Synthesis and Characterization with Antineoplastic, Biochemical, Cytotoxic, and Antimicrobial Studies of Schiff Base Cu(II) Ion Complexes

Copper(II) complexes containing two Schiff base ligands derived from 2-hydroxybenzaldehyde with 2-aminophenol and 3-aminophenol have been synthesized and characterized by means of analytical, magnetic, and spectroscopic methods. Bacteria, fungus, Entamoeba histolytica, and antineoplastic activities of the synthesized complexes have been determined by monitoring the parameters cell growth inhibition, survival time of tumour mice, time-body relation, causing of intraperitoneal cells and macrophages, alkaline phosphatase activity, hematological effect, and biopsy of tumour.


Introduction
Schiff base metal complexes based research works have been widely carried out from 1930, because of their biological and industrial applications [1][2][3][4][5]. The use of metal complexes as pharmaceuticals has shown promise in recent years particularly as anticancer agents [6]. Previously, synthesis and properties of thiocyanato complex of low valent metal ions containing different monodentate auxiliary ligands have been reported from our laboratory [7][8][9]. Worldwide spread of drug-resistant bacteria is now a critical problem in global health. To find new drug, recently, we studied few mixedligand complexes containing heterocyclic amine as secondary ligands and few Schiff base containing complexes [10][11][12]. In present study, mixed-ligand complexes of Cu(II) containing the Schiff base ligand derived from 2-hydroxybenzaldehyde with 2-aminophenol/3-aminophenol and bidentate auxiliary ligands were synthesized and characterized. The auxiliary ligands used were potassium thiocyanato, 2-aminopyridine, and 2-phenylenediamine. Antineoplastic, biochemical, cytotoxic, and antimicrobial activities of the complexes were also studied.

Experimental
2.1. Physical Measurement. The weighing operation was performed on a METTLER PM-200 electronic balance. Conductivity measurements were carried out in dimethyl sulfoxide (DMSO) using a WPACMS 35 conductivity meter and dipcell with platinized electrodes. The melting or decomposition temperatures of all the prepared metal complexes were observed in an electrothermal melting point apparatus model number AZ6512. The SHERWOOD SCIENTIFIC Magnetic Susceptibility Balance was used for the present investigation. Infrared spectra as KBr disc were recorded in a SIMADZU FTIR-8400 (Japan) infrared spectrophotometer, from 4000 to 400 cm −1 . The absorbances of the complexes were recorded on SHIMUDZU Spectrophotometer. Analyses of the complexes for carbon, hydrogen, and nitrogen were carried out by Microanalytical Services at the University of St. Andrews, Scotland. Transplantable tumour (Ehrlich's Ascites Carcinoma, EAC) used in this research was obtained from Indian Institute of Chemical Biology (IICB), Calcutta, India. In vivo antineoplastic activity of the test complexes was determined by measuring the effect of the test complexes   on tumour cell growth inhibition, survival time of tumour bearing mice, hematological parameters, and serum alkaline phosphatase activity of tumour bearing mice. Tumour growth was monitored by recording daily weight change. The concentration of haemoglobin was measured by the usual procedure using Sahli's haemometer.

Procedure of Preparation of Schiff Base (SB-1).
The Schiff bases were prepared by the condensation of 2hydroxybenzaldehyde with 2-aminophenol. 2-Hydroxybenzaldehyde (1.7 g, 0.014 mol) in absolute ethanol (20 mL) was added to an ethanolic (30 mL) solution of 2-aminophenol (1.5 g, 0.014 mol). The mixture was heated to reduce the volume to 25 mL, and then it was cooled in an ice-bath. The black crystalline product was isolated and washed with hot ethanol. The structure of SB-1 is shown in Figure 1. was added to the methanolic solution of C 13 H 9 NO 2 H 2 (SB-1) (0.005 mol, 80 mL). The resulting mixture was boiled on a water bath for 5 minutes and cooled. The complexes were separated, washed with hot ethanol, and dried in vacuo over P 4 O 10 .

Procedure of Preparation of Schiff Base (SB-2).
The Schiff base was prepared by the condensation of 2hydroxybenzaldehyde with 3-aminophenol. 2-Hydroxybenzaldehyde (1.7 g, 0.014 mol) in absolute methanol (20 mL) was added to an ethanolic solution (30 mL) of 3-aminophenol (1.5 g, 0.014 mol). The mixture was heated to reduce the volume to 25 mL and then it was cooled in an ice-bath. The black crystalline product was filtered and washed with hot ethanol. The structure of SB-2 is shown in Figure 2.

Elemental Analysis and Conductivity Measurement.
The analytical data and physical properties of the synthesized complexes are given in Table 1. The molar conductances in DMSO indicate that the complexes are all 1 : 1 electrolytes [13,14].

IR Studies
Complex A. The infrared spectral data are shown in Table 2. The

The Effect of Test Compounds and Bleomycin on Ehrlich Ascites Carcinoma (EAC) Cell Growth Inhibition.
Treatment with test compounds resulting in maximum cell growth inhibition on compounds A, B, and C as evident from 95.21%, 87.40%, and 89.49%, reduction of tumour cell, which was found to be equivalent to standard or nearly standard  antitumour agent bleomycin, which shows that cell growth inhibition is 94.90%. The results are shown in Table 4.

