untitled ISSN 215 Synthes biologic derived Nagesh Gu and Benni 1 Department of 2 Government D * Corresponding Tel.: +91.944.88 ARTICLE IN DOI: 10.5155/e Received: 24 No Received in rev Accepted: 19 De Published onlin Printed: 31 Mar KEYWORDS Thiazole Schiff base Mass fragmenta Antibacterial ac Transition meta 4‐(Diethylamin 1. Introduct Schiff ba ligands in m chemistry be and also im important ph directly corr proton trans nities for ind electronic fa homogeneou metals are in are essentia hetero atom models and function of bioinorganic complexes h providing sy metallo‐prot 53‐2249 (Print) sis, charac cal evalua d from 4‐( unvanthrao ikallu Hire M f Studies and Resea egree College, Seda g author at: Depart 830318. Fax: +91.08 FORMATION eurjchem.7.1.56‐65 ovember 2015 vised form: 18 Dece ecember 2015 ne: 31 March 2016 rch 2016 S ation ctivity al complexes no)salicylaldehyde tion ases have play main group a ecause of their mine ligands are hysical and biol related to inter sfer equilibria [ ducing substra actors and enha us or heterog nvolved in a nu al for life. The m (O or N) term play a cruci biological mac c chemistry, inte has centered o ynthetic model teins and metal / ISSN 2153‐225 ht Europ cterizatio ation of ne diethylam Yernale 1, M Mathada Mr arch in Chemistry, G am Road, Kalaburag tment of Studies an 8472.245632. E‐ma 5.1372 ember 2015 ed an importa and transition stability under e borderline L logical properti rmolecular hyd [2]. Schiff bases ate chirality, tu ancing the solub geneous cataly umber of biolog e metal ions c minals from pro al role in the cromolecules [ erest in Schiff b on the role of s for the meta lo‐enzymes [6] European Journ Europ 57 (Online)  20 ttp://dx.doi.org pean Jo Journal web n, mass s ew Schiff mino)salic Mahadev Dh uthyunjayas Gulbarga University agi, 585106, Karnat nd Research in Chem ail address: bhmms ABSTRACT A new Schiff yl) hydrazine were prepare suggests the cobalt(II) and complex, whe indicates that compounds w cobalt(II) com activity was p growth of bac vitro cytotoxi property. Cite this: Eur. ant role as che metal coordin a various cond Lewis bases [1] ies of Schiff bas drogen bondin s also offer opp uning metal‐cen bility and stabi ysts [3]. Tran gical processes w can coordinate oteins in a vari e conformation 4,5]. In the fie bases and their f such complex al‐containing si . al of Chemistry 7 pean Journal of C 016 Atlanta Pub /10.5155/eurjch ournal bpage: www. pectral fr base ligan cylaldehy anraj Udaya swamy 1,* y, Kalaburagi, 5851 taka, India mistry, Gulbarga Un swamy53@rediffma base ligand, 2‐ carboxamide (L d and character composition of d nickel(II) com ere L is the depr t all the metal co was evaluated us mplexes are fou performed by ag cteria and fungi. ic properties, t . J. Chem. 2016, elating nation ditions ]. The es are g and portu‐ ntered ility of nsition which e with ety of n and eld of metal xes in ites in met met carr dim com inve Man anti der inte of t med rep util pro cha am hyd 7 (1) (2016) 56‐ Chemistry lishing House LL hem.7.1.56‐65.1 of Che .eurjchem.co ragmentat nd and its yde and th agiri 2 106, Karnataka, Ind niversity, Kalabura ail.com (B.H.M. Mru (4‐(diethylamin L) and its copper ized by analytica the metal comp mplexes and 1:1 rotonated Schiff omplexes are no sing the DPPH as und to be good gar diffusion me Also, brine shri the copper(II) a 7(1), 56‐65 Furthermore, tal complexes tal complexes riers. Copper meric units a mpounds [7]. I estigations of d ny of these pos imicrobial and rivatives as li eresting and ha their structural dicinal and p resent an impo ized as startin oducts [12]. In this artic aracterization ino)‐2‐hydrox drazinecarboxam ‐65 LC ‐ All rights re 1372 emistry m tion, ther s metal(II hiazole mo dia agi, 585106, Karnat uthyunjayaswamy) no)‐2‐hydroxybe r(II), cobalt(II), al and spectrosc plexes to be 1:2 1 stoichiometry base ligand. The on‐electrolytes. T ssay. The Schiff b d antioxidants. T ethods. All the m imp bioassay wa and zinc(II) com the tridentate are increasin related to sy complexes of are considere In recent year different classes sess interesting d analgesic a igands with ave gained spec l chemistry, but pharmaceutical ortant class of ng materials in cle, we repor of new Schiff xybenzylidene mide (L) contai served ‐ Printed y mal study ) complex oiety taka, India. ). enzylidene)‐N‐(4 nickel(II) and z copic methods. T of the type [ML y of the type [M e conductance m The antioxidant base ligand (L) a The antibacteria metal(II) comple as also carried o mplexes showe Schiff base lig gly important ynthetic and n tridentate Sch ed as bioino rs, there have s of thiazole de g biological pro activities. Thia potential sulf cial attention no t also for their field [10,11] compounds be n the synthesi rt here the f base ligand, e)‐N‐(4‐pheny ining carbonyl, d in the USA y and xes 4‐phenylthiazol‐ zinc(II) complex The analytical da L2] for copper(I MLCl] for zinc(I measurement da t activity of all th and its copper(I al and antifung exes inhibited th out to study the d good cytotox ands and their for designing natural oxygen hiff base with rganic model e been intense erivatives [8,9]. operties such as azole and its fur atom are ot only because importance in . Schiff bases ecause they are s of industrial synthesis and , 2‐(4‐(diethyl ylthiazol‐2‐yl) azomethine 2‐ es ata I), II) ata he I), gal he in xic r g n h l e . s s e e n s e l d l Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 57 Scheme 1 and hydroxyl groups as chelating sites and its copper (II), cobalt (II), nickel (II) and zinc (II) complexes. 2. Experimental 2.1. Materials and methods All solvents and chemicals were of commercial reagent grade and used as they are received. The 4‐(diethylamino) salicylaldehyde purchased from Sigma Aldrich Chemical company and N‐(4‐phenylthiazol‐2‐yl) hydrazinecarboxamide is prepared as per literature methods [13,14]. 