Synthesis and Bio-Spectral Studies of Co(II) Complex of 5-Chloro-2,4-Dihydroxy Butyrophenoneoxime (CDHBOX)

Co(II) complex of 5-chloro-2,4-dihydroxy butyrophenoneoxime (CDHBOX) was synthesized from 5-chloro-2,4-dihydroxy butyrophenoneoxime by using standard protocol, and characterized by elemental analyses, melting point determination and spectral data. The ML2 (metal/ligand) stoichiometry of the complex was determined by spectrophotometric and potentiometric studies, and mass spectral data. The value of stability constant of the complex was found to be 6.94x 108 while its standard free energy of formation is 12.155 kcal/mol at 27ºC. Beer’s law is obeyed in the concentration range 2-15 ppm of Co. The value of molar extinction coefficient and sensitivity as per Sandell’s scale were found to 3.35x 103 L.mol-1cm-1and 0.017 μg Co/cm2 respectively. The value of activation energy and the Arrhenius constant Arrhenius constant were found as 4.949 kjmol -1 , 74.39. The IR studies reveal that the phenolic proton is lost on complexation and the oxygen of the phenolic (–OH) and nitrogen of the oximino (=NOH) groups coordinate with Co(II) ion. The electronic spectra and magnetic susceptibility measurement indicate that the complex is paramagnetic and tetrahedral in nature. The antimicrobial activity of different concentrations of ligand and its Co(II)-complex has been evaluated against Curvularia lunata, Fusarium oxysporum and Alternaria alternata fungi and Streproproteus, Staph, Escherchia coli, Klebsella, and pseudomonas bacteria. The results indicated that the ligand (CDHBOX) and its Co(II) complex have good anti-microbial properties. Absorbance measurements of a set of six solutions prepared in a similar way, and having the same concentration of all the reagents, show that the reproducibility of measurements is quite good with a standard deviation of 0.26%. The stability constant of the complex is found to be 6.94 x 10 8 , and the value of standard free energy of formation is 12.155 kcal/mol at 27ºC.


Physical measurements
The elemental analyses were carried by Elementar Vario EL III Model, and the estimation of metal was performed by AA-640-13 Shimadzu flame atomic absorption spectrophotometer. A Systronic spectrocolorimeter (Type 103) was used for the absorbance measurements and pH measurements were made on a Systronic(335) digital pH-meter, and the values corrected by using Van Uitert and Hass equation [20]. The electronic spectrum of the complex was recorded on Beckman DU-64 spectrophotometer. The IR spectra of the ligand and its metal complex were recorded on Perkin Elmer FT-IR spectrophotometer in KBr; their NMR spectra was recorded by high performance FT-NMR spectrometer. The FAB mass spectrum of the complex was recorded at USIC facility at IIT, Roorkee. Magnetic susceptibility measurement was carried out at room temperature by using powdered sample on a vibrating sample magnetometer PAR 155 with 5000 G-field strength, using Hg[Co(CNS) 4 ] as a calibrant. TG curve was recorded by Rigaku Model 8150 thermo-analyzer at the heating rate of 5 º min -1 . The instrument was calibrated by calcium oxalate for TG. The TG curve helped to identify the number of decomposition steps. The thermodynamic activation parameters such as E, ∆H, ∆S and ∆G were calculated from potentiometric data, using Coats and Redfern method [32].

Synthesis of 2,4-dihydroxy butyrophenone(DHB)
It was prepared by condensing resorcinol (0.1 mol) with butyric acid (0.1 mol) in the presence of anhydrous zinc chloride (0.1mol). The reaction mixture was kept at 160-165 o C for half an hour. It was then cooled and poured in ice cold 50% hydrochloric acid. The precipitated product 2,4-dihydroxy butyrophenone (DHB) was filtered, and crystallized from ethanol. The purity was checked by melting point (65 o C) and TLC.

Synthesis of 5-chloro-2,4-dihydroxy butyrophenone (CDHB)
5-chloro-2,4-dihydroxy butyrophenone (0.1mol) was dissolved in minimum quantity of ethanol in around bottomed flask fitted with an inlet tube and a reflux condenser. Dry chlorine gas was then passed into the flask through the inlet tube maintaining the temperature of the solution between70-80 0 C. The product was filtered, and crystallized from aqueous ethanol. The purity was checked by melting point (95 o C) and TLC.

