Received Date: May 10, 2012; Accepted Date: May 24, 2012; Published Date: May 26, 2012
Citation: Mavrova AT, Wesselinova D, Tsenov JA, Denkova P (2012) Cytotoxic Effects of Some N-Substituted-2-Amino-1H-Benzimidazoles. J Bioequiv Availab 4: 052-055. doi: 10.4172/jbb.10000112
Copyright: © 2012 Mavrova AT, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The cytotoxic activity of previously synthesized is (benzimidazol-2-yl) amines was evaluated on two cancer celllines:human epithelial colorectal carcinoma HT-29 (American Type Culture Collection HTB-38), breast cancer cells with epithelial-like morphology MDA-MB 231 (American Type Culture Collection ATCC HTB-26), and on normal spleen cells as well as. Indicative cytotoxic activity was ascertained for N-1H-benzimidazol-2-yl-1-propyl-1H-benzimidazol-2-amine 15, 1-propyl-N-(1-propyl-1H-benzimidazol-2-yl)-1H-benzimidazol-2-amine 21 and 1-methyl-N-(1-propyl-1H-benzimidazol-2-yl)-1H-benzimidazol-2-amine18 against the cellular line HT-29. The estimated IC50values were 0.91, 1.92 and 1.98 μM respectively. The same compounds exhibited comparatively high antiproliferative activity against MDA-MB-231 cells. The realized IC50 values were in the range 0.006 � 1.48 μM. Compounds 14, 16, 17, 18 and 21revealedstimulating activity to the normal spleen cells, wherethe EC50 values were between 0.5.10-4 μM for compound 21 and 0.013 μM for compound 14
2-aminobenzimidazoles; Bis (benzimidazol-2-yl) amines; Synthesis; Cytotoxicity; Proliferative effect; HT-29; MDA-MB- 231-cell lines
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  while carbendazim inhibits proliferation of human cancer cells, including drug- and multidrugresistant and p53-deficient cell lines . 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].
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.
The compounds 1-21 (Scheme 1) were synthesized as previously reported . 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 cooling the fusion was diluted with ethanol. The filtered pellet was recrystallized with ethanol.
The cells, incubated with the substances  were tested for proliferation ability by the MTS assay described in the protocol of “Promega” . 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 . All data points represent an average of three independent assays.
After the Student’s test the statistical errors were p ≤ 0.05.
EC50 and IC50 were calculated with the “Origin” computer program.
The synthesis of the studied compounds was accomplished as outlined in Scheme 1. The reaction of 1,2-diaminobenzene, carbon disulfide and sodium hydroxide in ethanol medium yielded 1H-benzimidazol-2-thiol 1. The fusion of 1-alkyl-benzimidazoles with sulfur at 180°C yielded in 1-alkyl-benzimidazole-2-thiols 2-4. The oxidation of the 1-(un)substituted-1H-benzimidazol-2-yl-tiols 1-4 with KMnO4 in 25% water solution of sodium hydroxide led to the formation of 1- (un) substituted-1H-benzimidazol-2-yl-sulfonic acids. The reaction of benzimidazol-2-yl-sulfonic acids 5-8, carried out with 25% ammonium hydroxide resulted in 2-aminobenzimidazoles 9-12. The bis (benzimidazol-2-yl) amines 13-21 were synthesized by heating benzimidazol-2-yl-sulfonic acids 5-8 and 2-aminobenzimidazoles 9-12 at 180°C.
Compounds 13-21 were evaluated for their anti proliferative effect on human colorectal cancer cell lineHT-29, breast cancer cells MDAMB- 231 and normal spleen cells using the MTS test .
The conversation of the MTS tetrazolium compound into blue colored formazan is due to NADPH or NADH from the examined cells .
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 and 3. The calculated and plotted result and the values of IC50 and EC50 are represented in Table 1.
|Compound||IC50 ± SE
|EC50 ± SE
|HT-29||MDA-MB-231||Normal spleen cells||HT-29||MDA-MB-231||Normal spleen cells|
|14||3.5.103 ± 0.35||1,65 ±0.07||-||-||-||0.013 ± 0.07|
|15||0.91 ± 0.11||0.135 ± 0.06||0.11 ± 0.62||-||-||-|
|16||-||-||-||0.01 ± 0.046||0.002 ± 0.05||0.047 ± 0.32|
|17||-||-||-||0.48 ± 0.009||1.48 ± 0.3||0.005 ± 0.15|
|18||1.98±0.43||1.48 ± 0.06||-||-||-||0.004 ± 0.13|
|19||-||1.87 ± 0.24||0.23 ± 1.96||0.005 ± 0.38||-||-|
|20||7.103 ± 0.05||16.7 ± 0.28||1.67 ± 0.02||-||-||-|
|21||1.92 ± 0.08||0.006 ± 0.27||-||-||-||0.5.10-4 ± 0.6|
Table 1: In vitro cytotoxicity and proliferative effects on HT-29, MDA-MB-231and normal spleen cells.