2.2. Synthesis of 2‐(4‐(diethylamino)‐2‐hydroxybenzylide ne)‐N‐(4‐phenylthiazol‐2‐yl)hydrazinecarboxamide (L) An equimolar mixture of 4‐(diethylamino)salicylaldehyde and N‐(4‐phenylthiazol‐2‐yl)hydrazinecarboxamide in ethanol (30 mL) was refluxed on water bath for about 5‐6 h with an addition of a catalytic amount of glacial acetic acid (2‐3 drops). The precipitate which separated during reflux was filtered, washed with hot ethanol and recrystallized from hot 1,4‐ dioxane to get Schiff base ligand, 2‐(4‐(diethylamino)‐2‐ hydroxybenzylidene)‐N‐(4‐phenylthiazol‐2‐ yl)hydrazinecarboxamide (L) (Yield: 78%). The pathway for the synthesis of Schiff base ligand (L) is presented in Scheme 1. 2‐(4‐(Diethylamino)‐2‐hydroxybenzylidene)‐N‐(4‐phenyl thiazol‐2‐yl)hydrazinecarboxamide (L): Color: Yellow. Yield: 78%. M.p.: 284‐286 °C. FT‐IR (KBr, , cm‐1): 3400 (OH) (br, phenolic), 3329 (NH) (amide), 3200 (NH) (thiazole), 1699 (C=O) (carbonyl), 1624 (C=N) (azomethine), 1297 (C‐O) (phenolic). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.3 (t, 6H, CH3), 3.2 (q, 4H, CH2), 6.1‐7.8 (m, 9H, ArH), 8.1 (s, 1H, HC=N), 10.6 (s, 1H, thiazole NH), 10.9 (s, 1H, CONH), 11.5 (s, 1H, Phenolic OH). MS (EI, m/z (%): 410 (M+1, 48). Anal. calcd. for C21H23N5O2S: C, 61.59; H, 5.66; N, 17.10. Found: C, 61.64; H, 5.66; N, 17.05%. 2.3. Preparation of metal complexes Schiff base ligand (L) (0.001 mol) in 20 mL of ethanol and a solution of respective metal chlorides (0.002 mol) in 20 mL of ethanol was refluxed on water bath for about 6‐7 h and pH of the reaction mixture adjusted to ca. 7.0‐7.5 using sodium acetate (0.5 g) and refluxing continued for about an hour more. The reaction mixture was cooled to room temperature and poured into distilled water. The separated precipitates were filtered off, washed with distilled water, then with hot ethanol to remove any traces of unreacted starting materials and finally dried in a vacuum over fused calcium chloride. Copper(II) complex: Color: Green. Yield: 59%. M.p.: >300 °C. FT‐IR (KBr, ν, cm‐1): 3319 (NH) (amide), 3253 (NH) (thiazole), 1663 (C=O) (carbonyl), 1595 (C=N) (azomethine), 1353 (C‐O) (phenolic), 553 (M‐O), 432 (M‐N). MS (EI, m/z (%): 880 (M+1, 3). Anal. calcd. for Cu(C21H22N5O2S)2: C, 57.29; H, 5.04; N, 15.91; Cu, 7.22. Found: C, 57.34; H, 5.06; N, 15.86; Cu, 7.15%. UV/Vis (DMF, cm‐1): 15696, 18146. Λm (S.m2.mol‐1): 29. µeff (BM): 1.85. Cobalt(II) complex: Color: Brown. Yield: 56%. M.p.: >300 °C. FT‐IR (KBr, , cm‐1): 3321 (NH) (amide), 3196 (NH) (thiazole), 1681 (C=O) (carbonyl), 1597 (C=N) (azomethine), 1346 (C‐O) (phenolic), 588 (M‐O), 450 (M‐N). MS (EI, m/z (%): 874 (M+1, 16). Anal. calcd. for Co(C21H22N5O2S)2: C, 57.59; H, 5.06; N, 15.99; Co, 6.73. Found: C, 57.64; H, 5.06; N, 15.96; Co, 6.79%. UV/Vis (DMF, cm‐1): 16692, 20493. Λm (S.m2.mol‐1): 18. µeff (BM): 5.06. Nickel(II) complex: Color: Brown. Yield: 52%. M.p.: >300 °C. FT‐IR (KBr, ν, cm‐1): 3322 (NH) (amide), 3213 (NH) (thiazole), 1687 (C=O) (carbonyl), 1546 (C=N) (azomethine), 1302 (C‐O) (phenolic), 541 (M‐O), 455 (M‐N). MS (EI, m/z (%): 874 (M+, 7). Anal. calcd. for Ni(C21H22N5O2S)2: C, 57.61; H, 5.06; N, 16.00; Ni, 6.70. Found: C, 57.66; H, 5.06; N, 16.06; Co, 6.67%. UV/Vis (DMF, cm‐1): 14897, 25117. Λm (S.m2.mol‐1): 17. µeff (BM): 2.93. Zinc(II) complex: Color: Orange. Yield: 74%. M.p.: >300 °C. FT‐IR (KBr, , cm‐1): 3341 (NH) (amide), 3198 (NH) (thiazole), 1617 (C=O) (carbonyl), 1604 (C=N) (azomethine), 1301 (C‐O) (phenolic), 533 (M‐O), 441 (M‐N), 353 (M‐Cl). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.3 (t, 6H, CH3), 3.3 (q, 4H, CH2), 6.1‐ 8.2 (m, 9H, ArH), 8.4 (s, 1H, HC=N), 11.1 (s, 1H, thiazole NH), 11.9 (s, 1H, CONH). MS (EI, m/z (%): 508, 510 (M+, 3). Anal. calcd. for ZnC21H22N5O2SCl: C, 49.52; H, 4.35; N, 13.75; Zn, 12.84. Found: C, 49.49; H, 4.36; N, 13.74; Zn, 12.81%. Λm (S.m2.mol‐1): 30. 2.4. Analysis and physical measurement The metal and chloride contents of the complexes were estimated gravimetrically as per standard method [15]. Carbon, hydrogen and nitrogen analysis was performed on Vario EL III CHNS analyzer. The IR spectra were recorded on Perkin Elmer Spectrum RX‐I FT‐IR spectrophotometer. 1H NMR spectra were recorded on Bruker 400 MHz spectrometer using DMSO‐d6 as solvent. ESI mass spectra were recorded on mass spectrometer equipped with an ESI source having a mass range of 4000 amu in quadruple and 20,000 amu in TOF. Electronic spectra were recorded on ELICO SL‐164 double beam UV‐Visible spectrophotometer (ca. 1×10‐3 M in DMF). Molar conductivity of metal complexes was measured on Elico‐ CM, 180 Conductivity Bridge (ca. 1×10‐3 M DMF). ESR measu‐ rement of solid copper (II) complex is carried out on BRUKER Bio Spin spectrometer working at a microwave frequency of 8.75‐9.65 GHz using DPPH as reference with field set at 3000 Gauss using tetracynoethylene as the ‘g’ marker (g = 2.00277). Thermogravimetric analyses data were measured on the Perkin Elmer thermal analyzer in a nitrogen atmosphere with a heating rate of 20 °C/min. 2.5. Biological evaluation 2.5.1. Antibacterial and antifungal activity The antibacterial and antifungal activity of the newly synthesized Schiff base ligand (L) and its copper(II), cobalt(II), nickel(II) and zinc(II) complexes was performed against S. aureus (MTCC 3160) and E. coli (MTCC 46) bacteria and A. flavus (MTCC 1883) and A. niger (MTCC 1881) fungi by agar diffusion methods [16,17]. The above microorganisms were 58 Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 obtained from the Department of Microbiology and Biotechno‐ logy, Gulbarga University, Kalaburagi, Karnataka, India which are previously procured from Institute of Microbial Technology Chandigarh (IMTC), India. For antibacterial activity, the