Antitumour activities of some schiff bases derived from benzoin, salicylaldehyde, amino phenol and 2,4 dinitrophenyl hydrazine

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1 20 Thai J. Pharm. Sci. 34 (2010) Original article Antitumour activities of some schiff bases derived from benzoin, salicylaldehyde, amino phenol and 2,4 dinitrophenyl hydrazine M. Jesmin 1*, M.M. Ali 1 and J.A. Khanam 2 1 Department of Applied Chemistry and Chemical Technology, Rajshahi University, Rajshahi-6205, Bangladesh. 2 Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205, Bangladesh. *Corresponding author. Tel: ; address: melejesminacru@yahoo.com Abstract: Three schiff bases PDH [N-(1-phenyl-2-hydroxy-2phenyl ethyledine)-2, 4 dinitrophenyl hydrazine], PHP [N-(1-phenyl, 2-hydroxy-2-phenyl ethylidine)-2 hydroxy phenyl imine] and HHP [N-(2-hydroxy benzylidine)-2 hydroxy phenyl imine] have been studied against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice. These compounds enhanced life span, reduced average tumour weight and inhibited tumour cell growth of EAC cell-bearing mice. They also restored the depleted haematological parameters like haemoglobin content, red blood cells (RBC) and white blood cells (WBC) counts towards normal. All these data have been compared by running parallel experiments with a standard anticancer drug, bleomycin. The macrophages which have some protecting action against EAC cells are found to be developed in mice when treated with the compounds alone. The compounds can therefore be considered as potent antitumour agents. Keywords: Antitumour agents; Ehrlich ascites carcinoma (EAC) cells; Haematology; Macrophages

2 M. Jesmin et al. 21 Introduction Schiff bases and their complexes are well known for their pronounced biological activities [1-3]. They are actively associated with antibacterial [1], anti-fungal [4, 5], herbicidal [6], antitubercular [7], anti-hiv [4] and anticancer activities [8]. In our recent published paper [9], we have reported the synthesis and characterization of three schiff bases namely PDH [N-(1-phenyl-2-hydroxy-2-phenyl ethylidine)-2, 4 dinitrophenyl hydrazine], HHP [N- (2-hydroxy benzylidine)-2 hydroxy phenyl imine] and PHP [N-(1-phenyl 2-hydroxy-2-phenyl ethylidine)-2 hydroxy phenyl imine] and studied their cytotoxic activities in vitro. In the present paper we have studied the anticancer activities of these three schiff bases (PDH, HHP and PHP) against Ehrlich ascities carcinoma (EAC) cells in Swiss albino mice. For this purpose, the effects of these compounds on average tumour weight, tumour cell growth inhibition and increase of life span of EAC cell bearing mice have been studied. This study also includes the effect of these compounds on haematological parameters in normal and EAC cell-bearing mice. Materials and Methods Chemicals and reagents All the chemicals and reagents used throughout the investigation were of reagent grade (BDH, England). Synthesis and characterization of PDH, PHP and HHP Synthesis of PDH, PHP, HHP and their characterization were done by the methods as described earlier [9]. Animals Six-to-eight weeks old male Swiss albino mice (25 ± 5 gm body weight) were collected from International Center for Diarrheal Disease Research, Bangladesh (ICDDRûB) and used throughout the studies. The mice were fed with standard mice food-pellets (collected from ICDDRûB) and water was given in adequate. Cell lines Ehrlich ascites carcinoma (EAC) cells were obtained by the courtesy of Indian Institute of Chemical Biology (IICB), Kolkata, India. The cells were maintained as ascites tumour in Swiss albino mice by intraperitoneal inoculation (i.p., by weekly) of 2 x 10 6 cells/mouse. Ethical clearance Protocol used in this study for the use of mice as animal model for cancer research was approved by the University Animal Ethical Committee (27/08/RUBCMB). Toxicity study An acute toxicity study relating to the determination of LD 50 was performed by the conventional method [10]. For this purpose these compounds were dissolved in 2% dimethyl sulfoxide (DMSO) and injected intraperitoneally to nine groups of mice (each containing six in number) in different doses (5, 9, 11, 13, 15, 15.5, 16, 18 and 20 mg/kg). LD 50 was evaluated by recording mortality after 24 hours. Cell growth inhibition In vivo tumour cell growth inhibition was carried out by the method as described by Sur and Ganguly [11]. For this study eleven groups of mice (six in each group) were