Synthesis and Anticancer Activities of Glycyrrhetinic Acid Derivatives

A total of forty novel glycyrrhetinic acid (GA) derivatives were designed and synthesized. The cytotoxic activity of the novel compounds was tested against two human breast cancer cell lines (MCF-7, MDA-MB-231) in vitro by the MTT method. The evaluation results revealed that, in comparison with GA, compound 42 shows the most promising anticancer activity (IC50 1.88 ± 0.20 and 1.37 ± 0.18 μM for MCF-7 and MDA-MB-231, respectively) and merits further exploration as a new anticancer agent.


Introduction
Breast cancer is one of the most common diseases amongst women throughout the world. About 521,900 women lost their lives because of it in 2012, 197,600 of which were from developed countries and 324,300 from developing countries [1]. In 2016, 249,260 new breast cancer cases and 40,890 breast cancer deaths are projected to occur in the United States [2]. A current hot research topic is how to develop novel therapeutics with improved selectivity and higher anticancer activity by chemical modifications of natural sources. Up to now, a large number of chemotherapeutic agents derived from natural products and used for the treatment of cancer, have shown satisfactory therapeutic effects, like vinblastine, vincristine, the camptothecin derivatives, e.g., topotecan, irinotecan and etoposide, were derived from epipodophyllotoxin and paclitaxel [3].
Terpenoids are the largest group of natural compounds found in plants. Among terpenoids, a large number of triterpenoids exhibit cytotoxicity against a variety of tumor cells as well as anticancer efficacy in preclinical animal models [4][5][6]. Those triterpenoids regulate tumor cell proliferation, transformation, survival, invasion, angiogenesis, metastasis, chemoresistance and radioresistance [7]. As a kind of triterpenoid, glycyrrhetinic acid (GA) has many valuable pharmacological properties, such as antiviral [8,9], anti-allergic [10], anti-inflammatory [11,12], anti-ulcer [13] and anticancer activity [14,15]. It has been reported that GA exhibited selective toxicity to varieties of tumor cells, making it an ideal lead compound for anticancer treatment [16,17]. Ferulic acid as a natural product that also has apparent biological activities like antibacterial [18], anti-inflammatory [19,20] and anticancer properties [21]. To improve the cytotoxicity of GA, many researchers have tried to enhance its potency by various derivatizations. Some studies have shown that the addition of lipophilic fragments to antitumor molecules could increase their anticancer activity [22][23][24][25]. In addition, natural products which were conjugated with amino acids provide improved bioacitivity [26][27][28]. In this study, based on the pro-drug principle and the previously reported therapeutic potential of GA, we designed and synthesized a series of novel GA derivatives in which the 30-carboxyl group was coupled with lipophilic fragments (ferulic acid analogs) and the 3-hydroxyl group was coupled with amino acids (L-methionine or L-selenomethionine) to improve the anticancer potency of GA.
In totally, forty derivatives of GA were successfully synthesized and and their structures characterized by 1 H-NMR, 13 C-NMR, MS and elemental analysis. Their in vitro anticancer activities were then tested, using a MTT assay, against two human breast cancer cell lines  and one normal human retinal pigment epithelial cell line . Most of the derivatives exhibited much stronger inhibitory activity than GA against those two breast cancer cell lines (but lower than the positive control doxorubicin) and relatively lower inhibitory activity against normal cells. More importantly, one derivative, compound 42 (see Section 2 below), showed significantly stronger cytotoxicity against both MCF-7 cells and MDA-MB-231 cells than GA itself. Our data suggested that coupling lipophilic fragments (especially ferulic acid methyl ester) and amino acids (especially L-selenomethionine) to GA is a promising approach to generate highly active anticancer compounds. Further SAR development is in progress to discover more potential lead antitumor drugs.

Chemistry
The synthesis of new GA derivatives 17-56 was carried out according to the steps shown in Scheme 1.
Molecules 2016, 21, 199 2 of 19 designed and synthesized a series of novel GA derivatives in which the 30-carboxyl group was coupled with lipophilic fragments (ferulic acid analogs) and the 3-hydroxyl group was coupled with amino acids (L-methionine or L-selenomethionine) to improve the anticancer potency of GA. In totally, forty derivatives of GA were successfully synthesized, and their structures characterized by 1 H-NMR, 13 C-NMR, MS and elemental analysis. Their in vitro anticancer activities were then tested, using a MTT assay, against two human breast cancer cell lines (MCF-7 and MDA-MB-231) and one normal human retinal pigment epithelial cell line . Most of the derivatives exhibited much stronger inhibitory activity than GA against those two breast cancer cell lines (but lower than the positive control doxorubicin) and relatively lower inhibitory activity against normal cells. More importantly, one derivative, compound 42 (see Section 2 below), showed significantly stronger cytotoxicity against both MCF-7 cells and MDA-MB-231 cells than GA itself. Our data suggested that coupling lipophilic fragments (especially ferulic acid methyl ester) and amino acids (especially L-selenomethionine) to GA is a promising approach to generate highly active anticancer compounds. Further SAR development is in progress to discover more potential lead antitumor drugs.