media were prepared by dissolving Peptone 10 g, NaCl 10 g, Yeast extract 5 g and Agar 20 g in 1000 mL of distilled water. Initially, the stock cultures of bacteria were revived by inoculating in broth media and grown at 37 °C for 18 h. The agar plates were prepared and wells were made in the plate. Further, each plate was inoculated with 18 h old cultures (100 μL, 1×10‐4 CFU) and spread evenly on the plate. After 20 min, the wells were filled with a test compound and standard antibiotic, ciproflaxacin at different concentrations (25, 50, 100, 250, 500 and 1000 µg/mL). All the plates were incubated at 37 °C for 24 h and the diameter of inhibition zone were measured. For antifungal activity, the media were prepared by using Czapek‐Dox Agar (Composition (g/L) sucrose 30.0; sodium nitrate 2.0; K2HPO4 1.0, MgSO4.7H2O 0.5; KCl 0.5; FeSO4 0.01; Agar 20). Initially, the stock cultures were revived by inoculating in broth media and grown at 27 °C for 48 h. The agar plates of the above media were prepared and wells were made in the plate. Each plate was inoculated with 48 h old cultures (100 μL, 1×104 CFU) and spread evenly on the plate. After 20 min, the wells were filled with different concentrations (25, 50, 100, 250, 500 and 1000 µg/mL) of test samples and antibiotic, fluconazole. All the plates were incubated at 27 °C for 96 h and the diameter of inhibition zone were noted. 2.5.2. In vitro cytotoxicity study For the study, Brine shrimp nauplii (Artemia salina) were used for cytotoxicity assay and followed the protocol given by Meyer et al. [10,18] with some modifications. This method is an efficient and inexpensive and has good correlation with cytotoxic activity. Brine shrimp nauplii eggs were hatched in a shallow rectangular dish (22 × 32 cm) filled with artificial sea water. An unequal partition was made in the dish with the help of punctured device. About 50 mg of eggs was sprinkled into the large compartment, which was darkened and the minor compartment was open to ordinary light. After two days nauplii were collected in a pipette from the lighted side. The stock solutions of the each test compound (1 mg/mL) were prepared by dissolving 10 mg of each test compound in 10 mL of DMSO, different concentrations of the test compounds were placed in separate vials and final volume is adjusted to 10 mL using artificial sea water. After two days, when shrimp larvae were ready, 10 nauplii were then placed in each vial, after 24 h incubation the vials were observed using a magnifying glass and the number of survivors in each vial was counted. Tests were performed in duplicate and the data were analyzed by a Finney computer program to determine the LD50 values [19]. The results were compared with a positive control Bleomycin, an anti‐cancer drug. 2.5.4. Antioxidant assay (free radical scavenging activity) The 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical scavenging activity evaluations is a standard assay in antioxidant activity studies. It is an efficient and rapid technique for screening the radical scavenging activity of specific compounds [20]. Various concentrations (12.5, 25, 50 and 100 μg/mL) of each test compound and standards butylated hydroxyanisole (BHA) and ascorbic acid (Vitamin C) were taken in different test tubes and adjust the volume to 100 μL by adding distilled DMF. To all the tubes add 5 mL methanolic solution of DPPH (0.1 mM), shaken vigorously and allowed to stand for 30 min at room temperature for incubation. After the completion of incubation period, the scavenging ability determines the antiradical power of an antioxidant by measuring the decrease in the absorbance of DPPH at 517 nm. In the radical form, DPPH shows a maximum absorbance at 517 nm, but on reduction by an antioxidant, the absorption decreases and pale‐yellow non radical form is produced through donation of a hydrogen atom to form a stable DPPH molecule. The lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The test was performed with three replicates to obtain mean±S.D. The percentage (%) of inhibition of free radical production from DPPH was calculated using the following mathematical equation: % Scavenging of DPPH = [(Control OD‐Sample OD)/Control OD]×100 (1) 3. Results and discussions 3.1. Chemistry The synthesized copper(II), cobalt(II), nickel(II) and zinc(II) complexes of L are colored solids, stable at room temperature and possess high melting point (>300 °C). The complexes are insoluble in water and common organic solvents; however, these complexes are soluble to a large extent in DMF and DMSO. Elemental analysis data agree well with the suggested composition of Schiff base ligand (L) and its metal(II) complexes. These data suggest that the metal to ligand stoichiometric ratio of the complexes is 1:2 of the type [ML2] for copper(II), cobalt(II) and nickel(II) complexes and 1:1 stoichiometry of the type [MLCl] for zinc(II) complex. The results of conductivity measurements are too low to account for any dissociation of the complexes in DMF (17‐30 ohm‐ 1·cm2·mole‐1). Hence, the complexes may be regarded as non‐ electrolytes [21]. 