5-Aryl-1-Arylideneamino-1H-Imidazole-2(3H)-Thiones: Synthesis and In Vitro Anticancer Evaluation

A novel series of N-1 arylidene amino imidazole-2-thiones were synthesized, identified using IR, 1H-NMR, and 13C-NMR spectral data. Cytotoxic effect of the prepared compounds was carried out utilizing three cancer cell lines; MCF-7 breast cancer, HepG2 liver cancer, and HCT-116 colon cancer cell lines. Imidazole derivative 5 was the most potent of all against three cell lines. DNA flow cytometric analysis showed that, imidazoles 4d and 5 exhibit pre-G1 apoptosis and cell cycle arrest at G2/M phase. The results of the VEGFR-2 and B-Raf kinase inhibition assay revealed that compounds 4d and 5 displayed good inhibitory activity compared with reference drug erlotinib.


Introduction
Cancer is the pathological uncontrolled proliferation of the abnormal cells that divide rapidly [1]. Moreover, cancer can involve any tissue of the body and have many different forms in each body area [2,3]. Therefore, there is a constant and growing interest to synthesize new biologically active molecules which might be useful in a cancer treatment.
Anticancer activity of the azole derivatives is most extensively studied and some of them are in clinical practice as antitumor active agents [4,5]. Imidazoles are an important class of heterocyclic molecules and imidazole derivatives are reported to have anticancer activity [6][7][8]. Misonidazole I displayed superior antiproliferative activity against a panel of cancer cell lines [9]. Tiazofurin II possesses good anticancer activity [10]. Lepidiline A and B are imidazole compounds which exhibit cytotoxicity against various types of human cancer cell lines at micromolar concentration [11]. Imidazole derivatives were found to exert their anticancer activity by acting mainly as antiangiogenic agents, inhibitors of B-Raf kinase, and as p38 MAP kinase inhibitors [12][13][14]. For example, compound III displayed potent antitumor activity against the MCF-7 cell line with an IC 50 value of 3.26 µM, as compared with SOR (IC 50 = 1.12 µM) [15]. Additionally, compound IV has been reported as a vascular endothelium growth factor (VEGFR-2) inhibitor and apoptotic inducer [15]. Moreover, it has been reported that imidazole molecule V inhibits the kinase activity of Raf in low nanomolecular concentrations [16]. Furthermore, compound VI was identified as a submicromolar inhibitor of B-Raf kinase [17] (Figure 1).
Based on the aforementioned aspects, the present study is concerned with the synthesis of novel imidazole molecules bearing arylidene amino substituents at the N-1 position. It is worthy to note that imidazole fragments are decorated at the C-5 position with phenyl groups carrying chloro or methoxy substituents to show the impact on anticancer activity. Finally, full details about the synthesis and evaluation of the antitumor activity in vitro are reported herein.
Furthermore, the novel imidazole derivatives 4a-e and 5 were synthesized by cyclocondensation of 2-arylhydrazinecarbothioamides 3a-e and substituted phenacylbromide in absolute ethanol in the presence of fused sodium acetate under reflux temperature (Scheme 1). The structure of novel imidazoles 4a-e was characterized using different spectroscopic methods. The 1 H-NMR spectra revealed the presence of additional aromatic signals attributed to phenyl ring B protons.

In Vitro Cytotoxic Activity against Three Cancer Cell Lines
The effect of imidazole molecules 4a-e and 5 on the viability of three cancer cell lines were studied using the MTT assay. The cytotoxicity was assessed using Docetaxel (DOC) as a standard antitumor drug. The three cancer cell lines were MCF-7 (breast cancer cell line), colon carcinoma (HCT-116), and HepG2 (hepatocellular carcinoma cell line). Treatment of MCF-7, HCT-116, and HepG2 cell lines with different concentrations of imidazole derivatives revealed moderate to good antitumor activities as concluded from their IC 50 values shown in Table 1. Compound 1-(2-hydroxybenzylideneamino)-5-(4methoxyphenyl)-1H-imidazole-2(3H)-thione (5) was the most potent cytotoxic compound against all of the tested tumor cell lines with IC 50 s < 5 µM. Moreover, the safety of compounds 4d and 5 was assessed against a normal breast cell line (MCF-10A) using the MTT assay. The results revealed weak cytotoxic effect towards MCF-10A and compound 4d showed less cytotoxic effect than standard drug DOC.

