Synthesis and Evaluation of Anticancer Activity of O-allylchalcone Derivatives

A large number of novel O-allylchalcones were synthesized by Claisen Schmidt condensation reaction of O-allylvanillin 3 with appropriate substituted acetophenones 4a-h. These model chalcones 5a-h and their precursor O-allylvanillin were screened for their in vitro cytotoxic activity against four human cancer cell lines. The most potent compound in this series with the IC50 values below or around 10 μM were 5f against THP-1 cells (10.42 μM) and 5g against THP-1 (4.76 μM), DU-145 (5.21 μM), HL60 (7.90 μM), Hep-G2 (10.12 μM) and MCF-7 (10.32 μM). *Corresponding author: Bathelemy Ngameni, Department of Pharmaceutical Sciences and Traditional Pharmacopoeia, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Cameroon, P.O. Box 8664, Tel: +237 76480440; Fax: +237 22221873; E-mail: bath_ngameni@yahoo.fr Received June 17, 2013; Accepted July 26, 2013; Published July 28, 2013 Citation: Ngameni B, Kuete V, Ambassa P, Justin K, Marlyse ML, et al. (2013) Synthesis and Evaluation of Anticancer Activity of O-allylchalcone Derivatives. Med chem 3: 233-237. doi:10.4172/2161-0444.1000144 Copyright: © 2013 Ngameni B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
There is a currently a good deal of interest in the health benefits of phytochemicals, in particular prenylated and allylated flavonoids. Chalcones (1,3-diaryl-2-propen-1-ones) and their derivatives are important intermediates of flavonoid synthetic pathway. Chalcones, one of the major classes of natural products with widespread distribution in fruits, vegetables, spices, tea and soy based foodstuff have also been the subject of great interest for their interesting pharmacological activities [1]. Chemically they can be considered open-chain flavonoids in which the two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl system. Chalcones have also been reported to possess many useful biological and pharmacological properties, including antibacterial [2,3], antimalarial [4,5], antifungal [6], antiviral [7,8], anti-inflammatory [9,10], and anticancer [11,12] properties. A good safety profile, possibility of oral administration [13] and easy synthesis are the major factors contributing to the increasing interest in exploring the pharmacological activities of chalcones. Chalcones comprise one of the main classes of natural small molecules with very promising anticancer activity, related to their ability to inhibit tubulin polymerization [14]. Most of the anticancer agents, of natural or synthetic origin exhibit enone function in their structure [15,16]. Also, synthesized chalcones holding allylic substitutions were recently reported as potent antimicrobial and antioxidant agents [17,18]. In addition, the substitution of ring B with electron withdrawing groups like methoxy or hydroxy group improve the antiproliferative activity against human colon HT-29 cancer cell line [19].
Prompted by all these observations, we report herein the synthesis of novel O-allylchalcones, bearing various substituents with potent activity against Human Hep-G2 hepatocarcinoma, breast carcinoma MCF-7, prostate carcinoma DU-145, and acute monocytic leukemia THP-1 and HL-60 cell lines. The structure-activity relationships are also discussed.

Materials and Methods
Chemistry IR spectra were determined with a Perkin Elmer FT-IR spectrophotometer. 1 H and 13 C NMR spectra were recorded with Bruker WM-300 in the CDCl 3 at 300 and 75 MHz, respectively using TMS as the internal standard. All chemical shifts are reported on δ scale. Mass spectra were obtained using a Varian MAT-311A. Thinlayer chromatography (TLC) was carried out using Merck silica gel 60 F-254 plates (layer thickness 0.25 mm) and all solvents were distilled prior to use.

Synthesis
Compounds 5a-h were synthesized by the condensation reaction of compound 3 with different substituted acetophenones 4a-h. The main intermediate 3 was prepared from vanillin 1 and allylbromide 2 in the presence of potassium carbonate in anhydrous acetone.

Biology
Cytotoxicity assay: Cell lines and treatment: The effect of synthesized compounds on cell growth was determined on five human tumor cells including Hep-G2 hepatocarcinoma, breast carcinoma MCF-7, prostate carcinoma DU-145, and acute monocytic leukemia THP-1 and HL-60 cell lines, obtained from National Cancer Institute, USA. THP-1 and HL-60 were maintained in RPMI medium while Hep-G2, MCF-7 and DU-145 were cultured in MEM medium. All media used were supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin. The cell lines were maintained under standard cell culture conditions at 37 o C and 5% CO 2 in a humidified environment.
The cytotoxicity of the samples against the five studied human cell lines was determined using Sulphorhodamine B (SRB) assay as previously described [20]. The cells were incubated at 37 o C in an atmosphere of 5% CO 2 and 95% relative humidity in a CO 2 incubator. Doxorubicin was used as positive reference. Suitable controls with equivalent concentration of DMSO were also included. The optical density (OD) was recorded using a 96 well plate reader, and growth inhibition was calculated [20]. A preliminary study was first carried out with compounds (Table 1, 100 µM) and doxorubicin (at 50 µM) to detect if samples were able to inhibit the proliferation of more that 50% of the cells. Then samples were serially diluted and tested against other cell lines for IC 50 determination. IC 50 is the concentration of sample required to inhibit 50% of the cell proliferation after 72 h incubation and was calculated by plotting the percentage survival versus the concentration, using Microsoft Excel. For all samples, each compound concentration was tested thrice in triplicates.