used. All the mice were injected with EAC cells (0.1 ml of 2 x 10 6 cells/mouse) intraperitoneally. Treatment was started after 24 hours of tumour inoculation and continued for 6 days. Groups 1 to 3 were treated by PDH at the doses of 0.5 mg/kg (i.p.), 1 mg/kg (i.p.) and 2 mg/kg (i.p.) respectively per day per mouse. Groups 4 to 6 were treated by PHP and 7 to 9 by HHP at the same doses as used for PDH. Group 10 received standard drug bleomycin (0.3 mg/kg, i.p.). Treatment with only normal saline (0.98%) was considered as untreated control (group 11). The mice of all the groups were sacrificed on the 7 th day after transplantation and tumour cells were collected by repeated intraperitoneal wash with 0.98% saline. Viable tumour cells per mouse of the treated groups were compared with those of control. The cell growth inhibition was calculated by using the following formula : % Cell growth inhibition = (1- T w Cw ) x 100 (1) where T w = Mean of number of tumour cells of the treated group of mice C w = Mean of number of tumour cells of the control group of mice.

3 22 Thai J. Pharm. Sci. 34 (2010) Average tumour weight and survival time The antitumour effects of PDH, PHP and HHP were assessed [12] by measuring average tumour weight, mean survival time (MST) and percentage increased of life span (% ILS). These parameters were measured under similar experimental conditions as stated in the previous experiment (cell growth inhibition). Treatment was continued for 10 days. Tumour growth was monitored daily by measuring weight change. MST of each group (6 in each) was monitored by recording the survival time. MST and % ILS were calculated by using the following equation. MST = Σsurvival time (days) of each mouse in a group (2) Total number of mice Percent increase of life span, % ILS = ( MST of treated group -1) Ó 100 (3) MST of control group Bioassay of EAC cells The procedure was a modification of the method of Fernandes and Klubes [13]. Four groups of mice (4 in each) were inoculated with 1 x 10 5 EAC cells. Groups 1 to 3 were treated with PDH, PHP and HHP respectively at the dose of 2 mg/kg (i,p) for six consecutive days. The group 4 served as control. On day 7, mice of all groups were sacrificed and tumour cells from each group were harvested in cold saline (0.98%), pooled and centrifuged. These cells were re-inoculated (10 5 cells/ mouse i.p) in to four fresh groups of mice (n=4) as before. No further treatment was done on these mice. On day 5, mice from each group were sacrificed and tumour cells per mouse were counted and compared with that of control. Haematological studies The haematological parameters viz. WBC, RBC and Hb content were determined by the standard methods [14] using cell dilution fluids and haemocytometer. For this purpose, blood was collected from the mouse by tail puncture. Six groups of mice (n=4) were taken for doses 0.5 mg/kg and 2 mg/kg (i.p) of the test compounds (PDH, PHP and HHP). Treatment started after 24 hours of tumour transplantation and was continued for 10 consecutive days. On days 5, 10, 15 and 25, the blood parameters were assayed. For normal mice, four groups (n=4) were taken for the purpose. The blood from the mice of group 1 was assayed on day 0 (without any treatment). Groups 1-3 were treated with PDH, PHP and HHP at the dose of 2 mg/kg (i,p.) respectively for the same days. Determination of the effect of PDH, PHP and HHP on normal peritoneal cells Effects of schiff bases on normal peritoneal cells were determined [15] by counting total peritoneal cells and number of macrophages. Three groups of mice (4 in each) were treated with PDH, PHP and HHP separately at the dose of 2 mg/kg (i.p) for three consecutive days. The untreated group was used as control. After 24 hours of last treatment, each animals were injected with 5 ml of normal saline (0.98%) into peritoneal cavity and then sacrificed. Intraperitoneal exuded cells and number of macrophages were counted with 1% neutral red by haemocytometer. Statistical analysis The experimental results have been expressed as the mean ± S.E.M. Data have been calculated by one way ANOVA followed by Dunnett çté test using SPSS software of 10 version. Results and Discussion The structures of three test compounds have been furnished in Figure 1. The toxicity of these compounds has been evaluated by measuring LD 50 values. For PDH, C = N H C OH H N NO 2 PDH NO 2 H C = N H C= N OH C OH HHP OH PHP OH Figure 1 Structure of three schiff bases

