Research Article Synthesis Characterization and Biological Activity Study of New Schiff and Mannich Bases and Some Metal Complexes Derived from Isatin and Dithiooxamide

Two new Schiff and Mannich bases, namely, 1-Morpholinomethyl-3(1′ -N-dithiooxamide)iminoisatin (LIH) and 1-diphenylaminomethyl-3-1′-N-dithiooxamide)iminoisatin (LIIH), were prepared from condensation reaction of new Schiff base 3-(1′-N-dithiooxamide)iminoisatin (SBH) with morpholine or diphenylamine respectively in presence of formaldehyde . The structures were characterized by IR, 1HNMR, mass spectrometry, and CHN analyses. Metal complexes of the two ligands were synthesized, and their structures were characterized by elemental analyses, atomic absorption, IR and UV-visible spectra, molar conductivity, and magnetic moment determination. All complexes showed octahedral geometries except palladium complexes which were square planar. The biological activity of the prepared compounds and some selected metal complexes was tested against three types of bacteria and against cell line of human epidermoid larynx carcinoma (Hep-2).


Introduction
Various Mannich Schiff bases of isatin have been found to be of biological importance [1] and have shown anticonvulsant [2], antibacterial [3,4], antimicrobial [5][6][7] and anti-HIV activities [8,9]. Dithiooxamide (dto) is an effective flexidentate complexing agent with varied coordination chemistry. Due to the intense chromophoric character, dto can be used in an imaging processes [10], coordination polymers [11], histological agents, and as a source for duplicating processes [12]. The transition metal complexes of dto and its derivatives are characterized by semiconductor, magnetic, and spectroscopic properties [12][13][14][15]. The aim of this work is to synthesize and study the coordination behavior of the two new Schiff and Mannich base ligands L I H and L II H shown in Scheme 1, from condensation reaction of a new Schiff base 3-(1 -N-dithiooxamide) iminoisatin (SBH) with morpholine or diphenylamine, respectively, in presence of formaldehyde in a mole ratio of (1 : 1 : 1), respectively, or from reaction of Mannich bases N-morpholinomethyl isatin (M I ) and Ndiphenylaminomethyl isatin (M II ) [16] with dithiooxamide.
The biological activity of the two ligands and some of their metal complexes was investigated against selected types of bacteria and against cancer cell line of human epidermoid larynx carcinoma (Hep-2).

Experimental/Materials and Methods
Melting points (uncorrected) were determined by using Gallenkamp MFB600-010f m.p apparatus. The purity of the synthesized compounds was checked by T.L.C. techniques using a mixture of chloroform and acetone (2 : 2 V/V) and various ratios of methyl acetate: acetone solvent mixture as eluents and iodine chamber for spot location. The HPLC of the Schiff base (SBH) and the derived two ligands were obtained by using HPLC (LKB), mobile phase CH 3 CN : H 2 O (80 : 20). Infrared spectra were recorded on a Perkin-Elmer 1310 IR spectrophotometer and Shimadzu corporation 200-91527 IR spectrophotometer using KBr and CsI disks. 1 H n.m.r spectra of the organic compounds were recorded on a 300 MHz n.m.r spectrophotometer (Joel) using TMS as internal reference. Mass spectra were recorded on a Joel 700 mass spectrometer. Elemental CHN analyses were obtained by using EA elemental analyzer (Fison Ision Instrument). Electronic spectra of the ligands and their metal complexes in the region 200-1100 nm were recorded on a Shimadzu UV-visible-160 spectrophotometer. The metal contents were determined by atomic absorption technique using a Varian-AA-775 atomic absorption instrument. Electrical conductivity of metal complexes was measured at room temperature in DMF (10 −3 M) using Elkta Lictfahigkeit conductivity meter (SIMENS). Magnetic moments (μ eff BM) for the solid metal complexes at room temperature were determined according to Faraday's method by using Johnson Mattey magnetic balance system division. Chloride content of metal complexes was determined by potentiometric titration using 1686titroprocessor-665 Dosinametrom (Swiss). All organic and inorganic materials were of high purity and used as received except ethanol, methanol, and DMF which were dried and distilled prior to use [17]. Palladium(II) chloride was converted to dichlorobis(benzonitrile)palladium(II) [18], and H 2 PtCl 6 ·6H 2 O was converted to potassium hexachloroplatinate(IV) hexahydrate [19] prior to use. Mannich bases Nmorpholinomethylisatin (M I ) and N-diphenylaminomethyl isatin (M II ) were prepared according to methods mentioned in the literature [16]. Complex formation was studied in solutions to obtain the molar ratio of the ligand to metal ion (L : M) using ethanol, or DMSO as solvents.   [16] in ethanol (10 mL) with continuous stirring, and the mixture was heated under reflux for 10 h. After leaving the mixture at room temperature for 24 h a precipitate was formed. The products were filtered, washed with warm ethanol, and crystallized; yield 20.3 and 21%, respectively (m.p. 215 and 283 • C, resp.).