From all tested substances compounds 15, 18 and 21 expressed toxic effect against HT-29 cells. The unsubstituted at 1-th position in the one of the two benzimidazole rings compound 15 showed the most pronounced toxic effect to HT-29 cells, IC50 = 0.91 μM, followed by the compound 21, IC50 = 1,92 μM and 18 - IC50 = 1,98 μM. Compounds 15, 18, 19, 20 and 21 exhibited relatively high anti proliferative activity to MDA-MB-231 cells. Most toxic were 1-propyl- N-(1-propyl-1H-benzimidazol-2-yl)-1H-benzimidazol-2-amine 21 and N-1H-benzimidazol-2-yl-1-propyl-1H-benzimidazol-2-amine 15 with IC50–0.0006 μM and IC50–0.135 μM respectively.
From the results, given in Table 1, it can be seen that compounds 14, 16, 17, 18 and 21 showed definitely stimulating effects to the normal spleen cells. The EC50 data varied from 0.05 × 10-3 μM for 1-propyl-N- (1-propyl-1H-benzimidazol-2-yl)-1H-benzimidazol-2-amine 21 to 0.013 μM for N-1H-benzimidazol-2-yl-1-ethyl-1H-benzimidazol- 2-amine 14. If the result for compound 21 is taken in mind it must be emphasized that 1-propyl-N-(1-propyl-1H-benzimidazol-2-yl)- 1H-benzimidazol-2-amine exhibit proliferative effect to the normal spleen cells even at higher dilutions in comparison to this at which the compound displays cytotoxic effect on HT-29 cells and MDAMB- 231 cells. As far as the properties of 1-methyl-N-(1-propyl-1Hbenzimidazol- 2-yl)-1H-benzimidazol-2-amine 18 should be pointed out that this compound possessed a relative high cytotoxicity both to HT-29 cells (IC50 = 1.9 μM) and to MDA-MB-231 (IC50 = 1.4 μM) cell line, but against normal spleen cells the same compound displayed proliferative activity at lower concentration (EC50 = 0.004 μM). The last result is important showing the selective capacity of substance 18. Surprising results demonstrated N-1H-benzimidazol-2-yl-1-ethyl-1H-benzimidazol-2-amine 14 revealing to be almost non-toxic to HT-29 and toxic to MDA-MB-231, but possessing a proliferative effect on normal spleen cells at a lower concentration (EC50-0.013 μM). Proliferative influence on all the experimental cells manifested 1-methyl-N-(1-methyl-1H-benzimidazol-2-yl)-1H-benzimidazol- 2-amine 16 and 1-ethyl-N-(1-methyl-1H-benzimidazol-2-yl)-1Hbenzimidazol- 2-amine 17.
The significant statistical deviations in all examinations were determined (p ≤ 0.05).
The preliminary screening in vitro showed that the studied compounds 15, N-1H-benzimidazol-2-yl-1-propyl-1H-benzimidazol- 2-amine 18 and 1-propyl-N-(1-propyl-1H-benzimidazol-2-yl)-1Hbenzimidazol- 2-amine 21 possessed comparatively high cytotoxic effect on HT-29 cells. IC50 values were in the range 0.91-1.98 μM. Strongest anti proliferative effect on MDA-MB-231 cells had compounds 15 and 21 with IC50–0.0006 μM and IC50–0.135 μM respectively. 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 2nd position in the structure of 2-aminobenzimidazole as well as the presence of a butyl group at 1st place are favorable to the reciprocal action of these molecules with thebiological agents.
The financial support by the National Research Fund of Bulgaria for the purchase of Bruker Avance II+ 600 NMR spectrometer in the framework of the Program“Promotion of the Research Potential through Unique Scientific Equipment” (Project UNA-17/2005) is gratefully acknowledged.