Effect of Test Compounds on Survival Time of EAC Cell
Bearing Mice. It was found that treatment of tumour induced test animals with the compounds A, B, and C resulting in the increase of life span 35.70%, 16.98%, and 22.32%, respectively, when compared to control mice (life span 21.37 days). It was noticed that the anticancer antibiotic bleomycin increased the life span by 87.3% when compared to control. The results are shown in Tables 5 and 6.

Effect of Test Compounds and Bleomycin on Average
Tumour Weight. Treatment of the test animals with test compounds, previously incubated with EAC cells, resulted in the inhibition of tumour growth. In case of treated group the body weight was growing slowly and by 53.99%, 46.78%, and 51.46% less in compounds A, B, and C, respectively, compared to control group after 20 days of tumour inoculation. But in case of bleomycin, this value is 52.63% compared to control group. DMSO does not show any change of body weight compared to control group.

The Effect of Test Compounds on Hematological Parameters in Normal and Tumour Bearing Mice.
Hematological parameters were studied in normal and tumour bearing mice. All were treated with test compounds for 12 days of tumour transplantation and after 12 days they were sacrificed and blood was collected for hematological examination. Number of mice were four. Results were shown in mean ± SEM and compared with normal (without EAC bearing mice) and control (EAC bearing mice) group as shown in Table 7.  The growth of tumour in mice induced by EAC cells effect in acute anemic condition as indicated by the significant decrease of the Hb% when compared to normal test animals under similar condition on day 12. The total white blood cell (WBC) count was also markedly decreased in the control group. In differential count of WBC, lymphocyte count was also found to be decreased and neutrophil count was increased on day 12 of tumour inoculation but no significant changes were observed in monocyte count on day 12 of the tumour inoculation as compared with normal mice. Effect of test compounds on hematological parameters of normal mice was also determined. No significant effect was found.

The Effect of Test Compounds on Serum Alkaline
Phosphate Activity. Serum alkaline phosphatase activities were studied in normal and tumour bearing mice. Tumour bearing mice were treated with test compounds for 5 days of tumour transplantation and after 5 days they were sacrificed and blood was collected for determination of serum phosphatase. Serum alkaline phosphatase activity level in tumour bearing was decreased due to tumorigenesis when compared to the normal. Treatment with the test compounds restores the enzyme activity towards normal significantly. The serum alkaline phosphatase activity of the test compounds is shown in Table 8.  Table 10.

Histopathological Effect of Test Compounds. Ehrlich
Ascites Carcinoma (EAC) cell inducing tumour at the site of injection was very prominent and showed fast growth, increased in size, and bulged out in skin. The histological feature shows necrosis at the centre and viable growing cells in the periphery. Some inflammatory reactions lymphocytic in nature with reduction of hair follicle were observed. The number of mitosis was observed which increases greatly. When treated with test compounds and bleomycin growth rate of tumour is reduced, inflammatory reaction has also increased to some extent (Table 11). Necrotic area is increased and hair follicles show their normal appearance.

Effect of Test Compounds on Total Protein in Peritoneal
Fluid. Inoculation of Ehrlich Ascites Carcinoma (EAC) cell in peritoneal cavity causes accumulation of fluid, which is rich in protein. But when treated with test compounds, the protein present in the peritoneal fluid is reduced and fluids accumulate in the peritoneal cavity very slowly (Table 12).

Zone of Inhibition of Antifungal Activity of Test Complexes.
The antifungal activity of the test complexes against different fungi was investigated by using the doses of 80 g/disc, where standard antibiotic disc of Nystatin (45 g/disc) was used for comparison purpose. The diameter was evaluated 4 mm, 2 mm, and 3 mm against tinea pedis; 8 mm, 23 mm, and 10 mm against Aspergillus niger; 22 mm, 6 mm, and 8 mm against Coniothyrium sp., respectively, for test complexes A, B, and C whereas diameter of zone of inhibition of Nystatin was found to be 18 mm, 28 mm, and 20 mm, respectively, against the organism. The antifungal activity (zone of inhibition) of the test complexes against respective fungi is presented in Table 13. The minimum inhibitory concentration (MIC) of the test complex A is 64 g/disc; for B and C it is 128 g/disc as listed in Table 14.

The Result of Antibacterial Activity of Test Compounds.
The antibacterial activity of the test complexes was determined by using the dose of 80 g/disc. The results of antibacterial activity measured in terms of zone of inhibition are shown in Table 15. The complexes showed minimum sensitivity against the following number of both Grampositive and Gram-negative bacteria and the results were compared with antibiotic disc of kanamycin. The histopathological investigation on the tumour showed a retardation of tumour growth, increase in the narcotic and inflammatory area, and increased hair follicles. The Schiff complexes showed significant antimicrobial activity compared to control. We definitely say that the synthesized complexes possess cytotoxic properties. Toxicological studies revealed that the complexes are much more toxic to liver and kidney. They altered all biochemical parameters of rat blood. The exact mode of action of the complexes is unknown to us. Further investigation is appreciated to investigate detailed mechanism of action and their effect in serum electrolyte before any clinical use, especially for the effective doses.