4 M. Jesmin et al. 23 PHP and HHP, the values have been found to be 15, 16 and 15.5 mg/kg respectively. The potency of these compounds as antitumour agents has been judged by measuring ùcell growth inhibitionû, ùreduction of average tumour weightû and ùenhancement of life spanû of tumour bearing mice. For these purpose we have selected 3 doses viz. 0.5 mg/kg, 1 mg/kg and 2 mg/kg of these compounds. Parallel experiments have also been carried out with a known effective anticancer drug ùbleomycinû at the dose of 0.3 mg/kg (i.p.) in order to judge the comparative effectiveness of these compounds. It has been found that the three schiff bases are quite capable of reducing average tumour weight. Of course in all cases, the tumour growth reduction ability increases with increased dose. With 2 mg/kg (i.p.), PDH shows a maximum 70.81% reduction of tumour weight when compared with that of control. On the other hand PHP shows 60.74% and HHP by 59.97% reduction at the same dose. Bleomycin at dose 0.3 mg/ kg (i.p.) reduces the same by 73.53%. The results have been presented in Tables 1-3. Analogous results have been obtained for EAC cell growth inhibition ability of these compounds (Table 4). With the increase of doses, the percentage of cell growth inhibition is found to increase noticeably. The values with 2 mg/kg (i.p.) are quite comparable to those of bleomycin at dose of 0.3 mg/kg (i.p.). PHP, on the other hand is also fairly active in inhibiting EAC cells. The order of activity with respect to cell growth inhibition may be written as PDH ~_ HHP > PHP The data for percentage increase of life span of EAC cell-bearing mice have been presented in Table 5. In comparison with the efficiency of bleomycin, PDH and HHP increase life span of EAC-bearing mice quite competently and particularly at the dose of 2 mg/kg (i.p.). The prolongation of the life of cancer cell bearing animals [16] is a very important and reliable criterion for judging the potency of compounds as anticancer drugs. From the results it can be argued that all the three compounds are effective antitumour agents. They significantly inhibit cell growth, reduce average tumour weight and enhance life span of EAC cell bearing mice. This fact is further supported from the data obtained from the hematological studies with these compounds on EAC treated mice. Figure 2 shows the effect of these compounds on red blood cells (RBC) content in blood of the EAC cellbearing mice. In all the experiments the RBC contents are found to decrease with time (days) elapsed. The detoriation of RBC is very fast in case of untreated EAC cell-bearing mice. The rate is found to be retarded noticeably upon treatment with bleomycin as well as with the three schiff bases. Figure 3 shows the effect of these schiff bases on white blood cells (WBC) content in blood of EAC cell bearing mice. With the growth of tumour, the WBC level is found to be increased rapidly. Bleomycin as well as the three compounds PDH, PHP and HHP have reduced the rate of increment of WBC significantly towards normal. The normalizing efficiency has been found to be as follows: Bleomycin > PDH > PHP HHP Figure 4 shows the effect of these compounds on haemoglobin content of the EAC-bearing mice. The haemoglobin content of EAC-bearing mice has been found to be reduced with time (days). All the compounds also have inhibited the rate of such reduction. Among them, PDH has been found to be most competent and quite comparable to that of bleomycin. The order of potency is obviously as follows. Bleomycin ~_ PDH > HHP > PHP The major problems usually in cancer chemotherapy which are being encountered, are myelosuppression and anemia [17,18] due to the reduction of both RBC content and haemoglobin percentage. This is probably owing to the deficiency of iron on haeomolytic or myelopathic condition [19]. Treatment with each of these three synthesized compounds has restored the haemoglobin content as well as RBC and WBC counts more or less to normal levels. This indicates that PDH, HHP and PHP possess protective actions on the haemopoietic system. It has been further found that the hematological parameters (RBC, WBC and heamoglobin content) depleted moderately with the treatment time (host toxic effect) but restored towards normal within a few days (Table 6). It should be mentioned here that the compounds tested in the present study have shown pronounced cytotoxic activity when screened using in vitro method [9].