3.2. IR spectral studies The IR spectrum of L showed a broad band at 3400 cm‐1 due to phenolic OH and medium intensity weak bands at 3329 and 3200 cm‐1 due to amide NH and NH attached to the thiazole moiety, respectively. The high intensity strong bands observed at 1699, 1624 and 1297 cm‐1 are due to carbonyl function ν(C=O), azomethine function ν(C=N) and phenolic C‐ O, respectively. In the IR spectra of all the metal complexes, it was observed that, the absence of absorption band due to phenolic OH at 3400 cm‐1 of ligand indicates the formation of a coordination bond between the metal ion and phenolic oxygen atom via deprotonation. This is further confirmed by the increase in absorption frequency about 4‐56 cm‐1 of phenolic ν(C‐O) which appeared in the region 1301‐1353 cm‐1 in all the metal complexes indicating the participation of oxygen atom of phenolic OH in the coordination. In the IR spectra of the metal complexes, medium intensity weak bands at 3319‐3341 and 3196‐3253 cm‐1 were due to amide NH and NH attached to thiazole moiety, respectively, which appeared almost at about the same position as in the case of ligand, thus confirming their non‐involvement in coordination. The shift of amide carbonyl ν(C=O) to lower frequency side about 12‐82 cm‐1 which appeared in the region 1617‐1687 cm‐1 in all the complexes confirms the coordination of oxygen atom of amide ν(C=O) with the metal ions as such without undergoing enolization [22,23]. The absorption frequency due to azomethine ν(C=N) function also shifted to the low frequency side about 20‐78 cm‐ 1 and appeared in the region 1546‐1604 cm‐1 suggesting the involvement of nitrogen atom of azomethine function in complexation with metal ions [14,24]. The coordination of metal ions with the Schiff base ligand was further confirmed by the appearance of new weak intensity, non‐ligand bands in the region 533‐588 and 432‐455 cm‐1 in all the complexes and 353 cm‐1 in the case of zinc(II) Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 59 complex are assigned to frequencies of ν(M‐O), ν(M‐N) and ν(M‐Cl) stretching vibrations respectively. 3.3. 1H NMR spectral studies The 1H NMR spectrum (Figure S1) of L displayed three singlets each at δ 11.5, 10.9 and 10.6 ppm are due the proton of phenolic OH, amide NH and NH attached to thiazole moiety, respectively. The signal due to azomethine proton (CH=N) resonated as singlet at δ 8.1 ppm. The signals due to nine aromatic protons (ArH) have resonated as multiplets in the region δ 6.1‐7.8 ppm and signals at δ 3.2 and 1.3 ppm are due to four and six protons of two CH2 and two CH3 groups respectively. The L upon complexation with zinc(II) ion showed the disappearance of signal due to the proton of phenolic OH confirms the involvement of bonding of phenolic oxygen to metal ion via deprotonation. The signals due to amide NH and NH of thiazole are appeared in the region δ 11.9 and 11.1 ppm, respectively. The signal due to azomethine proton (CH=N) resonated at δ 8.4 ppm. The signals due to nine aromatic protons (Ar‐H) have resonated as multiplets in the region δ 6.1‐8.2 ppm and signals at δ 3.3 and 1.3 ppm are due to four and six protons of two CH2 and two CH3 groups, respectively. When compared to the 1H NMR spectral data of L and its zinc(II) complex, all the signals due to protons have been shifted towards down field strength confirming the complexation of zinc(II) ion with the ligand. 3.4. ESI‐mass spectral studies 3.4.1. Mass spectral fragmentation pattern study of L The ESI mass spectrum of L exhibited M+ +1 peak at m/z 410 (48%). The molecular ion peak M+ 409, underwent fragmentation in two routes. In the first route, the fragmentation is observed due to loss of C9H7N2S radical of thiazole moiety and gave a fragment ion peak recorded at m/z 234 (14%). This fragment ion, on loss of CHN2O radical gave a fragment ion peak recorded at m/z 177 (10%). In a another route, the molecular ion undergone fragment due to the loss of C2H2 molecule giving a fragment ion peak recorded at m/z 383 (100%) which is also a base peak. The schematic mass spectral fragmentation pattern is in consistency with its structure which is provided in Scheme 2. 3.4.2. Mass spectral fragmentation pattern study of copper(II) complex The ESI mass spectrum of copper(II) complex showed M+ +1 peak recorded at m/z 880 (3%). The molecular ion peak M+ m/z 879 on loss of two C10H6N2OS molecule of thiazole moiety, CH3 radical and a H2C=CH2 species gave a fragment ion peak recorded at m/z 432 (25%). This on further loss of a C2H5 radical, NH2 radical and four hydrogen radicals gave a fragment ion peak recorded at m/z 383 (100%), which is also a base peak. The schematic mass spectral fragmentation pattern is in consistency with its structure which is illustrated in Scheme 3. 3.4.3. Mass spectral fragmentation pattern study of nickel(II) complex The ESI mass spectrum of nickel(II) complex showed a molecular ion peak recorded at m/z 874 (7%) which is equivalent to its molecular weight. The molecular ion peak underwent fragmentation in two routes. In the first route, the molecular ion peak on the loss of a deprotonated ligand (C21H22N5O2S) molecule, a benzene molecule, five hydrogen radicals gave a fragment ion peak recorded at m/z 383 (100%), which is also a base peak. In another route, the molecular ion on the loss of a 4‐phenylthiazole radical, a benzene molecule, three hydrogen radicals gave a fragment ion peak recorded at m/z 633 (3%). Further, this on loss of a diethylamine molecule of 4‐(diethylamino) salicylaldehyde moiety and hydrogen radical gave a fragment ion peak recorded at m/z 559 (3%). The schematic mass spectral fragmentation pattern is in consistency with its proposed structure which is presented in Scheme 4. 3.4.4. Mass spectral fragmentation pattern study of zinc(II) complex The ESI mass spectrum of zinc(II) complex showed a M+1 +1 peak recorded at m/z 508, 510 (3%, 1%), which is equivalent to its molecular weight. The molecular ion peak underwent fragmentation in two routes. In the first route, the sequential loss diethylamine molecule of 4‐(diethylamino) salicylaldehyde moiety, 2‐amino‐4‐phenylthiazole molecule and a coordinated chloride radical gave a fragment ion peak recorded at m/z 223 (19%). In another route, molecular ion peak on the loss of a hydrogen radical gave a fragment ion peak recorded at m/z 507, 509 (3%, 1%), this peak on further loss a hydrogen radical gave a fragment ion peak recorded at m/z 506, 508 (9%, 3%). This fragment ion on the loss of a C6H5 radical gave a fragment ion peak recorded at m/z 429, 431 (30%, 10%). Further, this on loss of two C2H5 radicals of 4‐ (diethylamino) salicylaldehyde moiety gave a fragment ion peak recorded at m/z 371, 373 (100%, 33%) which is also a base peak. This on simultaneous loss of and C2HN species of thiazole moiety and a coordinated chloride radical gave a fragment ion peak recorded at m/z 297 (15%). The schematic mass spectral fragmentation pattern of zinc(II) complex is in consistency with its proposed structure which is provided in Scheme 5. 