Potentiometric studies
Calvin and Bjerrum technique [27] was used to determine stability constant of the complex by evaluating n , n H and pL values at different temperatures, and concentrations by using standard formulae [28].
The following solutions were titrated against standard carbonate free sodium hydroxide ( 0.05 M ) to carry out potentiometric studies:

Spectrophotometric studies
A Systronic spectrocolorimeter (Type 103) was used for the absorbance measurement while pH value was adjusted on a Systronic (335) digital pH-meter. The nature of complex was determined by Vosburg and Cooper method [30], and its composition was known by using standard protocol [31].

Antibacterial screening
The antibacterial activitiy of the test compounds (CDHBOX and its Co-complex) was measured by paper disc diffusion method [22] using agar nutrient medium and 5mm diameter paper discs of Whatman No. 1 filter paper. The filter paper discs were soaked in a solution of known amount (0.05 to 0.40% w/v) of test compounds and a standard specimen (prepared in DMF), dried and laid on the surface of petri-plates which were already seeded with the test organism Streproproteus , Staph, Escherichia coli. Klebsella, and pseudomonas bacteria. All the agar dishes were then incubated in an incubator at 27±1ºC for about 48 hours. After incubation for the stated period, the growth of the micro-organism was measured in terms of inhibition zone(mm), formed in each disc in the form of a turbid layer, except in the region where the concentration of antibacterial agent is above the MIC. The size of the zone of inhibition depends upon sensitivity of the organism, nature of the culture medium, incubation conditions, rate of diffusion of the agent, and the concentration of the antibacterial agent.

Antifungal screening
The antifungal activity of different concentrations (0.05 to 0.40%w/v) of test compounds and a standard specimen ( prepared in DMF), was measured by determining the growth of test fungi Curvularia lunata, Fusarium oxysporum and Alternaria alternata by dry weight increase method. Richard liquid medium was used as culture medium [23] in the experiment. The test compound of varying concentration (0.05 to 0.40% w/v) was directly added in to the Richard liquid medium carrying the test fungus in a sterilized chamber, and was kept for seven days in an incubation chamber at 27±1ºC . Media with test solution served as treated while that without it as check. The resultant mycelial mats in each set were carefully removed, washed, dried and then weighed separately. The percentage inhibition was calculated by the following formula: Percentage inhibition of fungal growth =

Results and Discussion
The complexation reaction between metal ions and the ligand may be represented as

Spectrophotometric studies
Vosburg and Cooper method [30] shows that Co(II) ion forms only one complex with CDHBOX having max  at 410 nm in the pH range 7.5-9.0 .
The absorbance was measured at room temperature at regular intervals of time up to two weeks, and also at different temperatures varying from 300 K to 325 K. The results showed that the complex is stable for one week at 318 K without any change in absorbance. The optimum pH range for the complexation was 8.0. It was also found that a four fold excess of the reagent was necessary to attain the maximum colour intensity.
The composition of the complex was found to be 1:2 ( metal : Ligand ) by job , s method, mole ratio and slope ratio method  Absorbance measurements of a set of six solutions prepared in a similar way, and having the same concentration of all the reagents, show that the reproducibility of measurements is quite good with a standard deviation of 0.26%.
The stability constant of the complex is found to be 6.94 x 10 8 , and the value of standard free energy of formation is 12.155 kcal/mol at 27ºC.
Beer's law is obeyed in the concentration range 2-15 ppm of Co. The value of molar extinction coefficient and sensitivity as per Sandell's scale are 3.35 x 10 3 L mol -1 cm -1 and 0.017 µg Co/cm 2 respectively.

Effect of foreign ions
The effect of foreign ions on the spectrophotometric determination of cobalt was studied by adding these ions in quantities ranging from 50 to 2000 ppm to a solution containing a known amount of cobalt. After adjusting the pH of the solution at 8.0, cobalt was extracted as Co(II)-CDHBOX complex in the usual manner, and the absorbance of the organic layer measured. UO ions interfered seriously. A limit of 2.0% change in the absorbance was observed as the limiting concentration.

Magnetic moment and Electronic spectrum
The observed magnetic moment value (4.31 B.M.) of Co(II)-CDHBOX complex indicates the present complex is paramagnetic and has tetrahedral geometry. The bands occurring at 4056, 7285, and 18690 cm -1 in the electronic

spectrum of the complex correspond to the 4 A 2 (F) → 4 T 2 (F), 4 A 2 (F) → 4 T 2 (F) and 4 A 2 (F) → 4 T 2 (P) transitions
respectively. The values of B , , β and λ were also calculated. The values of β is comparable to that reported for a tetrahedral complex.