5 24 Thai J. Pharm. Sci. 34 (2010) RBC 10 6 Cells/ml ±0.108 ±0.021 ±0.091 ±0.028 ±0.047 ±0.047 ±0.047 ±0.064 ±0.091 ±0.091 ±0.047 ±0.047 ±0.075 ±0.020 ±0.051 ±0.091 ±0.091 ±0.069 ±0.073 ±0.028 ±0.070 ±0.029 ±0.047 ±0.091 ± Normal Control (EAC) PDH, 0.5 mg/kg PHP, 0.5 mg/kg HHP, 0.5 mg/kg PDH, 2 mg/kg PHP, 2 mg/kg HHP, 2 mg/kg Bleomycin 5 Days 10 Days 15 Days 25 Days Figure 2 Effect of PDH, PHP and HHP on RBC on days 5, 10, 15 and 25 of tumour cell inoculation (Data are expressed as the mean of 4 mice ± S.E.M. Treatment was continued for 10 consecutive days) For the evaluation of immunologic effect of PDH, PHP and HHP in fresh healthy mice, peritoneal macrophages have been counted. PDH, PHP and HHP have enhanced the number of peritoneal macrophages (Table 7) significantly in the following order. PDH > HHP > PHP This enhancement [20] might have produced some cytokinetic products such as tumour necrosis factor, interleukins, interferons etc. which in turn may be responsible in killing tumour cells. To find out the effect of PDH, PHP and HHP on the viability of EAC cells, transplantability of tumour cells has been determined and the results are shown in Table 8. PDH shows the maximum effect on the loss of transplantability of EAC cells when reinoculated in normal mice. The results have been found in the following order PDH > HHP > PHP Conclusion All the compounds tested here can be considered as potent antitumour agents. But only based on these data, it is not possible to suggest a plausible mechanism for antitumour effects at the present moment. The -NO 2 and -OH groups in benzene rings as well as the whole structure of the compounds probably influence the antitumour effects. It is expected that these compounds at different doses may be used against various cancer models and may prove to formulate safer drugs for future. Acknowledgements The authors are grateful to IICB (Kolkata, India) authority for providing the EAC cells and also to the ICDDRûB (Dhaka, Bangladesh) for kindly supplying Swiss albino mice and standard mouse pellets.

