IMIDAZOLE AND CARBAZOLE DERIVATIVES AS POTENTIAL ANTICANCER AGENTS: MOLECULAR DOCKING STUDIES AND CYTOTOXIC ACTIVITY EVALUATION

Carbazoles and imidazole represent two important classes of heterocycles which exhibit diverse biological activities such as antitumor properties. In this study, imidazole (C1-C3) and carbazole (C4 and C5) derivatives were evaluated for their cytotoxic activity against three human cancer cell lines namely, MCF7 (human breast cancer), HT29 (human colon cancer), and HeLa (human cervical cancer). Carbazole derivatives (C4 and C5) with IC50 < 10 μM showed greater cytotoxic effect than imidazole derivatives (C1-C3). Furthermore, all compounds exhibited better anticancer activity against MCF-7 than other two cell lines (HT-29, HeLa) and compound C4 was the most potent compound with the IC50 values of 2.5, 5.4 and 4.0 μM, against MCF-7, Hela and HT-29 cell lines, respectively. Physicochemical properties of compounds were calculated and their correlation with the IC50 values on MCF-7 cell line investigated. Surface area and polarizability of compounds showed good correlation by R = 0.8396 and R = 0.834, respectively. Docking studies of these compounds were also performed on the DNA as proposed target to comprehend their binding interactions and binding energies. The docking energy of compounds ranged from 11.32 to -13.48 kcal/mol. Compound C3 with energy of -13.48 kcal/mol had the highest docking energy. Docking results indicated that these compounds (C1-C5) had strong affinity in binding to the DNA.


INTRODUCTION
Cancer after the cardiovascular problems is one of the most spread and mortal disease in the world. Hence, the development of new and potent anticancer agents, is one of the fundamental goals in the medicinal chemistry [1].
Carbazole heterocyclic ring is a major constituent of many natural alkaloids and synthetic derivatives, possessing variant biological activities [2]. These activities including anticancer [3], anti-inflammatory and analgesic effects. Fujita et al. [4] reported that the carbazole derivatives, NP-10, NP-14 and HND-007, exhibit significant anticancer activity as a novel anti-microtubule agents ( Figure 1). Moreover, Kamble and co-worker synthesized carbazoles derivatives which indicate partial anticancer and significant antitubercular activities [2]. In addition to these reports, Lansiaux et al. synthesized and screened novel carbazole derivatives for cytotoxic along with topoisomerase II inhibitory activities which the best compound (53) displayed a high cytotoxicity (IC 50 = 150 nM), higher than etoposid (IC 50 = 490 nM) ( Figure 1) [5]. It is well known that imidazole heterocyclic nucleus is main part of many therapeutic agents with antifungal (econazole, miconazole, metranidazole, ketoconazole and itraconazole), and anticancer (dacarbazine, azathioprine and zoledronic acid) activity [6]. The importance of this nucleus has drawn attention of researchers to synthesize and develop new agents bearing imidazole ring. For example, Yurttas et al. synthesized new imidazole derivatives and evaluated their cytotoxicity against 60 human tumor cell lines along with antimicrobial and antifungal activities [7]. Furthermore, Salerno and co-workers designed and synthesized novel imidazole derivatives based on azalanstat and investigated their antiproliferative properties in prostate ((DU-145, PC3, LnCap) and breast cancer cell lines (MDA-MB-231, and MCF-7) [8].
Based on these premises and importance of imidazole and carbazole heterocyclic nucleuses in medicinal chemistry, we investigated imidazole and carbazole derivatives, which were synthesized recently [9][10][11], for cytotoxic activity.
Physicochemical properties of compounds, to indicate their correlation with the IC 50 values on MCF-7 cell line, were calculated. Docking study of these compounds was also carried out to determine the best binding mode and binding energies of these compounds with DNA as proposed targets.

Cell cultures and MTT assay
The cytotoxic activity of five carbazole and imidazole derivatives were evaluated using a 3-(4, 5-dimethylthiazol-2-y -2,5-diphenyl tetrazolium bromide (MTT) assay. In briefly, for the assay, three cell lines (MCF-7, HT-29 and HeLa) were freshly isolated and seeded in 96-well flat bottom tissue culture plate at a concentration of 1×10 5 cells/well containing 100 μL of RPMI-1640 (supplemented with 10% FBS) tissue culture medium. Then the microplate was incubated in incubator at 37 °C for 24 h. After discarding the medium, 100 μL of compounds with different concentration (0.1, 1, 10, 25, 50 75, 100 μM) were added to each deep plate. The plates were incubated for 48 h. After incubation, 100 μL of the supernatant media was removed and 100 μL of MTT solution (0.5 mg/mL) added and incubated at 37 °C for 3h. After the incubation, the MTT solution was discarded and 80 µL of DMSO was added to dissolve formazan. The optical density was determined at 540 nm using an ELISA plate reader (Bio-Tek, Winooski, VT, USA). The cell growth inhibition was calculated based on the following expression: IC 50 value was calculated by extrapolating a graph with % cell inhibition on Y-axis against concentration of test compound on X-axis.

Statistical analysis
The IC 50 values were calculated using Curve Expert 1.4. and were expressed as mean ± SD. One-way analysis of variance (ANOVA) were used for analysis of MTT cytotoxicity results and the level of P < 0.05 was considered to indicate statistically significant differences.

Physicochemical properties
Physicochemical properties of compounds including SA (surface area), volume, HE (hydration energy), Log P, polariability, reflectivity and mass, after minimizing of energy based on two methods (molecular mechanic (MM + ) and semiempirical (AM1) were calculated using HyperChem 8.