Infrared spectra and mode of bonding
The IR spectra of metal chelate and of free ligand were recorded both in the high frequency region (650-4000cm -1 ) and low frequency region (50-650cm -1 ). In general, vibrations which occur in the high frequency region, originate due to the ligand itself whereas those in the lower frequency region originate due to the metal-ligand bonds (Table 1). In CDHBOX, the broad band at 3320cm -1 has been assigned to the phenolic OH group. The band at 3270 cm -1 is due to =NOH group.
The band at 2970cm -1 and 2870 cm -1 are due to C-H stretching vibrations, the band at 1670 cm -1 is due to C=N stretching, the bands at 760 cm -1 are due to the presence of C-Cl stretching and the band at 990 cm -1 is due to N-O stretching. The band at 1190 cm -1 further confirms benzene ring substitution. The absence of band at 3320 cm -1 and a strong band of the free ligand at 1290 cm -1 is due to C-OH (phenolic) shift to higher frequency region in the complex which indicates deprotonation of the phenolic group, and coordination of the phenolic oxygen to Co(II) ion. The shifting of broad and low intensity band due to ν(O-H) mode of N-OH group from 3270 cm -1 to 3250 cm -1 suggests weakening of N-OH bond due to the formation of Co-N bond. The coordination of the oximino group through nitrogen is indicated by lowering of the C=N band from 1670 cm -1 in the ligand to 1630 cm -1 in the metal complex. Shifting of the N-O band at 990 cm -1 in CDHBOX to 1015 cm -1 in the metal complex further suggests the participation of nitrogen of the oximino group in the complexation with the formation of a Co-N bond. In the IR spectrum of the complex, the bands observed at 655 cm -1 and 470 cm -1 are assigned to the Co-N and Co-O stretching vibrations [24]. A band at 1368 cm -1 belonging to the benzene ν(C= C) is affected on complexation showing that the ligand is coordinated to metal through the oxygen of hydroxyl group of benzene ring [25]. It is observed that the aliphatic protons are not greatly affected on complexation [26].
It is clear from the above discussion that the free ligand interacts with Co(II) ion resulting in the formation of a metal-ligand complex. J a n u a r y 1 6 , 2 0 1 4

¹ H-NMR spectra
¹ H-NMR spectra of the ligand and its Co(II) complex were recorded in CDCl 3 . The absence of the phenolic OH proton signal (8.64δ) in the CDHBOX-Co(II) complex indicates coordination of phenolic oxygen to the Co(II) ion after deprotonation. The NMR spectral data of CDHBOX and its Co (II) complex are appended in Table 2.

Mass spectra
The FAB mass spectra of Co(II)-CDHBOX complex reveals its stoichiometric composition. The molecular [M + ] ion peak of the complex is shown at M/Z=415/419, suggesting the stoichiometry of the complex as ML2 .

Thermogravimetric studies
Thermo-gravimetric analysis (TGA) of Co (II)-CDHBOX suggests that complex is stable upto 300ºC. This indicates that the complex is not in the hydrated form. The initial decomposition shown in the TG curve was taken as a measure of the thermal stability of the complex. Sharp initial decomposition of the complex in the TG curve, is associated with a rapid loss in weight. The weight of Co(II)-CDHBOX complex decreases after decomposition, continuously upto 730ºC. On further heating, the weight of the residue remains constant and corresponds to CoO. The total mass loss is 85.60% (calculated value 85.66%) which is confirmed by comparing observed and calculated mass of the pyrolysis product.
The kinetic parameters were calculated graphically by employing the Coats-Redfern equation [32] log[-log(1-α)/T 2 ]=log[AR/θEº (1-2RT/Eº)]-Eº/2.303RT J a n u a r y 1 6 , 2 0 1 4 where, α is the mass loss up to temperature T, R is gas constant, Eº is the activation energy in Jmol -1 , θ is the linear heating rate, and the term (1-2RT/Eº)=1. A slope of the linear plot drawn between -log[-log(1-α)/T 2 ] and1/T gives the value of Eº as 4.949 kjmol -1 while its intercept (the Arrhenius constant) gives the value of A( Arrhenius constant) as 74.39 . Straight line of the graph confirms the first order kinetics for thermal decomposition of the complex .