6 M. Jesmin et al WBC 10 9 Cells/ml ±0.816 ±0.912 ±0.912 ±0.853 ±0.478 ±0.912 ±0.478 ±0.629 ±0.288 ±0.478 ±0.478 ±0.288 ±0.288 ±0.912 ±0.946 ±0.288 ±0.912 ±0.707 ±0.288 ±0.645 ±1.040 ±0.853 ±0.645 ± Normal Control (EAC) PDH, 0.5 mg/kg PHP, 0.5 mg/kg HHP, 0.5 mg/kg PDH, 2 mg/kg PHP, 2 mg/kg HHP, 2 mg/kg Bleomycin 0.3 mg/kg 5 Days 10 Days 15 Days 25 Days Figure 3 Effect of PDH, PHP and HHP on WBC on days 5, 10, 15 and 25 of tumour cell inoculation (Data are expressed as the mean of 4 mice ± S.E.M. Treatment was continued for 10 consecutive days)

7 26 Thai J. Pharm. Sci. 34 (2010) Haemoglobin, gm/dl Days Untreated control Bleomycin, 0.3 mg/kg PDH 0.5 mg/kg PHP, 0.5 mg/kg HHP, 0.5 mg/kg PDH, 2 mg/kg PHP, 2 mg/kg HHP, 2 mg/kg Figure 4 Effect of PDH, PHP and HHP on haemoglobin content on days 5, 10, 15 and 25 of tumour cell inoculation (Treatment was continued for 10 consecutive days)

8 M. Jesmin et al. 27 Table 1 Effect of PDH, PHP and HHP on average tumour weight at the dose of 0.5 mg/kg (i.p.) Tumour wt. of Tumour wt. of Tumour wt. of schiff base treated EAC untreated EAC bleomycin cell-bearing mice in gm at the dose of 0.5 mg/kg (i.p.) Days cell-bearing (0.3 mg/kg i.p.) mice in gm treated EAC cell-bearing mice in gm PDH PHP HHP ± ± 0.01*** 0.81 ± ± ± ± ± 0.02*** 1.24 ± 0.03*** 2.30 ± ± 0.11** ± ± 0.04*** 1.78 ± 0.07*** 3.30 ± ± 0.06*** ± ± 0.04*** 2.22 ± 0.06*** 3.90 ± ± 0.08* ± ± 0.03*** 3.05 ± 0.07*** 4.47 ± 0.05*** 4.10 ± 0.09*** ± ± 0.04*** 3.70 ± 0.04*** 5.15 ± 0.04*** 4.60 ± 0.06** ± ± 0.03*** 4.20 ± 0.04*** 5.87 ± 0.03*** 5.25 ± 0.04*** ± ± 0.04*** 5.00 ± 0.07*** 6.70 ± 0.09*** 6.15 ± 0.11*** ± ± 0.06*** 5.12 ± 0.07*** 7.05 ± 0.10*** 6.70 ± 0.07*** ± ± 0.06*** 5.87 ± 0.08*** 7.42 ± 0.10*** 7.13 ± 0.09*** Data are expressed as the mean of results in 6 mice ± S.E.M. Treatment was continued for 10 consecutive days. ***P < 0.001, **P < 0.01 and *P < 0.05 when compared with control. Table 2 Effect of PDH, PHP and HHP on average tumour weight at the dose of 1 mg/kg (i.p.) Tumour wt. of Tumour wt. of Tumour wt. of schiff base treated EAC untreated EAC bleomycin cell-bearing mice in gm at the dose of 1 mg/kg (i.p.) Days cell-bearing (0.3 mg/kg i.p.) mice in gm treated EAC cell-bearing mice in gm PDH PHP HHP ± ± 0.01*** 0.78 ± 0.03*** 0.62 ± 0.03** 0.65 ± 0.06*** ± ± 0.02*** 1.29 ± 0.03*** 1.65 ± 0.05*** 0.98 ± 0.03*** ± ± 0.04*** 1.62 ± 0.05*** 2.60 ± 0.01** 1.57 ± 0.08*** ± ± 0.04*** 2.09 ± 0.04*** 3.80 ± 0.07** 2.43 ± 0.04*** ± ± 0.03*** 2.30 ± 0.05*** 4.25 ± 0.04*** 3.28 ± 0.07*** ± ± 0.04*** 2.56 ± 0.03*** 4.70 ± 0.09*** 4.03 ± 0.05*** ± ± 0.03*** 3.04 ± 0.06*** 5.57 ± 0.07*** 4.45 ± 0.07*** ± ± 0.04*** 3.92 ± 0.05*** 6.25 ± 0.07*** 5.40 ± 0.05*** ± ± 0.06*** 4.32 ± 0.03*** 6.75 ± 0.04*** 6.15 ± 0.02*** ± ± 0.06*** 4.70 ± 0.05*** 7.27 ± 0.04*** 6.93 ± 0.06*** Data are expressed as the mean of results in 6 mice ± S.E.M. Treatment was continued for 10 consecutive days. ***P < 0.001, **P < 0.01 and *P < 0.05 when compared with control.

