Synthesis, Spectral Characterization and Biocidal Studies of Copper(II) Complexes of Chromen-2-one-3-carboxy Hydrazide and 2-(Chromen-3'-onyl)-5-(aryl)-1,3,4-oxadiazole Derivatives

Copper(II) complexes of chromen-2-one-3-carboxyhydrazide and 2-(chromen-3'-onyl)-5-(aryl)-1,3,4-oxadiazole derivatives have been synthesized. The structural features have been determined from their microanalytical, magnetic susceptibility, molar conductance, IR, UV Vis, 1H NMR and ESR spectral data. All the Cu(II) complexes exhibit the composition Cu(Ln)2X2; where L1= chromen-2-one-3-carboxy hydrazide, L2 = 2-(chromen-3'-onyl)-5-(2ʺ-hydroxy phenyl)-1,3,4-oxadiazole, L3 = 2-(chromen-3'-onyl)-5-(4ʺ-nitrophenyl)-1,3,4 -oxadiazole and L4 = 2-(chromen-3'-onyl)-5-(4ʺ-chlorophenyl)-1,3,4-oxadiazole; X = Cl-, Br-, NO3-, CH3COO-, ClO4- and CNS-. The N, O donor ligands act as a bidentate ligand in all the complexes. Distorted octahedral geometry for all the Cu(II) complexes is proposed. Molecular modeling studies have been made for the rapid structure building, geometry optimization and molecular display. These complexes show the conductance values, supporting their non-electrolytic nature. The monomeric nature of the complexes was confirmed from their magnetic susceptibility values. These complexes have been screened for their antimicrobial activities against some bacterial species like S.aureus, E.coli, Pseudomonas aeruginosa and few fungal strains C.albicans and Cryptococcus neoformans.


Introduction
The drugs containing a chromen-2-one nucleus have been reported in therapeutics for a wide variety of antibiotics 1 , anticoagulants 2 and skin pigmenting agents 3 . Hydrazides have been investigated due to their antibacterial, antifungal, antitumour and biocidal activity 4 . Hydrazides are highly useful starting materials and intermediates in the synthesis of heterocyclic molecules 5 . Metal chelates with hydazide derivatives have been the subject of several studies due to their structural aspects 6 . Many 1,3,4-oxadiazole derivatives show remarkable bactericidal and fungicidal activities 7 . The development of bacterial or fungal resistance to existing drugs is a major problem in anti microbial therapy and necessitates continuing research into new classes of antimicrobials 8 . The interesting properties of the metal chelates of chromen-2-one carboxy hydrazide and 1,3,4-oxadiazole derivatives have stimulated research in these compounds that find a variety of laboratory uses and many industrial applications 9,10 . For the study and designing of drug like molecules, the structurereactivity correlation is very important 11 . In order to develop new compounds of high potency, the biologically important moieties have been coupled and with the resulting molecules, metal chelates were synthesized and characterized.

Experimental
All chemicals used were of A.R. grade. The metal salts were prepared and analysed following the standard procedures 12 . The analyses for metal and anions were carried out by known methods 12 . Copper was estimated iodometrically by titration, chloride by Volhard's method, perchlorate by Kurz's method and acetate and nitrate by elemental analysis. C, H, N analyses were performed by microanalytical unit, CDRI, Lucknow. IR spectra were recorded on a Perkin-Elmer 783 IR-spectrophotometer. UV-Vis spectra of the complexes were recorded on Perkin-Elmer Lambda-25 spectrophotometer. The 1 H NMR spectra were recorded on a Bruker DRX-300 spectrometer at SAIF, Lucknow. The X-band ESR spectra of the complexes were recorded at room temperature in solid state in a Varian E112 X/Q band ESR-spectrometer at IIT Madras. Magnetic susceptibility was measured by vibrating sample magnetometer at IIT Madras.
Conductivity measurements were made on a Systronics direct reading conductivity meter. Molecular modeling studies were carried out in computer, 3D MAX, MAYA professional version at Bangalore. Antimicrobial studies were also carried out from sree chithra thirunal institute for medical sciences and technology, thiruvananthapuram.