4-Allyloxy-3-methoxybenzaldehyde or O-allylvanillin (3)
To 0.304 g (1.99 mmol) of vanillin in acetone (8 mL) was added K 2 CO 3 (0.1203 g) followed by allylbromide (0.12 mL, d = 1.43, 0.1772 g, 1.46 mmol). The reaction mixture was heated to reflux for 4 hours or left at room temperature for 48 hours. At the end of the reaction, the solvent was evaporated under reduced pressure and the residue is diluted in water (40 ml x3). The aqueous mixture was extracted with Ethyl Acetate (EA) (3 × 60 mL) and the extract was dried by anhydrous Na 2 SO 4 . After evaporation of the solvent and purification by column chromatography on silica gel eluting with Hexane-Ethyl Acetate (Hex-EA) system of increasing polarity, product 3 was obtained (725 mg, yield 70% in Hex-EA 87.5:12.5). 1

4-allyloxy-3-methoxychalcone (5a)
To a solution of acetophenone (0.18 mL, 3.41 mmol, d=1.0266) in methanol (30 mL) was added first O-allylvanillin (110 mg, 0.57 mmol) and then an aqueous solution of KOH (50%, 1 mL / mmol of acetophenone) or 3.41 mL. The reaction mixture was refluxed at 70°C for 5 hours or left at room temperature for 15 hours. At the end of the reaction the mixture was diluted with water (30 mL) and extracted with CH 2 Cl 2 (3×70 mL) and then the extract was washed with water (50 mL) and saturated with NaCl solution. The organic phase was dried with Na 2 SO 4 and the solvent evaporated under reduced pressure. After purification of the residue by Column Chromatography and Thin Layer Chromatography preparative on silica gel (Hex-EA 9:1), compound 5a was obtained pure (103.

Statistical analysis
The one-way ANOVA at 95% confidence level was used for statistical analysis.

Chemistry (synthesis)
The synthesis of chalcones 5a-h was accomplished by a onepot Claisen-Schmidt condensation [21,22] between the appropriate O-allylvanillin 3 and substituted acetophenones 4a-h, as shown in Scheme 2. O-allylvanillin 3 was prepared via the nucleophilic substitution of vanillin 1 and allylbromide 2 in the presence of potassium carbonate in anhydrous acetone (Scheme 1) [22].
In all the chalcones synthesized, only the trans double bond (on the basis of coupling constant) was obtained. All synthesized compounds were characterized by spectral data (mass, UV, IR and NMR) and were consistent with the structures proposed. The purity of these compounds was ascertained by TLC and spectral analysis.

Biological studies
These synthesized compounds were evaluated for their in vitro anticancer activity using Sulforhodamine B assays [20]. A preliminary assay against leukemia THP-1 cell line showed that compounds 3,  5a, 5d, 5e, 5f, 5g and 5h (at 100 µM) as well as doxorubicin at 50 µM were able to inhibit the proliferation of more than 50% cells ( Figure  1). These samples were consequently tested in other cell lines and the results are summarized in Table 1 When regarding the structure activity relationship, it appeared that the number and position of methyl group in cycle A of the synthesized chalcones influenced their activities, compound 5g with the -CH 3 group in position C-2 being more active on almost the five cell lines than compounds 5a (without any methyl group) and 5f bearing -CH 3 group in C-3 (Table 1). However, 5e with three -CH 3 groups was less active than compound 5g and 5f (only one -CH 3 group), but more active than 5a without a -CH 3 group, clearly confirming the influence of the methylation on the activity of the chalcones studied. Also, when comparing the activity of the two most cytotoxic compounds 5g and 5f with those of the methoxylated compounds 5d and 5h, it appeared that a single methylation induced an increase in activity compared to a single methoxylation of the chalcones studied. In addition, it is also clear that, the position of -CH 3 and that of -OCH 3 groups influence the antiproliferative activities of compounds 5d and 5h. Although the compounds studied did not show very good cytotoxicity, the study provides additional information on structure-activity relationships with chalcones, that could allow future synthesis of more potent derivatives.
In future, mechanistic studies such as the effects of compound 5g on cell cycle distribution, induction of apoptosis, caspases, and the effects on mitochondrial membrane potential will be carried out to explain the mode of action on this compound.

Conclusion
In conclusion, we report here a series of new O-allylchalcone derivatives prepared by a Claisen-Schmidt condensation reaction [22] and their ability to kill tumor cells in vitro. The mechanisms of cytotoxicity underlying this process remain to be fully elucidated. Previous studies reported in the literature reveal that, flavonoids such as chalcones are known microtubule inhibitors with antimitotic activity [14]. Detailed mechanistic studies and lead optimization of these O-allylchalcone derivatives are under investigation. It is intended that results from these studies will assist in elucidating their precise mechanisms of action and provide an approach to develop new potent O-allylchalcone hybrid prototypes for further optimization and development to get new leads for the treatment of cancer.