3.5. Electronic spectral studies The electronic spectra of green colored copper(II) complex displayed a low intensity single broad asymmetric band in the region 15696‐18146 cm‐1. The broadness of the band designates the three transitions 2B1g → 2A1g (ν1), 2B1g → 2B2g (ν2) and 2B1g → 2Eg (ν3), which are similar in energy and give rise to only one broad band and the broadness of the band may be due to dynamic Jahn‐Teller distortion. The obtained data suggest the distorted octahedral geometry around the copper(II) ion [25]. The brown colored cobalt(II) complex under present study displayed two absorption bands at 16692 and 20493 cm‐1. These bands are assigned to be 4T1g (F) → 4A2g (F) (ν2) and 4T1g (F) → 4T2g (P) (ν3) transitions, respectively, which are in good agreement with the literature values for octahedral geometry [26]. The lowest band, ν1 could not be observed due to the limited range of the instrument used, but it could be calculated using the band fitting procedure suggested by Underhill and Billing [27]. The calculated ν1 value is presented in Table 1. The transition values of ν1, ν2 and ν3 suggest the octahedral geometry for the cobalt (II) complex. The brown colored nickel (II) complex under present investigation exhibited two absorption bands in the region 14897 and 25117 cm‐1, which are assigned to 3A2g → 3T1g (F) (ν2) and 3A2g (F) → 3T1g (P) (ν3) transitions respectively in an octahedral environment [28]. The transition value of band ν1 was calculated by using a band fitting procedure [27]. The proposed octahedral geometry for copper(II), cobalt(II) and nickel(II) complexes was further supported by the calculated values of ligand field parameters, such as Racah inter electronic repulsion parameter (B’), nephelauxetic parameter (β), ligand field splitting energy (10 Dq) and ligand field stabilization energy (LFSE) [29]. The calculated B’ values for the copper(II) and nickel(II) complexes are lower than the free ion values, which is due to the orbital overlap and delocalization of d‐orbitals. 60 Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 Scheme 2 Scheme 3 The β values are important in determining the covalency for the metal‐ligand bond and they were found to be less than unity, suggesting a considerable amount of covalency for the metal‐ligand bonds. The β value for the nickel(II) complexes was less than that of the cobalt (II) complexes, indicating the greater covalency of the metal‐ligand (M‐L) bond. 3.6. Magnetic susceptibility studies The obtained magnetic moment value for copper(II) complex is 1.85 BM. The obtained value is slightly higher than the spin‐only value 1.73 BM due to one unpaired electron, suggesting the octahedral geometry for copper(II) complex [30]. Thus, the present complex is devoid of any spin interaction with distorted octahedral geometry. In octahedral cobalt(II) complex the ground state is 4T1g. A large orbital contribution to the singlet state lowers the magnetic moment values for the various cobalt(II) complexes which are in the range 4.12‐4.70 BM for tetrahedral and 4.70‐5.20 BM for octahedral geometry of the complexes, respectively [31]. In the present study the observed magnetic moment value 5.06 BM suggests the octahedral geometry. Further, the observed magnetic moment value for nickel (II) complex is 2.93 BM, which is also well within the expected range of 2.83‐3.50 BM, suggesting the consistency with its octahedral environment [32]. 3.7. ESR spectral study of copper(II) complex The ESR spectrum provides the evidence about the environment of the metal ion within the complex, i.e., the geometry and nature of the ligating sites of the ligand and metal ion. Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 61 Scheme 4 M++1 508, 510 (3%, 1%) H3C N H3C C O N H N C O Zn Cl H N N S m/z 507, 509 (3%, 1%) - H H3C N H3C C O N N C O Zn Cl H N N S m/z 506, 508 (9%, 3%) - C6H5 H3C N H3C C O N N C O Zn Cl H N N S m/z 429, 431 (30%, 10%) H3C CH2- 2 HN C O N N C O Zn Cl H N N S m/z 371, 373 (100%, 33%) S N NH2 H3C NH H3C - Cl C O N N C O Zn m/z 223 (19%) - Cl - C2HN HN C O N N C O Zn H N C S m/z 297 (15%) - H Scheme 5 In octahedral geometry with the g‐tensor parameter g > g|| > 2.0023, the unpaired electron lies in the dz2 orbital and g|| > g > 2.0023, the unpaired electron lies in the dx2–y2 orbital in the ground state [33]. The observed measurements of copper(II) complex is g|| (2.1467) > g (2.0349) > 2.0023 indicate that the complex is axially symmetric and copper site has a dx2–y2 ground state characteristic of octahedral geometry [34]. The g|| value is an important function for indicating the metal‐ligand (M‐L) bond character, for covalent and ionic character g|| value < 2.3 and g|| value > 2.3, respectively [33]. 