Potentiometric study
Proton-ligand stability constant (logK1) was calculated from the proton-ligand formation curve (the plot between n H and pH ), and metal ligand stability constant ( log K2 ) was calculated from the formation curve the plot between n and pL ) The thermodynamic formation constants were obtained by extrapolation of the observed formation constants to zero ionic strength on the graph between log of the stability constant and √ μ .
The thermodynamic parameters ∆H, ∆S and ∆G were calculated at different temperatures (Table 3) and concentrations [ Table 4] using the following equations: ∆G = -2.303 RT log K μ=0 , ∆H = 2.303 R x (T 2 x T 1 /T 2 -T 1 ) (logK2"/ K1') and ∆S = 2.303R log K + ∆H/T It is noted from the data that the accrued ligand (CDHBOX) behaved as a monoprotic acid due to deprotonation of the phenolic OH group ortho to the oximino group from which the proton was replaced by metal ion during complex formation. This was evident from the fact that metal titration curve was well separated from the ligand titration curve. The value of log βn and log K H decreases with the increase in ionic strength. It shows that the activity of metal ion for its interaction with other molecular species decreases with the increase in the ionic strength of the medium under consideration. The protonation constant of the ligand and stability constant of metal complex decreases with the increase in temperature. The complex has a negative entropy which indicates a more ordered activated state, compensated by enthalpies of activation leading to almost the same value for the free energy of activation [29].
The ionisation depends upon the dielectric constant () of the medium. A solvent of low value increases the electrostatic force between the ions, and hence facilitates the formation of molecular species resulting in the increase in H pk 1 value.

Antimicrobial activity
The fungicidal and bactericidal data of the graded concentrations (0.05 to 0.40%) of 2´-hydroxy-4´-methoxy acetophenoneoxime(CDHBOX) and its Co(II) complex against Curvularia lunata, Fusarium oxysporum and Alternaria alternata fungi and Streproproteus, Staph, Escherchia coli, Klebsella, and pseudomonas bacteria are recorded in Tables  6 and 7 and are displayed in the form of bar diagrams [ fig.4-11].The observed results reveal that antimicrobial activity of the compound is directly proportional to the concentration of the test compound. The activity for a given ligand or metal complex differs from fungus to fungus and from bacteria to bacteria. The Co(II)-CDHBOX complex has more antimicrobial activity as compared to the ligand (CDHBOX). The complex showed maximum fungicidal activity against Curvularia lunata and the least against Alternaria alternata , the overall order of fungicidal activity being Cl> Fu > Aa. The complex showed J a n u a r y 1 6 , 2 0 1 4 maximum bactericidal activitiy against Staph and the least against, pseudomonas the overall order of antibacterial activity being St> Kl Sp > E.coli >Pc. Table 5 Antifungal Activity data of CDHBOX(HL) and Co(II) complex ML2 against Alternaria alternate, Curvularia lunata, and Fusarium oxysporium .

Mechanism of 'Bioactivity'
The antimicrobial studies demonstrated that chelation increases antimicrobial activity. It has been suggested that metal chelation reduces polarity of metal ion mainly because of the partial sharing of its positive charge with the donar group, and the possibility of d-electron delocalization occurring within the chelate ring system formed on coordination. The process of chelation thus increases the lipophilic nature of the central metal atom which, in turn, favours its permeation through the lipoid layer of the membrane [33,34 ], and the mechanism of action is understood to be alkylation of essential cellular proteins. Thus, increase in antimicrobial activity is due to faster diffusion of the free ligand with electron withdrawing group, and metal complex as a whole through the cell membrane or due to combined activity of the ligand and metal [35]. This has been supported by the experimental findings, which suggest that the compounds having higher electron density have low antimicrobial activity. Oxime has high antimicrobial activity as compared to semicarbazone, phenylhydrazone and phenone itself. This is attributed to the formation of dimeric and pseudomacrocyclic species by way of intermolecular hydrogen-bonding [36].
Antimicrobial properties are also found to be related to thermodynamic stability [37] and selectivity.

Conclusion
Spectrophotometric studies suggested that Co(II) ion forms only one complex with the ligand CDHBOX having the composition ML 2.
The observed magnetic moment and electronic spectrum of the complex point to tetrahedral geometry and paramagnetic nature.
IR spectral studies indicate deprotonation of the phenolic group, and coordination of the phenolic oxygen, and participation of nitrogen of the oximino group in complexation of Co (II) ion with CDHBOX. The fact is also supported by NMR spectral data. FAB, mass spectrum of the complex reveals ML 2 stoichiometric composition for the complex TGA of the complex reveals its thermal stability in a graded manner while potentiometric study on the complex provides data to evaluate the proton-ligand stability vis a vis metal-ligand stability.
Finally, antimicrobial activity data suggest the complex to be more active than the ligand showing maximum activity against Curvularia lunata and Staph and the least against Alternaria alternata and respectively.
Structurally, the Co(II)-CDHBOX complex can be represented as .