Cytotoxic Effects of Some N-Substituted-2-Amino-1 H-Benzimidazoles

The 2-aminobenzimidazole represents a building block in the structure of several medicinally relevant small molecules. Therefore, the wide spectrum of biological activities (immunotropic, diuretic, anti histaminic, anti-inflammatory, antiviral) associated with the benzimidazolesis of great interest [1-6].The availability of the 2-aminobenzimidazole moiety in the structure of many antihelmithic and antiparasitic drugs support further the importance of the benzimidazole ring system in the development of new and better chemotherapeutical agents [7-11]. Nowadays many 2-aminobenzimidazole derivatives, which are known as microtubule inhibitors were evaluated for their anticancer activity and are appropriate as primary substances for the synthesis of novel anticancer drugs. It was established that benomyl and colchicine synergistically inhibits cell proliferation and mitosis of human cervical cancer (HeLa) cell line [12] while carbendazim inhibits proliferation of human cancer cells, including drugand multidrugresistant and p53-deficient cell lines [13]. Some albendazole derivatives demonstrated cytotoxic activity up to ten times higher than the parent drug (albendazole) against the HT-29-cell line and the definite prostate cancer cell line (PC-3). On the other hand many used in the praxis antiparasitic 2-aminobezimidazole derivatives revealed cytotoxicity against some leukemic and myeloma cells like SW707 (rectal), HCV29T (bladder), A549 (lung) and T47D (breast cancer) [14-18].


Introduction
The 2-aminobenzimidazole represents a building block in the structure of several medicinally relevant small molecules. Therefore, the wide spectrum of biological activities (immunotropic, diuretic, anti histaminic, anti-inflammatory, antiviral) associated with the benzimidazolesis of great interest [1][2][3][4][5][6].The availability of the 2-aminobenzimidazole moiety in the structure of many antihelmithic and antiparasitic drugs support further the importance of the benzimidazole ring system in the development of new and better chemotherapeutical agents [7][8][9][10][11]. Nowadays many 2-aminobenzimidazole derivatives, which are known as microtubule inhibitors were evaluated for their anticancer activity and are appropriate as primary substances for the synthesis of novel anticancer drugs. It was established that benomyl and colchicine synergistically inhibits cell proliferation and mitosis of human cervical cancer (HeLa) cell line [12] while carbendazim inhibits proliferation of human cancer cells, including drug-and multidrugresistant and p53-deficient cell lines [13]. Some albendazole derivatives demonstrated cytotoxic activity up to ten times higher than the parent drug (albendazole) against the HT-29-cell line and the definite prostate cancer cell line (PC-3). On the other hand many used in the praxis antiparasitic 2-aminobezimidazole derivatives revealed cytotoxicity against some leukemic and myeloma cells like SW707 (rectal), HCV29T (bladder), A549 (lung) and T47D (breast cancer) [14][15][16][17][18].
In the view of the above mentioned facts and as a continuation of our research in the field of benzimidazoles we undertook investigations on the effects of some bis (benzimidazol-2-yl) amines on human epithelial colorectal carcinoma HT-29, breast cancer cells, epitheliallike morphology MDA-MB 231 and normal spleen cells. Because of the structural similarity of benzimidazole nucleus to the purine basesof the DNA, we supposed that the benzimidazole derivatives would facile collaborate with biological substances. It could be anticipated that the incorporating of a second benzimidazole ring in the 2-aminobenzimidazole molecule can enhanced not only antiparasitic efficacy but can also lead to the appearance of proliferative or cytotoxic properties of the studied compounds. To find out appropriate medicines that would have specificity to cancer cells is indisputable.

Chemistry
The compounds 1-21 (Scheme 1) were synthesized as previously reported [11]. The tested compounds 13-21 were obtained by the reaction of the corresponding benzimidazole-2-sulfonic acids and appropriate benzimidazole-2-amines at 180°C for 30 minutes. After

MTS test
The cells, incubated with the substances [19] were tested for proliferation ability by the MTS assay described in the protocol of "Promega" [20]. Percentage of untreated control cells (100% viability), was calculated for each concentration. In the controls the calculated data, received after incubation of each cell kind only with DMSO [19]. All data points represent an average of three independent assays.

Statistics
After the Student's test the statistical errors were p ≤ 0.05. EC 50 and IC 50 were calculated with the "Origin" computer program.
Compounds 13-21 were evaluated for their anti proliferative effect on human colorectal cancer cell lineHT-29, breast cancer cells MDA-MB-231 and normal spleen cells using the MTS test [20].
The conversation of the MTS tetrazolium compound into blue colored formazan is due to NADPH or NADH from the examined cells [21].
The bis (benzimidazol-2-yl) amines 13-21 were DMSO-dissolved at a concentration of 500 ml. The stock solution was diluted further 10, 100, 1000 and10000 times. Untreated cells, cultured only in culture medium were the controls. After 24 h of incubation the samples were used in the MTS assay for cell survival and proliferation. All results are given in Figures 1, 2  From the results, given in Table 1, it can be seen that compounds    Simultaneously compounds 18 and 21exerted proliferative activity to the normal spleen cells. On the base of these promising screening results, it may be concluded that the selectivity of compounds 18 and 21 will be essential for anticancer drug development.The received experimental data substantiate the hypothesis that the introduction of another benzimidazole ring at 2 nd position in the structure of 2-aminobenzimidazole as well as the presence of a butyl group at 1 st place are favorable to the reciprocal action of these molecules with thebiological agents.