62 Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 Table 1. Electronic spectral data and ligand field parameters of copper(II), cobalt(II) and nickel(II) complexes. Metal Complexes Transitions in cm‐1 Dq (cm‐1) B′ (cm‐1) β β% ν2/ν1 LFSE (k.cal) ν1* ν2 ν3 Copper(II) 15696 ‐ 18146 ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ 29.00 Cobalt(II) 7782 16692 20493 891 924 0.951 4.84 2.14 15.27 Nickel(II) 9130 14897 25117 913 841 0.808 19.13 1.63 31.30 Table 2. Thermal degradation pattern of metal(II) complexes. Metal Complexes Decomposition temp. ( °C) Weight loss (%) Metal oxide (%) Inference Obs. Calc. Obs. Calc. Copper(II) 318 7.39 8.18 ‐ ‐ Loss due to C4H10N species of 4‐(diethylamino)salicylaldehyde 370 43.89 43.86 ‐ ‐ Loss due to two moles of 2‐amino‐4‐phenylthiazlole 450 5.71 6.61 ‐ ‐ Loss due to two CH3 groups. Up to 734 ‐ ‐ 10.19 11.48 Loss due to remaining organic moiety Cobalt(II) 209 16.29 16.45 ‐ ‐ Loss due to two moles of C4H10N species of 4‐(diethylamino)‐salicylaldehyde 282 18.58 21.06 ‐ ‐ Loss due to two phenyl groups of thiazole moieties 418 63.04 62.05 ‐ ‐ Loss due to C11H7N4O2S and C3H3N2S molecules. Up to 731 ‐ ‐ 11.14 12.29 Loss due to remaining organic moieties. Nickel(II) 160 8.14 8.23 ‐ ‐ Loss due to C4H10N species of 4‐(diethylamino)‐salicylaldehyde 300 20.16 19.43 ‐ ‐ Loss due to two benzene groups of thiazole moieties 400 58.58 57.79 ‐ ‐ Loss due to C11H14NO and C3H2N2S groups of 4‐(diethyl‐amino)salicylaldehyde and thiazole moiety respectively. 492 36.58 35.67 ‐ ‐ Loss due to NCO and NHNCO groups. Up to 731 ‐ ‐ 10.12 11.20 Loss due to remaining organic moiety. Zinc(II) 254 8.51 6.88 ‐ ‐ Loss due to a coordinated chlorine atom. 309 49.97 52.38 ‐ ‐ Loss due to a 2‐amino‐4‐phenylthiazlole and C4H10N group of 4‐(diethylamino) salicylaldehyde 388 52.48 52.34 ‐ ‐ Loss due to C7H4NO species. Up to 632 ‐ ‐ 11.27 12.20 Loss due to remaining organic moiety. Table 3. Powder X‐ray data of copper(II) complex. Peak 2  Sin Sin2 1000 Sin2 1000 Sin2/CF (h2+k2+l2) h k l d a in Å Obs. Calc. 1 27.027 13.513 0.233 0.0546 54.603 1.00 (1) 1 0 0 3.299 3.304 3.295 2 34.965 17.482 0.300 0.0902 90.248 1.65 (2) 1 1 0 2.566 2.566 3.293 3 50.782 25.391 0.428 0.1838 183.863 3.36 (3) 1 1 1 1.797 1.799 3.292 4 56.857 28.428 0.476 0.2266 226.634 4.15 (4) 2 0 0 1.619 1.617 3.295 5 60.449 30.224 0.503 0.2534 253.400 4.64 (5) 2 1 0 1.531 1.530 3.294 In the present case, g|| value is < 2.3, indicating an appreciable covalent character of the metal‐ligand (M‐L) bond. The geometric parameter (G) is the measure of extent of exchange interactions and is calculated by using the g‐tensor values by using the expression G = g|| ‐ 2.0023/g ‐ 2.0023. According to Hathaway and Billing [35], if the G value is greater than 4, the exchange interaction between the copper centers is negligible, whereas if its value is less than 4 and the exchange interaction is noticed. In the present investigations, it was found that the G value of the copper(II) complex is 4.43, indicate the exchange coupling effects are not operative. 3.8. Thermal studies The thermogram of copper(II) complex showed that the complex is stable up to 317 °C and no weight loss occurs before this temperature. The complex underwent degradation in three successive stages. The first stage of degradation occurred at 318 °C, due to the loss of C4H10N species of 4‐ (diethylamino) salicylaldehyde with a practical weight loss of 7.39% (Calcd. 8.18%). The resultant complex on further degradation gave a break at 370 °C by the loss of two moles of 2‐amino‐4‐phenylthiazlole with a practical weight loss of 43.89% (Calcd. 43.86%). Further, the resultant complex underwent third stage of decomposition at 450 °C due to the loss of two CH3 groups with a practical weight loss of 5.71% (Calcd. 6.61%). Further, complex showed gradual decompo‐ sition up to 734 °C and onwards due to the loss of remaining organic moiety. The final weight of the residue corresponds to cupric oxide. Similarly, cobalt(II), nickel(II) and zinc(II) complexes underwent a decomposition in three, four and three successive stages, respectively, and the final weight of the residue corresponds to the formation of respective metal oxides. The proposed stepwise degradation pattern of all the complexes at different stages are due to the loss of different organic moieties, with respect to temperature, which is illustrated in Table 2. 3.9. Powder X‐ray diffraction studies (Powder‐XRD) Though our newly synthesized metal complexes were soluble in some polar organic solvents like DMSO and DMF, crystals that are appropriate for single crystal studies are not achieved. In order to test the degree of crystallinity of metal complexes, we obtained the powder X‐ray diffraction pattern for all the metal (II) complexes. In the powder X‐ray diffraction pattern of all the complexes, it was observed that the trend of curves decreases from maximum to minimum intensity indicating the amorphous nature of the complexes. Powder X‐ray diffraction pattern of copper(II), cobalt(II), nickel(II) and zinc(II) complexes displayed a five, five, four and six reflections respectively, with maxima at 2θ = 60.449, 60.546, 60.431 and 64.433 ° corresponding to observed d values 3.294, 3.290, 2.947 and 2.567 Å, respectively. Bragg’s equation (n. = 2.d.sin) was used for calculating the inter‐ planar spacing (d). The calculated inter‐planar d‐spacing together with relative intensities with respect to most intense peak have been recorded. From all the highly intense peaks, unit cell calculations have been calculated for all the complexes and h2 + k2 + l2 values were also determined. The observed inter‐planar d‐spacing values have been compared with the calculated ones and it was found to be in good agreement with experimental values. The h2+k2+l2 values of copper (II) complex are 1, 2, 3, 4 and 5, also the calculated lattice parameter is a = b = c = 2.950 Å. From the Table 3, it was observed that the absence of forbidden numbers (7, 15, 23, etc.) indicates that the complex belongs to cubic symmetry. Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 63 Table 4. Antibacterial and antifungal activity results, zone of inhibition in mm. Bacteria/Fungi Ligand/Complexes Concentration (µg/mL) 25 50 100 250 500 1000 MIC S. aureus L 0 0 0 7 10 13 250 Copper(II) 0 0 8 10 16 20 100 Cobalt(II) 0 0 0 6 12 15 250 Nickel(II) 0 0 0 5 9 14 250 Zinc(II) 0 0 4 6 9 13 100 E. coli L 0 0 0 6 12 17 250 Copper(II) 0 0 7 11 17 21 100 Cobalt(II) 0 0 0 5 11 15 250 Nickel(II) 0 0 6 10 16 21 100 Zinc(II) 0 0 4 8 11 17 100 A. Flavus L 0 0 0 0 8 17 500 Copper(II) 0 0 9 15 21 29 100 Cobalt(II) 0 0 0 7 13 17 250 Nickel(II) 0 0 0 0 8 15 500 Zinc(II) 0 0 8 13 19 25 100 A. niger L 0 0 9 13 19 26 100 Copper(II) 0 0 7 13 17 24 100 Cobalt(II) 0 0 0 0 10 17 500 Nickel(II) 0 0 0 0 9 17 500 Zinc(II) 0 0 9 16 24 29 100 Ciproflaxacin * ** ** ** ** ** ** ** Fluconazole * ** ** ** ** ** ** ** * Broad spectrum antibiotics for bacterial/fungal strains. ** The inhibitions zones were too big to measure. Similar calculations were done for cobalt(II), nickel(II) and zinc(II) complexes and all the important peaks have been indexed and observed values of inter‐planar distances (d) have been compared with the calculated ones and it was found to be in good agreement. It was observed that the absence of forbidden numbers (7, 15, 23, etc.) in indicates that complexes belong to cubic symmetry. 3.10. Biological evaluations 3.10.1. Antibacterial and antifungal assay results The newly synthesized L and its metal(II) complexes have been evaluated for their antibacterial and antifungal activity. The MIC values of the tested compounds against the respective bacterial and fungal strains along with the standards are summarized in Table 4. In most of the cases, the metal complexes revealed good antimicrobial activity results than the free ligand. This activity found to be improved on coordination of the hetero atoms of the L with various metal ions. This enhancement in the antimicrobial activity of the complexes over the free ligand can be explained on the basis of chelation theory [36,37]. It is known that chelation enhances the ligand to act as more powerful and potent bactericidal/fungicidal agents by inhibiting the growth of bacteria/fungi, thus a zone of inhibition of metal complexes was found to be higher compared to the ligand [23]. The enhancement in the anti‐ microbial activity may be rationalized on the basis that ligands mainly possess azomethine (C=N) bond. Moreover, in metal complex, the positive charge of the metal ion is partially shared with the hetero donor atoms (N and O) present in the ligand and there may be π‐electron delocalization over the whole chelating system [38]. Hence the increase in the lipophilic character of the metal chelates favour their permeation through the lipoid layer of the bacterial cell membranes and blocking of the metal binding sites in the enzymes of microorganisms. In general, metal complexes are more active than the ligands because metal complexes may serve as a vehicle for activation of ligands as the principal cytotoxic species [39]. 3.10.2. In vitro cytotoxicity The brine shrimp bioassay is an excellent tool to be used for monitoring the biological activity in order to predict the capability to kill cancer cells [40]. A summary of the cytotoxic assay results of the L and its metal(II) complexes is presented in Table 5. Among all the tested compounds zinc(II) and nickel(II) complexes showed the highest cytotoxicity with LD50 value of 1.148×10‐4 and 1.216×10‐4, respectively. The L and its copper(II) and cobalt(II) complexes were found to be less active against A. salina. Table 5. Brine shrimp bioassay data. Compounds LD50 (M/mL) L 2.279×10‐4 Copper(II) 2.139×10‐4 Cobalt(II) 2.106×10‐4 Nickel(II) 1.216×10‐4 Zinc(II) 1.148×10‐4 Bleomycin 0.410×10‐4 3.10.3. Antioxidant assay (DPPH free radical scavenging activity) To evaluate the antioxidant property, we studied their free radical scavenging ability using DPPH radical which reacts with an electron or hydrogen donor to become a stable diamagnetic molecule viz. hydrazine. The solution therefore loses color depending on the number of electrons accepted. A substance capable of donating electrons or hydrogen atom is able to convert the purple color of DPPH to its non‐radical yellow color, form: 2,2‐diphenyl‐1‐picrylhydrazyl, a reaction which can be followed spectrophotometrically [41,42]. The results in different concentrations are presented in Figure 1. Amongst the tested compounds, L and its cobalt(II) and zinc(II) complexes have showed good scavenging activity. 4. Conclusion A series of copper(II), cobalt(II), nickel(II) and zinc(II) complexes were prepared with tridentate ONO donor Schiff base ligand (L) derived from N‐(4‐phenylthiazol‐2‐yl)‐ hydrazinecarboxamide and 4‐(diethylamino)salicylaldehyde and characterized them by various spectral techniques. Spectral analysis indicates octahedral geometry for copper(II), cobalt (II) and nickel(II) complexes have a 1:2 stoichiometric ratio of the type [M(L)2] and zinc(II) complex has a 1:1 stoichiometry ratio of the type [ZnLCl]. Also the mass fragmentation patterns of the complexes are in consistency with proposed structures. 64 The ant newly synth activity whe increased up metal atoms metal ion by lipophilic na metal enhan cell membra free ligand L to the metal ligand is due Hence, from that the Schi remarkable, compounds a the various may be used and higher e proposed th metal compl Acknowledg One of grateful to D for the awa 2014‐15/IF1 Indian Institu Instrumenta Analytical In providing s BioGenics R Hubli for bio References [1]. Hossein H H S imicrobial acti esized metal(II en compared to pon chelation o s. The chelatio y coordinating ature of the m ced its penetra ane of the micro showed good a l complexes an e to the presenc all these exten ff base ligand ( versatile and v about the study structures of t d as potent biol efficiency. Based e following geo exes. gements the authors ( Department of S ard of DST‐INS 120091]. The ute of Technolo tion Centre, nstrumentation spectral data. Research and T ological