Synthesis of Metal Complexes
To a solution of Schiff Mannich base ligand (2 mmole) in absolute ethanol (L I H) or ethanol and dimethylsulfoxide (1 : 1 v/v) (L II H) (5 mL) was added an alcoholic solution (5 mL) of the metal salt (chlorides, nitrates, or acetates) (1 mmol), and the mixture was heated under reflux with continuous stirring for 3 h. Precipitation of products took place after heating time of 30 min for (Co(II)), Ni(II), and complexes of L I H and L II H (C 2 , C 3 , C 8 , and C 9 , resp.), 1 h for (Mn(II), Cu(II), and Ir(III) complex of L I H and Cd(II) complex of L II H (C 1 , C 4 , C 5 , and C 12 , resp.), 1.5 h for Pt(IV) complex of L I H (C 7 ), 2 h for Pd(II) complex of L I H and Pt(IV) complex of L II H (C 6 and C 11 , resp.) and 3 h for Pd(II) complex of L II H (C 10 ). The products were filtered and purified from reactants by washing many times with ethanol and ether (C 1 -C 7 ) or with DMSO, ethanol and ether (C 8 -C 12 ), and vacuum dried. Purity of the products was detected by TLC, using silica gel as a stationary phase and a mixture of chloroform and acetone (2 : 2 V/V) or various ratios of methyl acetate: acetone solvent mixture as eluents.

Antibacterial Action.
Antibacterial activities of the prepared compounds were tested against three types of pathogenic bacteria, namely, Escherichia coli, Staphylococcus areus, and Proteus mirabilis using the antibiotic Ceftriaxone as a control. Bacterial cultures were prepared by streaking (0.1) mL of 10 6 CFU/mL broth of indicator strain on the whole surface of nutrient agar plate. In each plate four wells (pores) were created on the nutrient agar layer using sterile cork porer. In each hole was injected 50 μL of 10 −3 M of the studied compounds in DMSO by micropipette. The resulting cultures were incubated at 37 • C for 24 h. The inhibition zones caused by each compound were measured, and the results were interpreted according to diameter measurements.

Cytotoxic Activity.
A preliminary study of cytotoxic activity of some of the prepared compounds was performed against human epidermoid larynx carcinoma cell lines (Hep-2) of 52-year-old patient. Hep-2 monolayer cell lines were prepared by subculturing cell line into (RPMI-1640) medium supplemented with 10% heat deactivated fetal bovine serum. The resulting media were incubated at 37 • C for 48 h until confluent layer was achieved. Four concentrations of investigated compounds were prepared: 62.5, 125, 250, and 500 μg/mL using dimethyl sulfoxide (DMSO) as a diluent. Hep-2 cell line was plated into 96-well microtiter plates. Then 0.2 mL of each tested compound was added to each well in triplicates, and incubation was carried out for 48 h. Cultures were stained with 50 μL/well Neutral Red (NR) solution. The stained cultures were left in the incubator for further 2 h, washed with phosphate buffered saline solution followed by (0.1 mL) ethanol phosphate buffered solution (NaH 2 PO 4 : ethanol (1 : 1), vehicle ethanol). The cytotoxic effects of the applied compounds were measured in terms of optical density of viable cells at λ = 492 nm using a Micro ELISA reader.

