of nickel(II), copper(II) and cobalt(II) have been synthesized as per general methodology 1,16 as discussed previously (Scheme 3). The metal ion and ligands react in 1:2 molar ratios, where the ligands are attached to metal by using its two N atoms, like almost all of vic-dioximes act. All of this coordinated compounds are colored, amorphous solids, stable at room temperature as well. The discussed complexes are insoluble in commonly used organic solvents, but are soluble in DMF and DMSO. The recommended structures of the synthesized coordination compounds has beendemonstrated in Scheme 3 that are supported by spectroscopic, FTIR spectral date and elemental analysis studies. As per the suggested structures, the complexes can have syn-or anti-conformation 28 .The magnetic moment (µeff) statistics further favoured the mononuclear structures of the coordinated complexes. The magnetic moment for the nickel(II) complexes are diamagnetic at room temperature, since it is expected due to the d 8 1(a-c), 2(a-c),   3(a-c) and 4(a-c) has to be explained in Scheme 5. The newly synthesized complexes possibly occurs into four probable tautomeric forms i.e. keto-azo (T 1 and T 4 ), keto-hydrazo (T 2 ) as well as in enol-azo (T 3 ) form. As reported in the literature, utmost stabilized tautomeric form is a keto-hydrazo form (T 2 ) for azopyrazolone dyes. The complexes have an intramolecular hydrogen bonding N-H…O for keto-hydrazone (T 2 ) form and O-H…N for enol-azo (T 3 ) form.These results are consistent with the literature 23,[26][27][28][29]31 .

Biological activity
The synthesized compounds have been examined for their antimicrobial (antibacterial andanti-fungal)activity.

A-Candida albicans, B-Candida trophicalis, C -C a n d i d a g l a b r a t a , D -S t a p hy l o c o c c u s aureus, E-Bacillus subtilis, F-Escherichia coli, G-Pseudomonas aeruginosa
Results of the synthesized compounds1a,  1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a, 4b and 4c are outlined for antimicrobic activity with respect to four bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and three fungal as Candida albicans, Candida tropicalis and Candida glabrata are illustrated in Table 9.
Antimicrobial activity against "Gram -positive", "Gram-negative" bacteria and agaist fungus have been seen across all the synthesized compounds. Newly synthesized compounds 3b showed signifant gross broad-spectrum antimicrobial activity, i.e., against "Gram-positive","Gram-negative bacteria "as well as anti fungal. The effective concentration of these active compounds was 150 µg/well.

CONCLUSION
Investigations into the synthesis of new hydrazone oxime ligands having pyrazolone group and their metal complexes have been carried out in this study. The synthesized pyrazolone derivatives of Co(II), Ni(II) and Cu(II) complexes were isolated and their structures were characterized using physicochemical techniques. Also, tautomerism in the ligands was investigated by spectroscopic.Chemical propertirs of the complex compounds depend upon Tautomerism and also physical properties like colour fastness depend on it. The attachment of ligands to the metal ion in a neutral bidentate form with the azomethine nitrogen (C=N) and the carbonyl oxygen (C=O).The calculated magnetic moments of the coordinated complex compounds as 2.92 B.M for the nickel(II) and 1.65 B.M for the copper(II) are approximate the spin values only and propose a square planar geometry for the complexes. The compounds 3b exhibited broad-spectrum antimicrobial activity. Expeditions to explore their potentialities in the future for other biological assays are needed to investigate further. Furthermore, some Co(II) complexes act as potent anti-cancer agents due to their anti-proliferative effects; in this regard, the synthesized compounds should be investigated in the future as well. These complexes can be further explored in the dye/color industry due to the presence of chromophores.