9 28 Thai J. Pharm. Sci. 34 (2010) Table 3 Effect of PDH, PHP and HHP on average tumour weight at the dose of 2 mg/kg (i.p.) Tumour wt. of Tumour wt. of Tumour wt. of schiff base treated EAC untreated EAC bleomycin cell-bearing mice in gm at the dose of 2 mg/kg (i.p.) Days cell-bearing (0.3 mg/kg i.p.) mice in gm treated EAC cell-bearing mice in gm PDH PHP HHP ± ± 0.04*** 0.43 ± 0.04** 0.57 ± 0.05* 0.40 ± 0.03*** ± ± 0.02*** 1.10 ± 0.05*** 1.53 ± 0.07** 0.90 ± 0.04*** ± ± 0.04*** 1.56 ± 0.03*** 2.38 ± 0.01** 1.40 ± 0.04*** ± ± 0.04*** 1.98 ± 0.04*** 2.74 ± 0.11*** 2.20 ± 0.07*** ± ± 0.03*** 2.15 ± 0.04*** 2.92 ± 0.08*** 2.97 ± 0.06*** ± ± 0.04*** 2.53 ± 0.05*** 3.40 ± 0.06*** 3.85 ± 0.07*** ± ± 0.03*** 3.00 ± 0.06*** 3.94 ± 0.04*** 4.25 ± 0.09*** ± ± 0.04*** 3.42 ± 0.07*** 5.23 ± 0.23*** 4.83 ± 0.08*** ± ± 0.06*** 4.28 ± 0.03*** 5.63 ± 0.23*** 5.67 ± 0.13*** ± ± 0.06*** 4.52 ± 0.03*** 6.08 ± 0.13*** 6.20 ± 0.07*** Data are expressed as the mean of results in 6 mice ± S.E.M. Treatment was continued for 10 consecutive days. ***P < 0.001, **P < 0.01 and *P < 0.05 when compared with control. Table 4 Effect of the schiff bases and bleomycin (antitumour drug) on cell growth inhibition No. of EAC cells in mouse % cell growth Treatment Dose, mg/kg(i.p.) on day 6 after tumour cell inhibition inoculation x 10 7 Control (EAC cell-bearing mice) ± Bleomycin ± 0.007*** PDH ± 0.003*** ± 0.007*** ± 0.003*** PHP ± 0.014*** ± 0.020*** ± 0.003*** HHP ± 0.220*** ± 0.003*** ± 0.003*** Number of mice in each experiment were six (n=6); the results were shown as mean ± S.E.M. Treatment was continued for 6 consecutive days. ***P < when compared with control.

10 M. Jesmin et al. 29 Table 5 Effect of PDH, PHP, HHP and bleomycin on survival time and increase of life span of EAC cell-bearing mice Treatment Dose, mg/kg (i.p.) Mean survival time Mean ± S.E.M. (days) % Increase of life span Control ± Bleomycin ± 0.86*** PDH ± 0.89* ± 0.93*** ± 1.46*** PHP ± ± 1.03*** ± 1.67** HHP ± 0.93** ± 1.06*** ± 1.39*** Data are expressed as the mean of results in 6 mice ± S.E.M. Treatment was continued for 10 consecutive days. ***P < 0.001, **P < 0.01 and *P < 0.05 when compared with control. Table 6 Host toxicity study of PDH, PHP and HHP on RBC, WBC and heamoglobin content in normal mice at dose 2 mg/kg (i.p.) Experiment Days RBC (cells/ml x 10 9 ) WBC (cells/ml x 10 6 ) Hb content (mg/dl) Normal ± ± ± 0.18 Mice treated with PDH ± ± ± ± ± ± ± ± ± ± ± ± 0.06 Mice treated with PHP ± ± ± ± ± ± ± ± ± ± ± ± 0.08 Mice treated with HHP ± ± ± ± ± ± ± ± ± ± ± ± 0.05 Data are expressed as the mean of results in 4 mice ± S.E.M. Treatment was continued for 10 consecutive days.

