International Journal of Drug Research and Technology

Int. J. Drug Res. Tech. 2012, Vol. 2 (7), 479-485                ISSN   2277 - 1506


International Journal of Drug Research and Technology

   Available online at

Original Research Paper


Balram Soni1*, Mahendra Singh Ranawat1, Anil Bhandari2 and Rambabu Sharma2

1B.N.College of Pharmacy, Udaipur, Rajasthan-313 001, India

2Faculty of Pharmaceutical Sciences, Jodhpur National University,

Narnadi, Jodhpur, Rajasthan-342 003, India


One of the most important goals in medicinal chemistry is the development of new heterocyclic compounds with antitumor activity. Thus, a novel series of Schiff bases 1-11, the derivatives of 2-aryl-1H-benzimidazole has been synthesized and evaluated for their cytotoxic activity against the cells of human cancer cell lines, namely K-562 (myelogenous leukemia) and HL-60 (promyelocytic leukemia). Among the compounds tested against K-562 cell line, compounds 1, 7 and 10 showed potent activity and the compound 5 exhibited better activity against HL-60 cell line.

Keywords:  2-Aryl-1H-benzimidazole, Schiff bases, Leukemia, Antitumor activity.


Cancer, of which there are over 100 different forms, is a leading cause of death. Leukemia is the most common cancer worldwide and remains the most frequent cause of malignancy-associated death. The clinical success of cisplatin and related platinum based drugs, as anti-cancer drugs constitutes the most impressive contribution to the use of metals in medicine. However major problems associated with these mettalo-drugs include serious toxicity and other side-effects, and major problem with resistant.  New potent and selective anti-cancer drugs are urgently required.1 The most used classes of chemotherapeutic agents in cancer therapy comprise molecules that interact with DNA, such as groove binders, DNA alkylating agents, or intercalators. However, the activity of many antitumor drugs is based on intercalation, but selectivity could be improved by introduction of specific substituents on the intercalative aromatic core. Several antibiotics are DNA intercalators and are recognized as a new, promising class of antitumor agents.2 The benzimidazole nucleus is an important pharmacophore in drug discovery. Benzimidazoles are very useful intermediates/subunits for the development of molecules of pharmacological or biological interest. Substituted benzimidazole derivatives have diverse therapeutic applications as they exhibit antimicrobial3-4, antiproliferative5-7, anti-inflammatory8, sunscreen9, antidiabetic10, spasmolytic11, antihypertensive12, and antiviral13 activities. The biological relevance of these heteroaromatic groups is due to their being good bioisosteres of biomolecules.  Benzimidazoles are widely used as drugs such as, Omeprazole, Pantoprazole, Lansoprazole; proton pump inhibitor14, Albendazole, Mebendazole, Thiabendazole; antihelmintic15, Domperidone; antidopaminergic16, Pimozide; antipsychotic17, Pimobendan; ionodilator18 and Rifaximin; anticancer19.  Available data confirm that Schiff bases exhibit antineoplastic activity20. Recently we have prepared a set of 11 compounds and evaluated for their antimicrobial activity. The aim of this work was to evaluate in vitro cytotoxicity against the cells of human cancer cell lines.



The title compounds 1-11 were prepared according to the literature procedure21,22 and depicted in figure1.

Antitumor Activity

Cell lines

The following established in vitro human cancer cell lines were applied: K-562 and HL-60.


Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 (Roswell Park Memorial Institute) were used for culturing of cell lines.

Characterization of cell lines and culture media

Cultures were examined under an inverted phase microscope before start of experiments for the detection of any microbial and cross contamination. Cell lines used in experiment were free from any kind of microbial or fungal contamination.

Test compounds

Test solutions of compounds were prepared by dissolving the substances in 100 μl of DMSO completed with 900 μl of culture medium. Afterwards, 1:3 dilutions of test compounds were prepared in culture medium to reach the final concentrations of 100-0.005 µM. The solvent (DMSO) in the highest concentration used in test did not reveal any cytotoxic activity.

Reference compounds

Doxorubicin and methotrexate were used as reference compounds.

Cell lines, culture medium and growth conditions

Human K-562 and HL-60 cell lines were cultured in DMEM and RPMI-1640 respectively, at 37°C in an atmosphere of 5% CO2 and 75% relative humidity. Once reached ~90% confluency, cells were detached using 0.05% trypsin/EDTA and counted by means of trypan blue and haemocytometer.

Sub culturing of cell lines

In order to increase the viability and cell density of K-562 cell line, sub culturing was done by using complete media and additional 5% FBS. As a result, cell density was increased and viability was around 87.98% which was suitable for cytotoxicity screening. PDT (Population Doubling Time) for K-562 was 19.23 hrs. HL-60 cell line was sub cultured in the similar manner.

Seeding of cells

Cell lines in exponential growth phase were washed, trypsinized and re-suspended in complete culture media. Cells were seeded at a concentration of 5 × 104 cells / well in 100 μl culture medium in 96 well microtitre plate and incubated for 24 hrs during which a partial monolayer forms.