Docking procedure
The docking studies were performed using an in house batch script (DOCKFACE) [12][13][14][15][16] of AutoDock 4.2. HyperChem 8 was applied to optimized energy of each ligand with MM+ and AM1 minimization methods. Then the partial charges of atoms were calculated using Gasteiger-Marsili procedure. Non-polar hydrogens of compounds were merged and then rotatable bonds were assigned. Then mol2 format was converted PDBQT by MGL tools 1.5.6 [17]. The 3D crystal structure of DNA (PDB ID: 1BNA) as potential targets for our compounds were retrieved from protein data bank (http://www.rcsb.org/pdb/home/home.do). After removing water molecules, missing hydrogens were added and non-polar hydrogens were merged into their corresponding carbons using AutoDock Tools [18]. As search algorithms, Lamarckian Genetic Algorithm (LGA) was applied and performed by AutoDock 4.2. [17,[19][20][21]. Finally, the PDBQT file of the receptors was obtained using MGLTOOLS 1.5.6.
For Lamarckian GA, a maximum number of 2,500,000 energy evaluations, 27,000 maximum generations; 150 population sizes, a gene mutation rate of 0.02; and a crossover rate of 0.8 were applied. The grid maps of the receptors were calculated using AutoGrid tools of AutoDock 4.2. The grid box parameters for these targets were shown in the Table 1. The grid box parameters of 1BNA were 60×74×120 points in x, y, and z directions. gpf and dpf as grid and docking parameter files were built by AutoDock Tools.

In vitro cytotoxicity
In this study, all heterocyclic compounds were evaluated against three cell lines including MCF-7, HT-29 and HeLa by standard MTT assay method (Table 2). Comparison between these compounds on MCF-7 cell line indicated that after standard drug (doxorubicin), compound C4 had better cytotoxic effect with IC 50 =2.50 µM .
Compound C4 was the most potent compound against all cell lines MCF-7, Hela and HT-29 with the IC 50 values 2.5, 5.36 and 4.03 µM, respectively. As shown in Table 2, among compounds C1-C3 as imidazole derivatives, compound C1 with IC 50 28.7 and 6.63 µM against MCF-7 and Hela, respectively, showed significant anticancer activity than C2 and C3.
As mentioned above, imidazole and carbazole derivatives have attracted considerable attention because of variable biological activity such as anticancer activity. Thus, selected compounds which bearing imidazole and carbazole rings, were subjected to MTT assay and structure-activity relationship (SAR) of compounds was investigated. Comparison between carbazole derivatives C2 and C3, which contain NO 2 and NH 2 substitution, respectively, indicated that the NO 2 group had a greater effect on cytotoxic activity against MCF-7 cell line than NH 2 group.
Compound C4 and C5, similar to imidazole derivatives C2 and C3, showed that NO 2 group had higher anticancer effect than NH 2 group against all tested cell lines. Overall, all compounds have no significant effect against HT-29 cell line and none of them shown more cytotoxic effect than doxorubicin as positive control.
Carbazole derivatives (C4 and C5) showed higher cytotoxic activity against MCF-7 cell line in comparison to the other anticancer agents such as imidazolylphenylheterocyclic-2-

C1-C3
C4-C5  [22]. Furthermore, compound C4 and C5, had more cytotoxic activity on HeLa cell line than most of hybrid compounds of imidazole scaffold-based 2-benzylbenzofuran and carbazole alkaloide [23]. In addition,these compounds are effective on the greater number of cell lines than those mentioned above [22,24], which showed the broader spectrum activity of our studied compounds against different cancer cell lines.

Physicochemical properties
Physicochemical properties of compounds including surface area, volume, hydration energy, Log P, polariability, reflectivity and mass are presented in Table 3.  The IC 50 values on MCF-7 cell line and physicochemical properties of compounds were plotted and the results showed that surface area and polarizability had good correlation than the other physicochemical properties.
The IC 50 values on MCF-7 cell line showed good correlation with surface area and polarizability of compounds by R 2 = 0.8396 and R 2 = 0.834, respectively. The other physicochemical properties had low to moderate correlation with IC 50 value ( Figure 2).

Docking study
As shown in Table 4, the docking energy of compounds ranged from -11.32 to -13.48 kcal/mol.  Furthermore, 3D interactions of compound C3 into minor groove of DNA was shown in the Figure 3. The amino groups of compound C3 were involved in hydrogen bond interactions with adenosine 6 and guanosine 4. There was also exists an aren-H interaction between the imidazole ring and adenosine 5 of DNA.
Docking studies of these compounds were also performed on the DNA as proposed targets to found out their binding interactions and binding energies. Based on molecular docking results, it can be concluded that these compounds had stronger affinity to bound with DNA and compound C3 with energy of -13.48 kcal/mol had the highest affinity to bound with this target.

CONCLUSIONS
In the present work, the cytotoxic activity of the five imidazole and carbazole compounds were evaluated using MTT test on three cell lines namely breast cancer (MCF-7), cervical cancer (HeLa) and colon cancer (HT-29). The most cytotoxic activity in the entire set was related to carbazole derivative, compound C4. Based on these results, carbazole derivatives had better cytotoxic effects than imidazole derivatives and nitro substituted groups showed more cytotoxic activity than amino substituted groups. The docking simulations were carried out by means of AutoDock 4.2. to find the best pose of each ligand in the active site of the DNA and tubulin as proposed targets. Base on the docking binding energies, these compounds had stronger affinity in binding to the DNA, and the compound C3 had the highest docking energy when docked into the DNA. Although the molecular weight of these compounds is more than 500, biological results show that these compounds have acceptable anti-cancer effects. The range below 500 is one of Lipinski's rules that usually applies to compounds that can be administered orally. Therefore, one of the limitations of these compounds will probably be the route of their administration. The findings suggest that the new derivatives of imidazole and carbazole had the potential to be considered as cancer treatment and should be further explored in future cancer therapy.