Novel Lycorine Derivatives as Anticancer Agents: Synthesis and In Vitro Biological Evaluation

Lycorine, which is the most abundant alkaloid isolated from the Amaryllidaceae family of plants, reportedly exhibits promising anticancer activities. Herein, a series of novel lycorine derivatives were synthesized and evaluated for their in vitro inhibitory activities against seven different cancer cell lines, including A549, HCT116, SK-OV-3, NCI-H460, K562, MCF-7 and HL-60. The results indicated that compounds bearing diverse amine substituents at the C-2 position demonstrated good anticancer activities. The selectivity towards different cancer cell lines of the synthesized derivatives is discussed.


Introduction
Cancer is a proliferation disorder disease with apoptosis obstacles. It strikes more than one-third of the World's population and causes over 20% of all deaths [1]. The standard cancer treatment protocols include surgery, radiotherapy and chemotherapy. Unfortunately, chemotherapy is not effective in treating cancers associated with innate resistance to apoptosis and/or acquired resistance to drugs during treatment [2]. Discovery of novel effective anticancer medicines is therefore of great importance. As a source of drugs, natural products play an important role in drug discovery. Up to 80% OPEN ACCESS of approved anticancer drugs over the time frame from 1981 to 2010 derived from natural products [3,4]. Plant species belonging to the Amaryllidaceae family have been well known for centuries to exhibit diverse medicinal properties. Many of these plants have been used historically for the treatment of cancer, since the oil of the daffodil Narcissus poeticus L. was used by Hippocrates of Kos [5]. The application of narcissus oil in cancer management continued during the middle ages in China, North Africa and Central America [6]. The major components isolated from the Amaryllidaceae plant family are alkaloids. Hundreds of alkaloids have been isolated and extensively studied for their pharmacological properties since 1877 [7]. Lycorine is one of the most common alkaloids within Amaryllidaceae family of plants and reportedly exhibits promising anticancer properties [7][8][9][10][11][12].
Lycorine has displayed multiple inhibitory properties towards various cancer cell lines, including lymphoma [7], carcinoma [8], multiple myeloma [9], melanoma [10], leukemia [7,11], human U373 glioblastoma, human A549 non-small-cell-lung cancer, human OE21 esophageal cancer and human Hs683 anaplastic oligodendroglioma cell lines [12]. Previous studies revealed that lycorine exhibited significantly higher antiproliferative activities in cancer cells than in normal cells, and it could be used to combat cancer cells which may or may not be sensitive to proapoptotic stimuli [12]. Further studies provided a mechanistic insight into its anticancer properties. Evdokimov et al. reported that lycorine exhibited cytostatic effects by targeting the actin cytoskeleton rather than by inducing apoptosis in cancer cells [13]. However, lycorine was found to induce apoptosis as well as arrest cell cycle of HL-60, KM3, K562, Hey1B cells in different phases [7,[14][15][16]. Recently, the inhibitory effect on vasculogenic mimicry of melanoma cells [17] and suppression of neovascularization of ovarian cancer Hey1B cells by lycorine were reported [16]. Many factors thus seem involved in the anticancer properties of lycorine and a clear mechanism remains to be determined. Nevertheless, the anticancer properties of lycorine have drawn significant attention from chemists [5,6,[11][12][13]. In the present study we report the synthesis of some novel lycorine derivatives and the evaluation of their in vitro anticancer activities.

Chemistry
The hydrochloride salt of lycorine (1) is commercially available, but its solubility is poor in most organic solvents. Therefore, 1, 2-diacetyllycorine (2) obtained from the acetylation of 1 with Ac 2 O/Py was used as the key intermediate for further modifications.
Several derivatives were synthesized to investigate the influences of different substituents at the C-2 position. As outlined in the Scheme 1, the intermediate 3 was prepared via the selective deacetylation of the C-2 hydroxyl group using conc. HCl in methanol since the C-2 hydroxyl group is more reactive than the C-1 hydroxyl group [13]. The C-2 hydroxyl of 3 was then coupled with diverse acyl chlorides to afford derivatives 7a-e. Alternatively, the replacement of the C-2 hydroxyl group by chloride using phosphorus oxychloride as halogenating agent provided 1-acetyl-2-chlorolycorine (4), which was further treated with sodium methoxide to produce 1,2-α-epoxyllycorine (8). Compounds 9a-e were obtained via the opening of the epoxide using the corresponding amines. The nucleophilic attack of the epoxide should occur from the least hindered side with high regioselectivity to afford 9a-e, according to a previous report [18]. This conclusion was confirmed using NOESY analysis. Taking 9b as an example, a correlation peak was found for H11b/H1, but no correlation between H11b/H2 was observed ( Figure 1). Oxidation of the double bond in D-ring with m-CPBA led to derivatives 5 and 6. Scheme 1. Synthesis of lycorine derivatives.