Cell Cycle Analysis
The most active compounds, 4d and 5 on MCF-7 were studied using DNA flow cytometric analysis. Treatment of MCF-7 cells with IC 50 concentration dose value of compounds 4d and 5 resulted in a significant alteration in cell cycle profile. There was a significant increase in the percentage of cell population at the G2/M phase from (8.79%) control to 35.55% and 25.63% upon treatment with compounds 4d and 5, respectively. Additionally, a significant increase in the percentage of cells at pre-G1 phase from 1.61% (control) to 22.36% and 22.81% upon treatment with compounds 4d and 5, respectively. Therefore, it can be concluded that compounds 4d and 5 inhibit the cell proliferation of MCF-7 cells via cell cycle arrest at the G2/M phase and induction of apoptosis ( Figure 2).

Annexin V-FITC/PI Apoptosis Induction Analysis
Several imidazole molecules have been reported in the literature as apoptosis inducers in several tumor cell lines [20]. To evaluate the mode of cell death induced by compounds 4d and 5, Annexin V/FITC and propidium iodide (PI) assays were performed. MCF-7 cells were treated with compounds 4d and 5 at their IC 50 concentration dose value for 48 h. The results showed that the selected candidates induce apoptosis of MCF-7 cells at both early and later stages. As shown in Figure 3, the percent of early apoptosis was increased from 0.43% (control) to 6.12% and 2.66% for compounds 4d and 5, respectively. Additionally, the percent of late apoptosis was increased from 0.23% (control) to 10.23% and 13.64% upon exposure to compounds 4d and 5, respectively. The results proved that the antiproliferative activity of compounds 4d and 5 is attributed to its apoptosis inducing activity in MCF-7 cells.

VEGFR-2 Kinase Inhibitory Activity
To investigate the possible molecular mechanism underlying the potent anticancer activity of compounds 4d and 5. Their inhibitory activity against VEGFR-2 kinase was determined using an ELISA assay. The results in (Figure 4) showed that the selected imidazole molecules 4d and 5 elicited potent activity against VEGFR-2 kinase with IC 50 values 247.81 and 82.09 ng/mL, respectively, compared with erlotinib as the reference drug. According to these results, imidazole molecules 4d and 5 possess good inhibition of VEGFR-2 kinase activity.

B-Raf Ihibitory Activity
Raf is a protein kinase that initiates a cascade of other protein kinases by acting on the protein kinases MEK-1 and MEK-2 [21]. Therefore, constitutive activation of this signaling pathway is observed in variety of cancers. In the present assay, the activity potential of compounds 4d and 5 was evaluated against B-Raf V600E using an ELISA assay.
The results showed that compounds 4d and 5 revealed good inhibitory activities against B-Raf V600E with IC 50 values of 13.05 and 2.38 µg/mL, respectively, compared with the value of 0.98 µg/mL for erlotinib ( Figure 5).

Molecular Docking Study
Compounds 4d and 5 were docked into the VEGFR-2 crystal structure (PDB code: 3U6J) [22]. Compound 4d interacted with the active site of 3U6J by hydrogen bonding with the amino acid Lys 868 by its methoxy group. In addition, the hydrophobicity induced by compound 4d resulted in a docking score of −20.69 kcal/mol. On the other hand, compound 5 interacted through hydrogen bonding with the amino acids Lys 868 and Ile 1025 by its methoxy and hydroxy groups, respectively, and its hydrophobicity resulted in docking score of −21.58 kcal/mol ( Figure 6). The interaction result is consistent with the obtained IC 50 values against the VEGFR-2 enzyme. Accordingly, from the obtained results, compound 5 was more potent than compound 4d and with higher VEGFR-2 inhibitory activity.

General
1 H and 13 C-NMR spectra were measured with a Bruker 400 DRX, Avance NMR spectrometer (Chichago, Elk Grove Village, USA) using the DMSO-d 6 solvent. The IR data were obtained with a shimadzu 470 spectrometer (Kyoto, Kyoto, Japan). Melting points of the prepared derivatives were measured with an electro thermal melting point apparatus and were not corrected. The microanalyses were implemented on a Perkin Elmer CHN elemental analyzer (Haan, Germany). All chemicals were purchased from Aldrich chemical company and all reagents were of analytical grade. The prepared imidazole molecules were soluble in DMSO and not soluble in water at any concentration.

General Procedure for the Synthesis of 2-Arylhydrazinecarbothioamide (3a-d)
To a mixture of aromatic aldehyde (0.01 mol) and thiosemicarbazide 2 in absolute ethanol (50 mL) and a few drops of glacial acetic acid were added. The reaction mixture was heated under reflux for 4 h. After cooling, the separated solid was collected by filtration, dried, and crystallized from ethanol to give 3a-d.

General Procedure for the Synthesis of Imidazole Derivatives 4a-e and 5
A suspension of thiosemicarbazone derivative 3a-e (0.01 mol) and freshly prepared substituted phenacyl bromide (0.01 mol) in 30 mL of absolute ethanol and freshly fused sodium acetate (0.03 mol) was refluxed for 6 h. After concentration and cooling, the solid that formed was collected and crystallized from proper solvent to give the target imidazole compound.