11 30 Thai J. Pharm. Sci. 34 (2010) Table 7 Effect of schiff bases on the enhancement of normal peritoneal cells of mice Treatment Dose, mg/kg (i.p.) Macrophages Total peritoneal cells x (10 6 ) Control (Normal) ± ± 0.04 Normal + PDH ± ± 0.13*** Normal + PHP ± ± 0.12** Normal + HHP ± ± 0.11*** Data are expressed as the mean of results in 4 mice ± S.E.M. Treatment was continued for 3 consecutive days. ***P < and **P < 0.01 when compared with control Table 8 Bioassay of schiff bases with EAC cells Treatment Dose, mg/kg(i.p.) No. of EAC cells (x 10 7 ) Cell growth inhibition after inoculation with drug treated EAC cells Control ± PDH ± 0.004*** 83.59% PHP ± 0.003*** 80.08% HHP ± 0.002*** 81.40% Data are expressed as the mean of results in 4 mice ± S.E.M. ***P < when compared with control. References [1] I. Sakiyan, E. Logoglu, S. Arslan, N. Sari, and N. Sakiyan. Antimicrobial activities of N-(2 hydroxy 1-naphthalidene) amino acid (glycine, alanine, phenyl alamine, histidine, tryptophane) schiff bases and their manganese complexes, Biometals 17: (2004). [2] Z. H. Chohan, M. Arif, Z. Shafiq, M. Yaqub, and C.T. Supuran. In vitro antibacterial, antifungal and cytotoxic activity of some isonicotinylhydrazide schiffûs bases and their cobalt (II), copper (II), nickel (II) and zinc (II) complexes, J. Enzyme Inhib. Med. Chem. 21: (2006). [3] Y. P. Wang, Y. N. Xiao, C. X. Zhang, and R. M. Wang. Studies on the interaction of DNA and water soluble polymer supported schiff base complex, J. Macromol. Sci. Part A, 38: (2001). [4] S. N. Pandeya, D. Sriram, G. Nath, and E. DeCLecq. Synthesis, antibacterial, antifungal and anti HIV activities of schiff mannich bases derived from isatin derivatives and N-[4-(4 chlorophenyl) thiazole-2-yl] thio semicarbazide, European J. Pharm. 9: (1999). [5] H. Chen, and J. Rhodes. Schiff base forming drugs, mechanisms of immune potentiation and therapeutic potential, J. Mol. Med. 74: (1996). [6] B. S. Molla, B. S. Rao, K. Shridhara, and P. M. Akberali. Studies on arylfuran derivatives, Part XI. Synthesis, characterization and biological studies on some mannich bases carrying 2,4 dichloropheyl furfural moiety, Farmaco 55: (2000). [7] M. R. Islam, A. H. Mirza, Q. M. N. Huda, and B. R. Khan. The synthesis of some anti tubercular agents; aldehyde thiosemicarbazones and 1,3,4 thiadiazoline derivatives, J. Bang. Chem. Soc. 2: (1989). [8] M. M. Ali, M. Jesmin, M. K. Sarker, M. S. Salahuddin, M. R. Habib, and J. A. Khanam. Antineoplastic activity of N-salicylidene glycinato-di-aquanickel(ii) complex against Ehrlich Ascites Carcinoma (EAC) cells in mice, Int. J. Biol. Chem. Sci. 2: (2008). [9] M. Jesmin, M. M. Ali, M. S. Salahuddin, M. R. Habib, and J. A. Khanam. Antimicrobial activity of some schiff bases derived from benzoin, salicylaldehyde, amino phenol and 2,4 dinitrophenyl hydrazine, Mycobiology, 36: (2008). [10] J. T. Litehifield, and F. Wilcoxon. A simplified method of evaluating dose effects experiments, J. Pharmacol. Exp. Ther. 96: (1949).

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