Synthesis and antitumor activity of novel N-substituted carbazole imidazolium salt derivatives

A series of novel N-substituted carbazole imidazolium salt derivatives has been prepared and investigated for their cytotoxic activity against five human tumor cell lines by MTS assay. The results indicated that the existence of 5,6-dimethyl-benzimidazole ring, substitution of the imidazolyl-3-position with a 2-bromobenzyl or naphthylacyl group, as well as alkyl chain length between carbazole and imidazole ring were important for the antitumor activity. Compound 61, bearing a 2-bromobenzyl substituent at position-3 of the 5,6-dimethyl-benzimidazole, showed powerful inhibitory activities and was more selective to HL-60, SMMC-7721, MCF-7 and SW480 cell lines with IC50 values 0.51–2.48 μM. Mechanism of action studies revealed that this new compound could remarkably induce cell cycle arrest and apoptosis in SMMC-7721 cells. This work provides alternative novel way for future drug development based on carbazole and imidazolium salt scaffolds.


General Experimental
Melting points were obtained on a XT-4 melting-point apparatus and were uncorrected. Proton nuclear magnetic resonance ( 1 H-NMR) spectra were recorded on a Bruker Avance 300/400 spectrometer at 300/400 MHz. Carbon-13 nuclear magnetic resonance ( 13 C-NMR) was recorded on Bruker Avance 300/400 spectrometer at 75/100 MHz. Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane (TMS) for all recorded NMR spectra. Low-resolution Mass spectra were recorded on a VG Auto Spec-3000 magnetic sector MS spectrometer. High Resolution Mass spectra were taken on AB QSTAR Pulsar mass spectrometer. X-Ray data was determined using a Bruker APEX JASCO P-1020 polarimeter.
Silica gel (200-300 mesh) for column chromatography and silica GF 254 for TLC were produced by Qingdao Marine Chemical Company (China). All air-or moisturesensitive reactions were conducted under an argon atmosphere. Starting materials and reagents used in reactions were obtained commercially from Acros, Aldrich, Fluka and were used without purification, unless otherwise indicated. To a mixture of carbazole 1 (1.5 g, 9 mmol) and NaOH (520 mg, 13 mmol) in DMF (30 mL) at 0 °C was added alkyl dibromide (27 mmol). The reaction mixture was stirred at room temperature for 5 h. Reaction progress was monitored by TLC, then diluted with water (50 mL), and extracted with ether (20 mL×3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na 2 SO 4 and concentrated. The residue was purified by column chromatography (silica gel, petroleum ether 60-90 o C : EtOAc = 15:1) to afford 2-4 in 68-72% yield as white powder.

X-ray crystal structure of compound 24 and 30
X-ray crystal structure of compound 24.

MTS assay.
Cytotoxicity was determined by performing MTS assay. Briefly, 100 ml of cells suspension were seeded in 96-well cell culture plates and allowed to adhere overnight.
The cells were treated with drugs for 48 hours, and then 20 ml of CellTiter 96 ® AQ ueous One Solution Reagent (Promega, Madison, USA) was added and the cells were further incubated at 37 o C for 1-2 h. Cell viability was measured by reading the absorbance at a wavelength of 490 nm. Concentrations of 50% inhibition of growth (IC50) were calculated on the basis of the relative survival curve.

Cell cycle analysis.
To analyze the DNA content by flow cytometry, cells were collected and washed twice with PBS. Cells were fixed with 70% ethanol overnight. Fixed cells were washed with PBS, and then stained with a 50 μg/ml propidium iodide (PI) solution containing 50 μg/ml RNase A for 30 min at room temperature. Fluorescence intensity was analyzed by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

S74
The percentages of the cells distributed in different phases of the cell cycle were determined using ModFIT LT 2.0.