3 Quantum Chemical Calculations for some Isatin Thiosemicarbazones

Derivatives of isatin are reported to be present in mammalian tissues and body fluids (Casas et al., 1996; Agrawal & Sartorelli, 1978; Casas et al., 1994; Medvedev et al., 1998; Boon, 1997; Pandeya & Dimmock, 1993; Rodríguez-Argüelles et al., 1999; Casas et al., 2000) and possess antibacterial (Daisley & Shah, 1984), antifungal (Piscopo et al., 1987), and anti-HIV (Pandeya et al., 1998, 1999) activities. N-methylisatin--4’, 4’ – diethylthiosemicarbazone were also reported to have activity against the viruses such as cytomegalo and moloney leukemia viruses (Sherman et al., 1980; Ronen et al., 1987). With the help of combinatorial method, the cytotoxicity and antiviral activities of isaitin--thiosemicarbazones against the vaccine virus and cowpox virus-infected human cells were evaluated (Pirrung et al., 2005).

Isatin-thiosemicarbazones may coordinate through the deprotonated nitrogen atom, sulphur atom of thiosemicarbazone group, and carbonyl oxygen atom with the metal, depending on its nature.Zinc (II) and mercury (II) complexes of isatin-3-thiosemicarbazones were reported to be coordinated through imino nitrogen and thiolato sulfur atoms and was suggested to have tetrahedral structures (Akinchan et al., 2002).

Antibacterial activity
The antibacterial activities were determined by using the agar well diffusion method (Rahman et al., 2001).The wells were dugged in the media with a sterile borer and an eighthour-old bacterial inoculum containing 0>ca.104-106 colony forming units (CFU)/mL were spread on the surface of the nutrient agar.The recommended concentration of the test sample (2 mg/mL in DMSO) was introduced into the respective wells.Other wells containing DMSO and the reference antibacterial drug imipenum served as negative and positive controls, respectively.The plates were incubated immediately at 37 o C for 20 h.The activity was determined by measuring the diameter of the inhibition zone (in mm), showing complete inhibition.Growth inhibition was calculated with reference to the positive control.

Antifungal assay
To test for antifungal activity, the agar dilution method, a modification of the agar dilution method of Washington and Sutter (980), was employed (Ajaiyeoba et al., 1988).Test tubes having sterile SDA were inoculated with test samples (200 mg/mL), and kept in a slanting position at room temperature.Test fungal culture was inoculated on the slant and growth inhibitions were observed after an incubation period of 7 days at 27 o C. Control agar tubes were made in paralellel and treated smilarly, except for the presence of test sample.Growth inhibition was calculated with reference to positive control.
Fukui functions, which are common descriptors of site reactivity and can be expressed by the following equations, were calculated by AOMix program (Gorelsky, 2009;Gorelsky & Lever, 2001).
Where, k represents the sites (atoms/molecular fragments) for nucleophilic, electrophilic and radical agents and  k are their gross electron populations.An elevated value of k f implies a high reactivity of the site k.

Results and discussion
The optimized structures of the compounds 18, 5, 19, and 20 ligands, and the optimized structures of their corresponding Ni(II) and Zn(II) complexes are shown in Figure 1.

Compound 14
Compound 9 Fig. 1.Optimized structures of compounds 18, 5, 19, 20, 14, 9 Values of the optimized geometrical parameters for all compounds are presented in Tables 2 and 3.The bond lengths, bond angles, and dihedral angles for compounds 18, 5, 19, and 20 are almost the same as in isatin group.Mulliken charges of most atoms, except N and S, belong to thiosemicarbazone group are the same value.Therefore comparision among ligands, and deprotonated forms of ligand and complexes were made for compound 19.In the deprotonated forms of 19, as in N11-N12, C6-C7, and C13-C14, the bond lengths decrease, while C7-C9, C5-N8, and C4-C6 bond lengths increase.In Zn (II) complexes, C4-C6, C6-C7 bond lengths are similar to those of ligands.N11-N12 bond length in the deprotonated form of the ligands decreases, whereas in the complexes this bond length increases due to a transfer of charge from N atoms to metal.In the complex form, C-S bond length increases from 1.667 Å to 1.759 Å in Ni (II) complex, and 1.744 Å in Zn (II) complex.
The calculated bond lengths of the Zn-S and Zn-N bonds for compound 9 were found to be 2.323 and 2.107 Å.As presented in Table 3, Mulliken charges of C5, C6, N8, C9, and N12 were similar to each other in the neutral form.Most of the changes are on the N14 atom because of the changes in substituent attached to the N14 atom.The Mulliken charge of N14 atom was -0.405 ē for H 2 IBT, -0.392 ē for H 2 ICHT, -0.463 ē for H 2 IPT, and -0.437 ē for H 2 ICPT.Mulliken charges of atoms, both in the indole ring and thiosemicarbazone group, undergo a significant change, depending whether ligand is in the deprotonated form or in complexation process.