Results and Discussions
6.1. Synthesis. The synthesis of the two new ligands has been achieved by following two different pathways A and B as is illustrated by Scheme 2. Pathway A involves the synthesis of Schiff base precursor of isatin (SBH) followed by condensation with the secondary amine, morpholine or diphenylamine, in presence of formaldehyde to form L I H and L II H, respectively. Pathway B involves the formation  of Mannich base precusor of isatin (M I and M II ) followed by condensation reaction with dithiooxamide. The second method showed lower yield and longer reaction time.
The 1 H n.m.r spectrum of the Schiff base precursor SBH in CD 2 Cl 2 ( Figure 1) is characterized by the appearance of chemical shift related to the NH 2 protons of dto moiety at δ2.022 ppm [11,20,21] and the appearance of NH proton of isatin ring at δ12.012 ppm [5][6][7][22][23][24][25] which is quite agreeable with the suggested structure of SBH. The 1 H n.m.r spectrum of L II H in CD 2 Cl 2 exhibited chemical shifts of NH 2 protons at 2.17 ppm while that of L I H in DMSO ( Figure 2) gave chemical shifts at δ1.567 ppm. This was attributed to tautomerism of L I H in DMSO to iminosulfhydryl structure in equilibrium with dithioamide structure, as a result of solvent polarity [26,27]. Such behavior was confirmed by the appearance of the signal assigned to imino NH group at lower field. The spectrum of L II H ( Figure 3) exhibited chemical shifts of aromatic protons of isatin ring and diphenylamine at δ6.89-7.11 and at δ7.68-7.59, respectively, while those of methylene group appeared at high fields [22][23][24].
The mass spectra of the two Mannich and Schiff base ligands as well as SBH are shown in Figures 4, 5,   The IR spectra of the three organic compounds exhibited the disappearance of stretching modes assigned to C-3 carbonyl of isatin ring and appearance of stretching modes of azomethine group of Schiff base products at 1614 cm −1 [35]. Stretching vibrations of C-2 carbonyl group of isatin ring for SBH and the two Mannich Schiff base ligands were observed at 1733-1730 cm −1 [35]. The presence of bands assigned to NH 2 asymmetric symmetric stretching vibrations indicates that the formation of Schiff bases was through one NH 2 group only. Both ligands exhibited the absence of stretching vibrations assigned to NH of isatin ring, and instead vibrational modes of N-CH 2 groups were observed at 2813-2304 cm −1 [35]. Bands observed at 1149, 1328 in the spectrum of L I H were attributed to C-O-C and C-N-C vibration of morpholine ring, respectively [35][36][37][38].

Physical Properties and Analytical Data of Metal
Complexes. The color, melting points, yields, and elemental analyses of the prepared metal complexes of isatin Schiff Mannich base ligands are described in Table 1. Most results were in agreement with the suggested formula. Some deviations in elemental analyses may be attributed to incomplete combustion of the complexes. The low yield resulted from extensive purification of products from the starting materials as was indicated from TLC results.  Table 2. The Mn(II), Co(II) and Ni(II) complexes of L I H (C 1 -C 3 , resp.) and Co(II) complex of L II H (C 8 ) exhibited shifts of the thioamide groups to lower frequencies indicating the involvement of thiocarbonyl sulfur atoms in coordination with these metal ions [39,40]. The spectra of C 1 and C 2 demonstrated further shift of NH 2 group vibrational modes to lower frequencies as a result of bonding. On the other hand the spectra of Cu(II), Ir(III), and Pt(IV) complexes of L I H (C 4 , C 6 , C 7 , resp.) and Pd(II), Pt(IV),  and C 10 -C 12 . The appearance of stretching modes assigned to NH and C=N of −C=NH groups was observed at 3371-3100 and 1640-1620 cm −1 , respectively [15,35,39,41]. The stretching vibrations of azomethine group of the Schiff base ligands were shifted to lower frequencies in all spectra except those of C 6 , C 10 , and C 12 , whereas stretching vibrations of carbonyl group were shifted to lower frequencies in all spectra except C 1 , C 4 , C 6 , and C 10 indicating additional     coordination of metal ions to C=N and C=O groups [36][37][38]. Bands related to coordinated water vibrations were observed in the spectra of C 1 , C 4 , and C 12 at (3490, 756, 640), (3480, 800, 730), and (3500, 864, 710) cm −1 , respectively, and to lattice water vibrations at frequency range 3519-3464 cm −1 in the other complexes. The bands related to nitrate ions were observed in the spectra of C 2 and C 3 at (1522, 1478), (1765, 1641) cm −1 and were assigned to monodentate and free ion behaviors, respectively, whereas that of C 8 appeared at 1750-1660 and 1406-1380 cm −1 showing monodentate and bidentate behaviors, respectively [42]. Bands attributed to acetate group vibrations were observed in the spectra of C 9 and C 12 at (1645, 1340) and (1590, 1465) cm −1 , respectively, indicating monodentate and bidentate bridging behaviors, respectively [42]. Additional bands were observed at lower frequencies (600-250 cm −1 ) and were attributed to M-N, M-O, M-S, and M-X (X = acetate, NO − 3 , Cl − ) stretching modes [42].

Thermal Analysis.
Steps of thermal decomposition of the Co(II) and Ir(III) complexes of L I H (C 2 , C 6 ) following TG and DTG curves under nitrogen atmosphere and heating range 50-800 • C are described in Table 3, and their thermographs are shown in Figures 7 and 8, respectively. At low temperatures, the initial weight losses were determined from TG curves referred to loss of water of crystallization [43][44][45]. The final stage of thermal decomposition of C 2 gave the metal oxide whereas the Ir complex (C 6 ) gave the free metal as a final residue [43][44][45]. Table 4 describes the energies of bands observed in the spectra of metal complexes and their assignments together with magnetic moments and molar conductivity in DMF (10 −3 M). The spectral parameters 10 Dq, Dq/B, B, and β as well as energies of unobserved ligand field bands were obtained by applying observed band energies and band ratios on Tanabe-Saugano diagrams of the specified metal ion [46][47][48].