Treating cells with test compounds

After 24 hrs of seeding, the cultivated K-562 cells were exposed to various concentrations of selected test compounds (100-0.005 µM) and standard doxorubicin. HL-60 cells were treated with various concentrations (10, 30, 100 µg) of compounds (2, 4, 5) and standard methotrexate. Control wells were received only maintenance medium. The plates were then incubated at 37 °C in a humidified incubator with 5% CO2, 75% relative humidity for a period of 24 hrs.

MTT assay for evaluating cell viability

MTT assay was performed 24 hrs after transfection. 10 μl MTT labeling mixture was added to each well containing cell, mixed by shaking briefly on an orbital shaker and incubated the plates for 4 hrs at 37 °C with 5% CO2 and 75% relative humidity. The plates were centrifuged and the supernatant was discarded. 100 μl of DMSO as solubilizing agent was added to each well and incubated overnight. Measured the absorbance on an ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm to obtain sample signal.

Data interpretation

After 24 hrs, the cytotoxicity data was evaluated by determining absorbance and calculating the correspondent compound concentrations. Linear regression analysis with 95% confidence limit was used to define dose-response curves and to compute the concentration of chemical agents needed to reduce absorbance of the formazan by 50% (IC50).

Percentage cell growth inhibition or percentage cytotoxicity was calculated by following formula:

% viability = (AT AB) / (AC AB) × 100


AT = Absorbance of treated cells (drug)

AB = Absorbance of blank (only media)

AC = Absorbance of control (untreated)

From % viability (% cell survival), % cytotoxicity was calculated by following equation:

% cytotoxicity = 100 % cell survival

The results of cytotoxicity against K-562 and HL-60 cell lines are depicted in Tables 1.

Determination of IC50 Value

The cytotoxic activity of test compounds against cancer cell lines was determined by calculation of IC50. IC50 values were determined from plot of Dose Response curve between log of compound concentration and percentage growth inhibition. IC50 values were derived using curve fitting methods with GraphPad Prism as statical software (Ver. 5.02). IC50 values were calculated using the linear regression program origin. The average of two (duplicates manner) were taken in determination. Graph was plotted by keeping log concentration of drug on X axis and %cytotoxicity on Y axis. The dose-response profile for compounds on human K-562 cell line is shown in Figure 2.

The IC50 values of compounds against HL-60 cell line were calculated by plotting a graph of %cell viability verses compound concentration as shown in Figures 3, 4 and 5.


Figure 1: General structure of previously synthesized compounds

Figure 2: Dose-response profile for compounds on human K-562 cell line in vitro. The cells were treated with compounds at different concentrations and % of cell inhibition (IC50) was calculated.









Figure 3: IC50 value of compound 1.D.I against HL-60 cell line










Figure 4: IC50 value of compound 1.D.IV against HL-60 cell line










Figure 5: IC50 value of compound 1.D.V against HL-60 cell line


Table 1: Results of cytotoxicity testing


IC50 (K-562)

IC50 (HL-60)








































NT= Not Tested


Antitumor screening results (IC50 value), presented in Table 1, revealed that all compounds showed noticeable cytotoxic activity. The results of cytotoxic activity in vitro were expressed as an IC50 value. The IC50 value for each compound was calculated from dose-response curve using linear regression analysis. Among the synthesized compounds evaluated for their in vitro cytotoxic activity against human K-562 cell line, the compounds 1, 7 and 10 showed potent cytotoxic activity when looking with IC50 value of standard doxorubicin i.e. 7.25. From the results, it was observed that IC50 value near by standard indicate good cytotoxic effect and more than 100 indicate no cytotoxicity. Among the compounds tested against human K-562 cell line, compounds 9 showed good activity whereas compounds 8 and 6 showed moderate activity. Compounds 11 and 3 were found to be less active against human K-562 cell line. Among the compounds tested against human HL-60 cell line, compound 5 showed better activity with an IC50 of 30 μg/ml whereas compounds 2 and 4 showed less activity. The obtained results indicate on these compounds as good candidates for further studies in vitro against a broad panel of human tumor cell lines, with an aim to select the most active compound for further preclinical in vivo studies.


Summarizingly, a series of benzimidazole derivatives were screened for their in vitro cytotoxic activity. From the cytotoxic activity study, it was observed that compound with unsubstituted aromatic rings showed cytotoxic activity somewhat comparable to standard doxorubicin. When substitution was made in the aromatic rings, activity is decreased or increased depending on the nature and position of substituents in the aromatic rings. Although the results obtained with the different substituents do not permit a conclusive analysis of SAR on cytotoxic activity. However, it appears that the presence of a 2-chloro on aromatic ring and 2-NO2 on benzylidene amino group (compound 10) in most cases gives better cytotoxic activity against human K-562 cell line.  


Authors thank the Dean, J.N.U., Jodhpur and Principal, B.N. College of Pharmacy, Udaipur, for laboratory facilities and for better cooperation. Thanks are due to CIL Punjab University, Chandigarh and CDRI, Lucknow, India for spectral analysis. Authors thank the principal, Ganpat University, Mehsana, Gujarat to carry out the anticancer activity.