Antitumor Activity against Three Cancer Cell Lines
Antitumor activity of the newly prepared imidazole compounds 4a-e and 5 were carried out against MCF-7, HepG2, HCT-116, and MCF-10A using the MTT assay method. Cells at a density of 1 × 10 4 were seeded in 96-well plates at 37 • C for 48 h under 5% CO 2 . After incubation, the cells were treated with different concentrations of the prepared molecules and incubated for 24 h. MTT dye was added at the end of 24 h of drug treatment and incubated for 4 h at 37 • C. Next, 100 µL of dimethyl sulfoxide was added to each well to dissolve the purple formazan formed. The color intensity of the formazan product, which represents the growth condition of the cells, is quantified using an ELISA plate reader at 570 nm. The experimental conditions were carried out with at least three replicates and the experiments were repeated at least three times.

Cell Cycle Analysis of Compounds 4d and 5
MCF-7 cells (2 × 10 5 /well) were harvested and washed twice in PBS. After that, cells were incubated at 37 • C and 5% CO 2 . The medium was replaced with DMSO (1% v/v) containing the tested compounds, then incubated for 48 h, washed twice in PBS, fixed with 70% ethanol, rinsed again with PBS, and then stained with DNA fluorochrome PI for 15 min at 37 • C. DNA content was analyzed by flow cytometry on a FACS Calibur flow cytometer (Becton and Dickinson, Heidelberg, Germany).

Annexin V FITC/PI Apoptosis Detection Staining Assay
Apoptosis in MCF-7 cells was investigated using fluorescent Annexin V-FITC/PI detection kit by flow cytometry assay. Briefly, MCF-7 cells (2 × 10 5 ) after incubation for 12 h. Cells were treated with compounds 4d and 5 at their IC 50 concentration for 48 h, then the cells were harvested and stained with Annexin V-FITC/PI dye for 15 min in the dark at 37 • C. Flow cytometry analyses were performed using a FACS Calibur flow cytometer (Becton and Dickinson, Heidelberg, Germany).

In Vitro VEGFR-2 Kinase Assay
VEGFR-2 inhibitory activity for compounds 4d, 5, and erlotinib was evaluated using human VEGFR-2 ELISA kits according to the manufacturer's instructions. A total of 100 µL of standard solution and tested molecules were pipetted into the 96-wells and VEGFR-2 was bound to the wells by the immobilized antibody. The wells were washed and biotinylated antihuman VEGFR-2 antibody was added. After washing away unbound biotinylated antibody, 100 µL of conjugated streptavidin solution was pipetted into the wells. After washing, 100 µL of TMB substrate solution was added to the wells for 30 min at 37 • C. Stop solution (50 µL) was added, and the intensity of the color was measured immediately at 450 nm.
3.5.5. In Vitro B-Raf Kinase Assay RAF inhibitory activity for compounds 4d, 5, and erlotinib was evaluated using human B-RAF ELISA kits according to the manufacturer's instructions. Next, 100 µL of standard solution and tested molecules were pipetted into the 96-wells and B-RAF was bound to the wells by the immobilized antibody. The wells were washed and biotinylated antihuman B-Raf antibody was added. After washing away unbound biotinylated antibody, 100 µL of conjugated streptavidin solution was pipetted into the wells. After washing, 100 µL of TMB substrate solution was added to the wells for 30 min at 37 • C. Stop solution (50 µL) was added, and the intensity of the color is measured immediately at 450 nm.

Molecular Docking Study
A molecular docking study was performed using the MOE software program (MOE 2009.10). The VEGFR-2 crystal structure (PDB code: 3U6J) was obtained from a protein data bank (Supplementary Materials).

Conclusions
In conclusion, a novel series of imidazoles bearing arylidene amino substituents at the N-1 position were synthesized in order to investigate their anticancer activity. The structure of the prepared imidazole derivatives was confirmed utilizing spectral data such as IR, 1 H-NMR, and 13 C-NMR along with elemental analyses. The prepared imidazoles molecules were screened for their cytotoxic activity against three cancer cell lines namely, MCF-7 breast cancer, HepG2 liver cancer, and HCT-116 colon cancer cell lines. Compound 5 was the most potent of all against the three cell lines. DNA flow cytometric analysis showed that, compounds 4d and 5 exhibit pre-G1 apoptosis and cell cycle arrest at the G2/M phase. The results of the VEGFR-2 and B-Raf kinase inhibition assays revealed that compounds 4d and 5 displayed good inhibitory activity compared with the reference drug erlotinib. Finally, imidazole compounds 4d and 5 could serve as lead chemical entities for further structural optimization as anticancer agents.