Chemistry
The synthesis of new GA derivatives 17-56 was carried out according to the steps shown in Scheme 1. All these derivatives are new compounds which were not previously reported. The lipophilic fragments 5-12 were obtained by the treatment of 1-4 with methanol or ethanol catalyzed by concentrated sulfuric acid. To avoid the formation of byproducts, we used the t-butyloxycarbonyl (Boc-) group to protect amino acids and thus obtained compounds 15-16, which were used in the next step without further purification.
Compounds 17-24 were obtained through the formation of an ester bond between compounds 5-12 and GA after stirring for 12 h at room temperature in dry dichloromethane (DCM) catalyzed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylamino-pyridine (DMAP). We applied the same method to introduce the N-Boc group to protect amino acids at position C-3 and obtained compounds 25-40. To get compounds 41-56, deprotection was performed by treating the compounds with dry HCl gas in DCM.

Anticancer Activities
The anticancer activities of compounds 17-24, 41-56 against MCF-7, MDA-MB-231 and hTERT-RPE1 cells were determined in vitro by an MTT assay. Doxorubicin was included in the experiments as positive control, and the IC 50 of GA is also presented to compare the anticancer activities. The data was calculated and presented as IC 50 values in All values are given as means˘standard deviation. a IC 50 is the drug concentration effective in inhibiting 50% of the cell growth measured by MTT method. b The drug doxorubicin (ADR) was used as positive control in this study.

General Information
All the chemicals and reagents were commercially available and used without further purification. Routine thin-layer chromatography (TLC) was performed on silica gel plates (silica gel GF254 from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and visualization was performed using UV. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker-400 instrument (Bruker, Billerica, MA, USA) at room temperature with TMS as an internal standard and CDCl 3 or DMSO-d 6 as solvents. Chemical shifts are expressed in δ (ppm) and coupling constants (J) in Hz. Mass spectra were recorded with a MSQ Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). Melting points were measured by an SGWX-4 micro melting point apparatus (Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, China) and are uncorrected.

General Method for Synthesizing Compounds 5-12
Ferulic acid (1) or trans-4-hydroxycinnamic acid (2) or isoferulic acid (3) or trans-3-hydroxycinnamic acid (4) and five drops of H 2 SO 4 (95%) were refluxed in methanol or ethanol for 12 h. The reaction mixture was concentrated in vacuo and the residue was dissolved in ethyl acetate. The organic layer was washed with a 5% aqueous NaHCO 3 solution and water. After drying over anhydrous Na 2 SO 4 , the ethyl acetate was removed in vacuo. The residue was purified by column chromatography on silica gel using ethyl acetate/petroleum ether mixtures as eluents to afford compounds 5-12.

General Method for Synthesizing Compounds 15-16
The appropriate amino acid (13 or 14, 1 equiv.) and sodium bicarbonate (3 equiv.) was dissolved in a 1:1 mixture of water and 1,4-dioxane. Di-tert-butyl dicarbonate (1.2 equiv.) was added and the mixture was stirred at room temperature for 12 h. The 1,4-dioxane was removed under reduced pressure and the mixture was extracted with ethyl acetate. Then the solution was acidified using 1 M hydrochloric acid solution and extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and the solvent was evaporated. The crude protected amino acids 15-16 were used without any further purification.

General Method for Synthesizing Compouds 17-24
GA (485 mg, 1 mmol) was dissolved in dry DCM (30 mL) and stirred at room temperature for 5 min. Then EDCI (230 mg, 1.2 mmol), DMAP (24 mg, 0.2 mmol) and compounds 5-12 (1 mmol) were added to the solution, and then the reaction mixture was stirred at room temperature for 12 h. The organic layer was washed with 1 M HCl solution and concentrated in vacuo. The residue was purified by column chromatography on silica gel with ethyl acetate/petroleum as eluent to yield pure compounds 17-24.