studies. , N.; Mohsen, M. J. C Y O N H3C H3C M C H N N H C O N H N S M = Cu(II), ivity results s I) complexes ha o the free ligan or coordination n process redu g with ligand, w etals. This lipo ation through th oorganism. In t antioxidant acti nd increased in ce of hydroxyl sive observatio L) and its meta aluable informa y of bonding m the metal comp logical agents w d on physicoche ometry/structu (Nagesh Gunva Science and Te SPIRE SRF [DS authors exten ogy Bombay, So Cochin Unive n Facility, Pan Authors are Training Centr Chem. 2013, 70182 Yernale et al. / Eu Figure 1 H C N H N C O H N M O N C C Co(II) and Ni(II) Figure 2. Propos howed that a ave exhibited h nd. This activity n of the ligand uces the polar which increase ophilic nature o he lipoid layer he present stud ivity when com n the activity o groups in the l ons, it was conc al complexes gav ation of coordin odes and elucid plexes and also with reduced to emical evidence ure (Figure 2) anthrao Yerna chnology, New ST/AORC‐IF/UP nd their than ophisticated Te ersity, Sophist njab University also thankfu re in Biotechn 26, 1‐8. uropean Journal 1. Antioxidant activ N S H3 H3 sed structures of th all the higher y was d with rity of es the of the of the dy the mpared of the igand. cluded ve the nation dating o they oxicity es, we of the ale) is Delhi PGRD/ ks to st and icated y, for ul for ology, [2]. [3]. [4]. [5]. [6]. [7]. [8]. [9]. [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] of Chemistry 7 (1 vity results. O N H3C H3C he metal complexe Ikechukwu, P. E Reda, A. A. A.; A 8686‐8699. Richard, H. H.; 2314. Yuanyuan, C.; Biol. Chem. 201 Joseyphus, R. S 204. Yoshihisa, K.; Hiroshi, S.; Yuy 923‐931. Xu, Z.; Ba, M.; Zhang, X.; Li, Z.; 42. Leyla, Y.; Yusuf . Yernale, N. G.; 2014, 314963, . Maya, S. S.; Sush . Kiran, S.; Yoge 2012, 729708, . Basavarajaiah, 2010, 49B, 111 . Nagesh, G. Y.; R 2015, 1079, 42 . Vogel, A. I, Q Instrumental A . Threlfall, E. J.; Microb. Drug re . Prescott, J. F. Antimicrobial s Antimicrobial T Press, 12‐26. . Meyer, B. N.; Fe E.; McLaughlin. . Finney, D. J. P Kingdom, 1971 . Singh, R. P.; M 2002, 50, 81‐82 . Geary, W. J. Coo . Roy, S.; Mandal J. Coord. Chem. . Nagesh, G. Y.; Pharm. Sci. Rev. . Chandra, S.; Gu 1) (2016) 56‐65 H C O N H N C O H N Zn Cl es. E.; Peter, A. A. Mole Abdel‐Nasser, M. A Pierre, K.; Edwar Erik, R. F. ; Mark, 12, 287, 13356‐133 S.; Sivasankaran, N Naoyuki, M.; Mich ya, H.; Makoto, H.; Zhou, H.; Cao, Y.; ; Zhu, L.; Guo, Y.; G f, O.; Hulya, K. G.; U Mruthyunjayaswa 1‐17. hobhan, C. RSC Adv ender, K.; Parvesh, 1‐9. S. M.; Mruthyunja 17‐1126. Raj, K. M.; Mruthyu 3‐432. Quantitative Inorg Analysis, 2th edition Fisher, I. S. T.; Wa esist. 1999, 5, 195‐ .; Baggot, J. D.; susceptibility testi Therapy in Veterin errigni, N. R.; Putn Planta. Med. 1982 Probit Analysis, C 1. urthy, K. C. N.; Jay 2. ord. Chem. Rev. 197 l, T. N.; Das, K.; But 2010, 3, 2146‐215 Mahadev, D. U.; M . Res. 2015, 31, 19 pta, L. K. Spectroch N S ecules 2015, 20, 97 A. A. Int. J. Electroc rd, I. S. Chem. Rev R. C. ; Krzysztof, 370. N. M. Arabian J. Che hihiro, K.; Takash Masahiro, M. Che Tang, C.; Yang, Y. Guo, C. Eur. J. Med. C lviye, A. J. Chem. 20 amy, B. H. M. Bioi vances 2012, 2, 45 , P.; Gulab, S. Bioi ayaswamy, B. H. M unjayaswamy, B. H anic Analysis Inc n, London, 1962. ard, L.; Tschape, H 199. Walker, R. D.; ing and interpreta nary Medicine, Iow nam, J. E.; Jacobsen 2, 45, 31‐34. ambridge Univers yaprakasha, G. K. 71, 7, 81‐122. tcher, R. J.; Rheingo 57. Mruthyunjayaswam 0‐197. him. Acta A 2005, 6 788‐9802. chem. Sci. 2013, 8, v. 1996, 96, 2239‐ P.; Philip, D. K. J. em. 2010, 3, 195‐ hi, H.; Daisuke, Y.; em. Pap. 2014, 68, ; He, R.; Liang, Y.; Chem. 2014, 6, 27‐ 015, 464379, 1‐7. inorg. Chem. Appl. 47‐4592. inorg. Chem. Appl. M. Indian. J. Chem. H. M. J. Mol. Struct. cluding Elemental .; Gerner‐Smidt, P. eds. Ames, I. A, ation of results. In: wa State University n, L. B.; Nichols, D. sity Press, United Agric. Food Chem. old, A. L.; Kar, S. K. my, B. H. M. Int. J. 62, 1102‐1106. , ‐ . ‐ ; , ; ‐ . . . . l . , : y . d . . . Yernale et al. / European Journal of Chemistry 7 (1) (2016) 56‐65 65 [25]. Liu, H.; Wang, H.; Gao, F.; Niu, D.; Lu, Z. J. Coord. Chem. 2007, 60, 2671‐2678. [26]. Rai, R. A. J. Inorg. Nucl. Chem. 1980, 43, 450‐453. [27]. Underhill, A. E.; Billing, D. E. Nature, 1966, 210, 834‐835. [28]. Bayoumi, H. A.; Alaghaz, A. M. A.; Aljahdali, M. S. Int. J. Electrochem. Sci. 2013, 8, 9399‐9413. [29]. Satyanarayana, D. N. Electronic Absorption Spectroscopy and Related Technique, University Press India Limited, New Delhi, 2001. [30]. Singh, D. P.; Kumar, R.; Malik, V.; Tyagi, P. Trans. Met. Chem. 2007, 32, 1051‐1055. [31]. Baranwal, B. P.; Gupta, T. Synth. React. Inorg. Met‐Org. Chem. 2004, 32, 1737‐1754. [32]. Rao, T. R.; Archana, P. Synth. React. Inorg. Met‐Org. Chem. 2005, 35, 299‐304. [33]. Thaker, B. T.; Tandel, P. K.; Patel, A. S.; Vyas, C. J.; Jesani, M. S.; Patel, D. M. Indian J. Chem. 2005, 44A, 265‐270. [34]. Abdallah, S. M.; Zayed, M. A.; Mohamad, G. G. Arab. J. Chem. 2010, 3, 103‐113. [35]. Hathaway, B. J.; Billing, D. E. Coord. Chem. Rev. 1970, 5, 143‐207. [36]. Chohan, Z. H.; Arif, M.; Akhtar, M. A.; Supuran, C. T. Bioinorg. Chem. Appl. 2006, 83131, 1‐13. [37]. Nagesh, G. Y.; Mruthyunjayaswamy, B. H. M. J. Mol. Struct. 2015, 1085, 198‐206. [38]. Abd El‐Wahab, Z. H.; Mashaly, M. M.; Salman, A. A.; El‐Shetary, B. A.; Faheim, A. A.; Spectrochim. Acta A 2004, 60, 2861‐2873. [39]. Petering, D. H.; Sigel, H. Metal Ions in Biological Systems, Marcel Dekker, New York, 1980. [40]. Hartl, M.; Humpf, H. U.; Food Chem. Toxicol. 2000, 38, 1097‐1102. [41]. Mahendra, R. K.; Vivekanand, B.; Nagesh, G. Y.; Mruthyunjayaswamy, B. H. M. J. Mol. Struct. 2014, 1059, 280‐293. [42]. Satyendra, M.; Shailendra, K. J.; Avadhesha, S. Electrochim. Acta 2015, 151, 574‐583.