Electronic Spectra and Suggested Structures.
All metal complexes exhibited spectra related to octahedral arrangement of ligand atoms around the metal ions except those of palladium(II) as they gave square planar geometries.
The high values of magnetic moments of Co(II), Ni(II), and Cu(II) complexes are attributed to spin-orbital coupling [49]. All complexes were of high-spin octahedral geometries except Pt(IV), Ir(III), and Cd(II) complexes which were diamagnetic and so were Pd(II) complexes. The spectrum of the Cd(II) complex (C 12 ) exhibited charge transfer bands only, which is a common phenomenon for d 10 metal complexes where d-d transitions are excluded [47,48]. Conductivity measurement of metal complexes in DMF solution (10 −3 M) showed nonelectrolytic nature of Mn(II), Co(II), and Pt(IV) complexes of L I H (C 1 , C 2 , and C 7 , resp.) and Co(II), Ni(II), Pd(II), and Cd(II) complexes of L II H (C 8 -C 10 and C 12 , resp.) [50]. Electrolytic nature of 1 : 1 was exhibited by Pd(II), Ir(III) complexes of L I H (C 5 and C 6 ) and Pt(IV) complex of L II H (C 11 [50]. According to the above-mentioned data and those of elemental analyses and i.r. spectra, the structures of the metal complexes can be suggested as illustrated in Scheme 3.

Biological Activity
7.1. Antibacterial Activity. The growth inhibition of the prepared Schiff and Mannich base ligands and some selected metal complexes were studied against three types of pathogenic bacteria, namely, Proteus mirabilis, Escherichia coli, and Staphylococcus aureus by using DMSO as a solvent and the antibiotic Ceftriaxone as a control. Cultures were incubated at 37 • C for 24 h. The inhibition zones were measured, and results are described in Table 5. The Schiff base precursor (SBH) and L I H were potent against all types of bacteria with the latter being more active than Ceftriaxone, while L II H was inactive. Complexes of L I H with Co(II), Ni(II), Pd(II), and Ir(III) ions (C 2 , C 3 , C 5 , C 6 ) showed no activity while the Pt(IV) complex (C 7 ) was as active as the original ligand against all types. Among the selected metal complexes of L II H, the Pd(II) complex (C 10 ) was highly potent against all bacterial cultures. These results indicate that the degree of growth inhibition is highly dependent on the structure of ligands, metal complexes, and type of metal ion [51,52]. Although the inhibition zones of L I H, C 7 and C 10 were larger than that caused by Ceftriaxone, other categories, like toxicity of these compounds, still have to be studied in detail.  Intralig π → π * * Calculated. in optical absorbance of NR in the treated cells compared with the controls which refers to complete disruption of cell functions [53]. The cytotoxic effect of metal complexes of L I H was found to increase in the order of Pt(IV) < Pd(II) << Ir(III) as is shown in Figure 9. The Pt(IV) complex (C 7 ) was much less active than the parent ligand, and its performance was found to decrease with concentration that it was totally inactive at 500 μg/mL. The Pd(II) complex (C 5 ) followed the same trend of concentration as the Pt(IV) complex but it was toxic enough to cause cell death. The Ir(III) complex (C 6 ) was exceptionally active, and its cytotoxic activity was found to increase with concentration. Figure 10 illustrates the difference in dye distribution in the tissue culture sections of Hep-2 before and after treatment with this complex in comparison with the control. Both the Pd(II) and Cd(II) complexes of L II H (C 10 and C 12 ) were less toxic than the parent ligand, and their activity slightly increased with concentration. The Cd(II) complex (C 12 ) caused 30-60% decrease of ligand activity. The complex was similarly inactive against growth of the three bacterial cultures although Cd ++ ion is well known as an environmental carcinogen at very low concentrations in human and animals [54]. This indicates that the toxicity of Cd ++ ion can be decreased on complexation with some ligands.

Cytotoxic
The present study describes a narrow scope of the cytotoxic activity of the studied compounds. Extension of this study, in future work to involve other cancer cell lines and other metal complexes, using normal human cell lines as control, may reveal more important data.

Conclusions
Condensation of dithiooxamide with isatin or its N-Mannich bases occurred from one amino end of the compound which allowed for tautomerism of the resulted compound in solutions as was confirmed by 1 H.n.m.r spectrum of the product and from the IR spectra of some of its coordination compounds. The potent chelating behavior of the two new Mannich and Schiff bases led to the formation of bi-and polynuclear metal complexes. The preliminary study of biological activity showed some controversy in performance between bacterial growth inhibition and cytotoxic activities against Hep-2 cell line. The Ir(III) complex of L I H which showed the highest cytotoxic effect was almost inactive against bacterial growth.