Synthesis of ligands
Chromen and its derivatives were synthesized by Pechmann reaction method 13 . All the ligands (L1, L2, L3 and L4) were prepared using the methods reported earlier 14 . 3-Ethyl carbethoxy coumarin ester was prepared by condensation of salicylaldehyde with diethyl malonic ester using conc. H 2 SO 4 according to Pechmann reaction 13 . The compound obtained above (14 mmole) and hydrazine hydrate (16 mmole) were dissolved in a sufficient quantity of ethanol to give a clear solution and refluxed for 3 h. The contents were concentrated to a small volume. On cooling, the crystals of chromen-2-one-3-carboxy hydrazide (L1) separated out which were filtered and recrystallized from ethanol to give TLC pure colourless needles 15 .
Ligands Chromen-2-one-3-carboxy hydrazide (10 mmole) in phosphorous oxychloride (15 mL) and the acids [salicylic acid or p-nitro benzoic acid or p-chloro benzoic acid] (10 mmole) were added into a 100 mL R.B. flask. The reaction mixture was refluxed for 5 h, cooled to room temperature and poured on to crushed ice. On neutralization with 2% NaHCO 3 solution, a solid mass separated out, which was filtered, washed with water and recrystallized from methanol to give L2 or L3 or L4 which were purified by TLC. The melting point of the four ligands were L1=136 ºC, L2=310 ºC, L3=203 ºC and L4=138 ºC. All the ligands were confirmed by m.pts, IR and 1 H NMR spectra.  Volume of the solution was then reduced by keeping on a hot water bath and after cooling, pH of the mixture was raised to ~7.2 with one or two drops of alcoholic ammonia solution.
The metal complexes that precipitated were filtered, washed with ethanol and dried in a desiccator over anhydrous calcium chloride.

Results and Discussion
Analytical data given in Table 1 suggest that the metal-ligand stoichiometry is 1:2 and the complexes are monomeric in nature. Molar conductivity data obtained in methanol, acetonitrile and nitrobenzene suggest that all the complexes are of non-electrolyte type 16

Electronic spectra
The electronic spectra in methanolic solution were taken in the range 11111 to 50000 cm -1 and the absorption bands along with their assignments are given in Table 1. Cu(II) is a typical case where strong Jahn Teller distortion prevents the formation of regular octahedral complexes. The electronic spectra of the present copper(II) complexes show a broad band with a maximum of ~16160 cm -1 . This band may be due to the square planar or tetragonally distorted octahedral geometry around Cu(II) 18 . The band is likely to be the result of combination of 2 B 1g → 2 A 1g , 2 B 1g → 2 B 2g and 2 B 1g → 2 E 1g transitions in D 4h symmetry 19 . The high intensity band observed around ~24000 cm -1 for all the complexes in the u.v. region may be assigned to a ligand to metal, charge transfer excitation.

Magnetic moment
The effective magnetic moments of the Cu(II) complexes at room temperature are also shown in Table 1