1.      Hranjec, M; Kralj, M; Piantanida, I; Sedic, M et al. (2007), “Synthesis, Phytochemical synthesis, DNA binding and antitumor evaluation”, J Med Chem, Vol. 50, 5696-5711.

2.      Devereux, M; Kellett, A; Malachy, M; Shea, DO et al. (2007), “Synthesis, X-ray crystal structures and biomimetic and anticancer activities of novel copper (II) benzoate complexes”, J Inorg Biochem, Vol. 101, 881-892.

3.      He, Y; Risen, L; Wu, B; Yang, J et al. (2004), “Synthesis and biological evaluation of novel benzimidazoles as potential antibacterial agents”, Bioorg Med Chem Lett, Vol. 14, 1217-1220.

4.      Durmaz, R; Gunal, S; Kucukbay, H and Orhan, E (2003), “Synthesis, antibacterial and antifungal activities of electron-rich olefins derived benzimidazole compounds”, IL Farmaco, Vol. 58, 431-437.

5.      Kumar, CSA; Kumar, YCS; Swamy, SN; Thimmegowda, NR et al. (2008), “Synthesis, characterization and evaluation of benzimidazole derivatives and its precursors as inhibitors of MDA-MB-231 human breast cancer cell proliferation”, Bioorg Med Chem Lett, Vol. 18, 432-435.

6.      Jacob, MR; Kerwin, SM; Kumar, D and Reynolds, MB (2002), “Synthesis and evaluation of anticancer benzoxazoles and benzimidazoles related to UK-1”, Bioorg Med Chem, Vol. 10, 3997-4004.

7.      Kumar, PP; Paramashivappa, R; Rao, AS and Rao, PVS (2003), “Design, synthesis and biological evaluation of benzimidazole/benzothiazole and benzoxazole derivatives as cyclooxygenase inhibitors”, Bioorg Med Chem Lett, 13, 657-660.

8.      Kohno, T; Ohtaka, H; Tsukamoto, G; Yoshino, K et al. (1980), “Synthesis and anti-inflammatory activity of some 2-(substituted-pyridinyl) benzimidazoles”, J Med Chem, Vol. 23, 734-738.

9.      Davies, H; Jeremy, R and Stevenson, C (1999), “Photosensitization of guanine-specific DNA damage by 2-phenylbenzimidaozle and the sunscreen agent 2-phenylbenzimidazole-5-sulphonic acid”, Chem Res Toxicol, Vol. 12, 38-45.

10.  Bhise, UN; Kumar, SBV; Ramanatham, V; Vaidya, SD et al. (2008), “Synthesis, antibacterial, anti-asthmatic and antidiabetic activities of novel N-substituted benzimidazoles”, Eur J Med Chem, Vol. 43, 986-995.

11.  Francisco, AC; Gabriel, NV; Hermenegilda, MD and Ismael, LR (2006), “Design, microwave-assisted synthesis and spasmolytic activity of 2-(alkyloxyaryl)-1H-benzimidazole derivatives as constrained stilbene bioisosteres”, Bioorg Med Chem Lett, Vol. 16, 4169-4173.

12.  Jat, JL., Jat, RK and Pathak, DP (2006), “Synthesis of benzimidazole derivatives: As antihypertensive agents”, E-J. Chem, Vol. 3(13), 278-285.

13.  Chimirri, A; Clercq, ED; Monforte, AM; Rao, A et al. (2002), “Synthesis and anti-HIV activity of 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole structurally-related 1,2-substituted benzimidazoles”, IL Farmaco, Vol. 57, 819-823.

14.  Razdan, B (2010), “Medicinal Chemistry, 1, CBS Publisher, New Delhi, 335-337.

15.  Ainapure, SS; Bhandarkar, SD and Satoskar, RS (1995), “Pharmacology and Pharmacotherapeutics, 2, Popular Prakashan, Bombay, 716-719.

16., Accessed: June 2011.

17., Accessed: June 2011.

18.  Alamgir, M; Black, DSC and Kumar, N (2007), “Synthesis, reactivity and biological activity of benzimidazoles”, Top Heterocycl Chem, Vol. 9, 88.

19.  Awadallah, AM; Seppelt, K and Shorafa, H (2006), “Synthesis and X-ray crystal structure of pyrrolo [1, 2-a] benzimidazoles”, Tetrahedron, Vol. 62, 7744-7746.

20.  Khalid, I; Lamba, HS; Ramanpreet, W and Syeda FN (2011), “Benzimidazole derivatives –an overview”, IJRPC, Vol. 1(3), 565-574.

21.  Bhandari, A; Ranawat, MS; Soni, B and Verma, R et al. (2012), “Synthesis and antimicrobial activity of some novel N-substituted-2-substituted benzimidazole derivatives” J Pharm Res, Vol. 5(7), 3523-3526.

22.  Bhandari, A; Ranawat, MS; Soni, B; Sharma, P et al. (2012), “Synthesis and evaluation of some new benzimidazole derivatives as potential antimicrobial agents”, Pharmacie Globale, Vol. 3(9), 1-4.