Synthesis and Biological Evaluation of Novel Acenaphthene Derivatives as Potential Antitumor Agents

Twelve novel acenaphthene derivatives have been synthesized. The structures of all compounds were confirmed by 1H-NMR, MS and elemental analysis. Their antitumor activities were evaluated in six human solid tumor cell lines, namely non-small cell lung cancer (H460), human colon adenocarcinoma (SW480), human breast cancer cell (MDA-MB-468 and SKRB-3), human melanoma cell (A375) and human pancreatic cancer (BxPC-3). Among them, compound 3c shows the best antitumor activity against SKRB-3 cell line, as high as the positive control adriamycin.


Introduction
Acenaphthene derivatives have gained great importance due to their diverse biological properties including antitumor [1,2], antifungal [3], antimicrobial [4], anti-inflammatory [5,6] and insecticidal [7] activities. In order to obtain novel acenaphthene derivatives with a wide spectrum of pharmaceutical applications, we report herein the synthesis of a series of acenaphthene derivatives containing thiazole backbone and the results of their preliminary in vitro antitumor evaluation.

Chemistry
The synthetic route for the target compounds is shown in Scheme 1. The prepared acenaphthene derivatives 3a-h and 4a-d are listed in Table 1.

Scheme 1. Synthetic route for 3a-h and 4a-d.
Reagents and conditions: a) ethanol, reflux, 2-5 h, NaHCO 3 ; b) acyl chloride, ethyldiisopropylamine, pyridine, reflux, 6-8 h. Commercially available acenaphthene was first reacted with bromoacetyl bromide in the presence of aluminum chloride to give crude 5-bromoacetylacenaphthene (1) (containing a little 3-bromoacetyl acenaphthene). The raw product was purified using a silica column with ethyl acetate/petroleum ether (1:15 v/v) as eluent. 3-Bromoacetylacenaphthene was eluted first and 5-bromoacetylacenaphthene was elueted next. The compounds 3a-h were prepared by reacting 1 and 2-thiourea (2a) or aryl thioureas 2b-h under reflux using ethanol as solvent. The HBr produced was neutralized with NaHCO 3 solution and the crude product was recrystallized from appropriate solvent to yield 3a-h (purity >97%, HPLC). Compound 3a was further reacted with an appropriate acyl chloride in the presence of ethyldiisopropylamine using pyridine as solvent. Compounds 4a-b (purity >97%, HPLC) were obtained by recrystallization of the crude products from appropriate solvents without purification by column chromatography.

Antiproliferative Activities
In the present study, the antiproliferative activity of the synthesized compounds was tested in vitro on six human tumor cell lines, including non-small cell lung cancer (H460), human colon adenocarcinoma (SW480), human breast cancer cell (MDA-MB-468 and SKRB-3), human melanoma cell (A375) and human pancreatic cancer (BxPC-3) by the MTT assay with adriamycin (ADM) as a positive control. The inhibition rates under drug concentration of 20 μM are summarized in Table 1. Among the acenaphthene derivatives, 3c shows better inhibition against breast cancer cells line of MDA-MB-468 and SKRB-3 (inhibition rate are 55.5 ± 3.8% and 66.1 ± 2.2% respectively), while the corresponding results for ADM are 63.4 ± 0.4% and 68.1 ± 1.3%. These results suggest that 3c has potent antitumor activity.

General
The human cancer cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco's modified Eagle medium (DMEM) and RPMI 1640 were purchased from Gibco (Grand Island, New York, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA). All chemicals were commercially available and used without further purification unless otherwise stated. Column chromatography was carried out on silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). Thin layer chromatography (TLC) was performed on TLC silica gel 60 F254 plates. The purity of compound screened in biological assays was determined to be ≥97% by HPLC analysis with a photodiode array detector (Waters, Milford, MA, USA). An atlantis C 18 (150 mm × 4.6 mm, i.d. 5 μm) (Waters, Milford, Mass, USA) was used with a gradient elution of methanol and HPLC-grade water as mobile phase at a flow rate of 1 mL/min. 1 H-NMR were recorded at 400 MHz on a Varian spectrometer (Varian, Palo Alto, CA, USA) model Gemini 400 and chemical shifts (δ) are reported in parts per million relative to tetramethylsilane (TMS) used as an internal standard, where (δ) TMS = 0.00 ppm. Mass Spectra (MS) were measured by Q-TOF Priemier mass spectrometer utilizing electrospray ionization (ESI) (Micromass, Manchester, UK). Elemental analyses were carried out on a Carlo Erba-1106 analyzer. Melting points were determined on a SGW X-4 microscopic melting point (Shanghai Precision & Scientific Instrument Co., Ltd, China). (1) 5-Bromoacetylacenaphthene was synthesized according to a literature method [8] with some modifications. Briefly, aluminum chloride (22.8 g, 171 mmol) was added slowly while stirring to a solution of acenaphthene (18.6 g, 211 mmol) and bromoacetyl bromide (12.6 mL, 145 mmol) in CH 2 Cl 2 (120 mL) at -20 °C. The mixture was allowed to warm to room temperature and stirred for 30 min, neutralized with 5% NaHCO 3 solution, extracted with chloroform (3×50 mL), washed with water (2×50 mL) and dried over Na 2 SO 4 . The solvent was evaporated and residue was purified with column chromatography (ethyl acetate: petroleum ether = 1:15) to give compound 1 as an off-white solid. Yield: 13.8 g, 41.5%. 1

General Procedure for Preparing Compounds 4a-d
A mixture of 3a (0.50 mmol), acyl chloride (0.55 mmol) and ethyldiisopropylamine (0.3 mmol) was refluxed in pyridine (10 mL). After completion of the reaction, the reaction mixture was evaporated. Water (20 mL) and ethyl acetate (50 mL) were added. The organic layer was washed with 5% Na 2 CO 3 DMSO was added to dissolve formazan crystals. Absorbance was measured at 570 nm using an ELISA reader (Thermo). The effects of the compounds on the proliferation of cancer cells were expressed as the % cell growth inhibition, using the following formula: % inhibition = (A 570 of control -A 570 of treated cells)/A 570 of control cells × 100%.

Conclusions
In this work, twelve novel acenaphthene derivatives containing thiazole backbone were synthesized and the in vitro antitumor activity was evaluated. Among all of these derivatives, compound 3c showed more potent cytotoxicity towards SKRB-3 cell than the positive control adriamycin, which indicates the potential of 3c as a future anticancer agent.