IR spectra
The IR spectral bands and their assignments for the important groups of the free ligands and the complexes are helpful in deciding the stereochemical environment around Cu(II) ion in the complexes. The IR spectrum of the ligand L1 shows bands at 3458 and 3305 cm -1 , which are assigned to the two >NH stretching vibrations of the carboxy hydrazide group 23 . The band at 3458 cm -1 due to -NH stretching vibration of the terminal -NH 2 group is unaffected in the complexes, indicating the non-coordination of -NH 2 group to the metal atom 23 . The significant band in ligand (L1) undergoing change on complexation is that at 3305 cm -1 representing the stretching mode of hydrazide >NH group. This band is shifted by around 45-60 cm -1 to lower frequencies in the complexes confirming its involvement in co-ordination. Also, the bands at 1526 cm -1 and 731 cm -1 representing the in-plane and out-of-plane bending modes of >NH group show pronounced downshift (~30 cm -1 ) in the complexes 24 . The ligand spectrum exhibits two bands occurring at 1700 cm -1 and 1650 cm -1 , which may be due to the ring carbonyl (ν C= 0 , coumarin) and hydrazide carbonyl stretching frequencies. On chelation, the ring carbonyl band is red shifted by 30-40 cm -1 and the other carbonyl band is unaffected. The appearance of bands at 1272, 1156, 1030 and 980 cm -1 in the spectrum of Cu(II) complexes of chromen-2-one-3-carboxy hydrazide suggests the bidentate nature of the ligand 23 . IR spectra of the ligands L2, L3 and L4 exhibit a strong band at 1710 cm -1 representing the ν (C=O) chromen carbonyl group. On chelation this band is shifted to lower frequencies (∼ 20-40 cm -1 ) showing its involvement in complexation 25 .
The significant band around 3580 cm -1 representing the oxadiazole ring stretching frequency is highly altered and shifted to about 40-50 cm -1 to lower frequencies in the oxadiazole containing complexes 26 . This is further confirmed by the bands at 1600 cm -1 and 840 cm -1 representing the in-plane and out-of-plane bending modes of ν C=N group in the oxadiazole ring showing pronounced downshift ∼30 cm -1 in the complexes 26 .
The bands around 1480, 1370, 1030 and 970 cm -1 are assigned to the oxadiazole ring (nucleus) vibrations 26 . The bands at 1030 cm -1 can be assigned to υ C-O and the bands in the region 970-945 cm -1 are assigned to ν (N-N) groups. A strong band around 820 cm -1 representing the stretching mode of the disubstituted benzene is also present in all the oxadiazole ligands and their Cu(II) complexes. These bands are unaffected in the complexes, showing their non-involvement in complexation 27  The bands at 1422 cm -1 and 1312 cm -1 assigned to ν 2 vibration of coordinated nitrate in the complex 29 . The spectrum of the perchlorate complex shows strong bands at 1147 (ν 3 ) and 621 cm -1 (ν 4 ) with pronounced splitting 30 . In the IR spectra of Cu(II) acetate complex, strong bands appear at 1570 cm -1 and 1395 cm -1 assignable to coordinated acetate group vibrations 31 . The thiocyanate complex of Cu(II) shows a strong band at 2040 cm -1 along with medium bands at 828 and 460 cm -1 indicating N-coordinated thiocyanate in the complex 32 . The univalent nature of the halide ion evidenced by IR spectra of the chloride complexes and their non-electrolyte nature suggest that the chloride ion is coordinated to the metal 33 .

ESR spectra
The x-band ESR spectrum of Cu(L3) 2 Cl 2 complex recorded at 300K, at a microwave frequency 9.33 GHz with a field strength of 4000G, is characteristic of copper(II) in axial field symmetry, g || = g z and g ┴ = gx = g y . Calculated values of g || and g ┴ are 2.215 and 2.08 respectively. The trend g || > g ┴ > ge (free ion value) observed for this complex shows that the unpaired electron is located in the d x 2 -y 2 orbital of the copper(II) ion and the spectrum obtained is characteristic of axial symmetry 34 . A || value calculated for the complex is 200x10 4 cm -1 since the hyperfine component shows no hyperfine split; A ┴ = 0 35 . From the observed value, it is clear that g || > g ┴ >2.00 and A || > A ┴ which suggests that the complex is present in distorted octahedral geometry. Molecular orbital coefficients, α 2 (covalent in -plane σ -bonding) and β 2 (covalent in-plane π -bonding) were calculated and it is found that α 2 > β 2 which shows that the σ -bonding is more covalent than that of the in-plane π -bonding 36 .

Molecular modeling studies
Molecular modeling studies are carried out in computer, using 3D MAX software and energy minimized configurations are shown below. To describe the physical, chemical and biological properties, the structure is very important. For the study of the binding modes of drug like molecules and for the designing of molecular systems, the structure -reactivity correlation is important.

Antimicrobial activity
Carboxy hydrazide of chromen-2-one and 2-(chromen-3'-onyl)-5-(aryl)-1,3,4-oxadiazole derivatives and their Cu(II) metal complexes were screened for antibacterial activity and antifungal activity against the bacterial strains S. aureus, E. Coli and Pseudomonas aeruginosa and the fungal strains C. albicans and Cryptococcus neoformans by disc diffusion technique. The test solutions were prepared in DMSO and DMF at different concentrations (50-500 µg /mL). Chloromycetin and Amphotericin B discs were used as standard drugs for evaluating the antibacterial and antifungal activity. Nutrient agar was used as culture medium. The zones of inhibition formed were measured in mm. Cu(II) metal complexes were found to be more active than their ligands. In general, metal complexes are more active than their ligands because the metal complexes may serve as a vehicle for activation of ligands as the principal cytotoxic species 37 . The results of the biological activities show that these complexes are very active and so they can be used as drugs against the above mentioned bacterial and fungal diseases. Table 3 below shows the antimicrobial data of few Cu(II) complexes. ATCC Staphylococcus aureus ATCC pseudomonas aeruginosa