The possible tautomeric forms of ligands
The optimized structure for 18 ligand, calculated with B3LYP/6-311G(d,p), is shown in Figure 2. Electronic and zero point energies for compounds 18, 5, 19, and 20 were given in Table 4.The most stable tautomeric form is A for studied ligands.In the form A, calculated electronic and zero point energy for compounds
Metal acetates with the ligands (H 2 L) lead to isolation of complexes of formula M(HL) 2 (Rodriguez-Argiielles et al., 1999).As seen from the C, H, N analyses, the synthesized complexes are with the formula M(HL) 2 (if the ligand is written as H 2 IPT, then complex has the formula of M(HIPT) 2 ) and form 2:1 ligand-to-metal complexes which two of the ligands were anionic.
The UV transitions and their excitation energies and ossillator strengths for zinc (II) and nickel (II) complexes of compounds 5, 18, 19, and 20 were calculated by using TDB3LYP with the 6-31G(d,p), 6-311G(d,p), BP86-CEP-31G basis sets.The transitions for nickel (II) complexes were calculated with both multiplicity states, 1 and 3, with two unpaired electrons.The results are summarized in Tables 11-14.The UV spectra, calculated with two unpaired electrons for nickel (II) complexes, were not in agreement with the experimental results.The band assigned at around 372, due to C=N transitions, were observed at 432, and 450 nm in the UV spectra of zinc (II) and nickel (II) complexes, respectively.Band at 434 nm (experimental) for compound 18 was found to be at 436 nm in the calculated of spectra of BP86-CEP-31G.Bands located around 360 nm in zinc (II) complexes and 374 nm in nickel (II) complexes due to ligand LMCT transition, suggested a metal-sulfur bond formation (Akinchan et al., 2002).Nickel (II) complexes showed three transitions around 450, belonging to transition 3 A 2g  3 T 2g (F), 635 including transition A 2g  3 T 2g (F), and 842 in transition 3 A 2g  3 T 1g (P), these transitions are characteristic of hexacoordinated nickel (II) complexes.5.93(0.10)E41BP86-CEP-31G* 3.17(0.12)E23.24(0.32)E33.66(0.42)E44.83(0.24)E94.88(013)E10 5.05(0.25)E115.24(0.10)E13The proton signal of N8-H group seen at  9. 77, 8.85, 10.83, 10.88, respectively, in the 1 H-NMR spectrum for compounds 5, 18, 19, and 20, also appeared in the spectrum of their zinc (II) complexes at  10.81, 10.80, 11.01, and 11.06, respectively.The upfield shift was due to the lack of intermolecular hydrogen bonding in its corresponding complex (Akinchan et al., 2002).A stronger hydrogen-bond interaction will shorten the O-H distance, will elongate the N-H distance, and will also cause a significant deshielding of the proton leading to a further downfield NMR signal (De Silva et al., 2007).The peak due to N12-H in the 1 H-NMR spectra of compounds 5, 18, 19, and 20 disappeared in their corresponding complexes.
The experimental and theoretical NMR results of compounds 11, 6, 9, and 10 are shown in Table 16.Geometrical optimizations of above mentioned studied complexes were performed by using the B3LYP method, and 6-31G(d,p), 6-311G(d,p) standard basis sets at the gas phase, and DMSO solution.The calculated peak, due to N12-H in the 1 H-NMR spectra of compounds 5, 18, 19, and 20 disappears in their corresponding zinc (II) complexes, as the calculation was performed by considering the corresponding ligand, deprotonated from N12 belong to thiosemicarbazone group in its complex form.

Antifungal activity
All the compounds 1-16 were also tested against Candida albicans, Aspergillus falvus, Microsporum canis, Fusarium solani, and Candida galbrata, according to the literature protocol (Lee et al., 1988) and found that compounds showed a varying degree of percentage inhibition.These results were compared with the standard drugs miconazole and amphotericin B, as shown in Table 18.Compound 5 demonstrated activity an against Aspergillus flavus (40% inhibition).
Compound 10 was found to be active against Candia albicans with 40% inhibition.
Table 18 shows results of antifungal assay on compounds 1-16 (concentration used 200 g/mL of DMSO, and Percentage Inhibition).All the compounds in this series showed nonsignificant activity against all the bacteria.

Acknowledgement
The financial support for this study was provided by the TUBİTAK Fund (Project Number: 108T974), and the High Education Commission of Pakistan.

Table 5
HOMO (highest occupied molecular orbital), and the E LUMO (lowest unoccupied molecular orbital).The highest occupied and the lowest unoccupied molecular orbital

Table 6 .
Calculated values for the highest occupied and the lowest unoccupied molecular orbital energies HOMO and LUMO, and the five molecular orbital energy the nearest frontier orbitals for (a) 18, (b) 5, (c) 19, and (d) 20