Biological Activity Results
In vitro anticancer activities of the synthesized lycorine derivatives were evaluated using the MTT colorimetric assay against a panel of seven human cancer cell lines [19], including non-small-cell-lung cancer (A549), colon carcinoma (HCT116), ovarian carcinoma (SK-OV-3), large-cell-lung cancer (NCI-H460), human myelogenous leukemia (K562), promyelocytic leukemia (HL-60) and human breast adenocarcinoma (MCF-7) cell lines. The results were expressed as IC 50 for the inhibitory activities and are summarized in Table 1.  [12]. In our study, 8 also presented excellent potency against all tested cell lines. However, compared with 2 and 3, epoxides 5 and 6 derived from 2 and 3, respectively, showed much reduced activity (>20 μM) against almost all seven cell lines. The results indicated that an epoxide ring at C3-C4 didn't benefit the anticancer activities and the presence of the double bond of A-ring appeared to be essential for the potency.
Esters 7a-e were synthesized to investigate the influence of modifications of the C-2 hydroxyl group. In general, 7a, 7b, and 7c exhibited moderate potency against all of tested cell lines, except K562 and HL-60, while 7e demonstrated moderate potency against all of the tested cell lines except MCF-7 and HL-60. Interesting selectivity was observed with ester substituents as well. Generally, esterification of the C-2 hydroxyl group with aliphatic or aromatic acids slightly increased the activities for the A549 and SK-OV-3 cell lines, but decreased the activities for HCT116, NCI-H460, K562 and MCF-7 at different levels (2, 7a, 7b, 7c, 7e vs. 3). For the HCT116 and NCI-H460 cell lines, both the aliphatic (2, 7a, 7b, 7c) and the aromatic substituent (7e) at the C-2 position appeared to be tolerated well, but for the K562 cell line, an aromatic substituent (compound 7e) exhibited better activities than linear aliphatic substituents (2, 7a, 7b). However, the results were the opposite for the MCF-7 cell line. It is noteworthy that 7d with a carboxylic acid terminus lost its activity against all of the tested cell lines. The decrease in activities for 7d might result from the ionic nature of the carboxylate group and the significant change of cLog P. In a previous report [20], cell permeability was an important determinant for anticancer activities of lycorine derivatives. It is likely that this observation reflects the difficulty of cell penetration for highly hydrophilic lycorine derivatives like 7d. In another variation, the hydroxyl group at the C-2 position was replaced by a chlorine to afford 4.
This replacement led to the dramatic decrease in potency against all of the tested cell lines (>20 μM).
To further examine the influence of substituents at the C-2 position, a variety of amines (alkylamine, piperidine, pyrrole) were introduced to yield 9a-e via the opening of the epoxide of 8. Overall, these derivatives exhibited higher potency than esters 7a-e. From the perspective of hydrogen-bond acceptor/donor, tertiary amino substituents at the C-2 position of compounds 9a-c could be an acceptor, while the secondary amino substituents of compounds 9d-e could be both an acceptor and a donor. No clearly difference was observed between the activities of 9a-c and 9d-e towards all of tested cell lines. Thus, a hydrogen donor at this position might be less important for the anticancer activities than a hydrogen acceptor. Lycorine exhibited similar good activities towards all of tested cell lines, while amine substituents at the C-2 position improved the selectivity to different cancer cell lines. Aliphatic substituents (compounds 9a, 9b, 9d) exhibited better activities than aromatic substituent (9c) for A549 and HCT116 cell lines. For SK-OV-3, 9a (IC 50 = 4.5 μM) represented comparable activity with lycorine (IC 50 = 3.0 μM). The compound with a linear aliphatic substituent 9d provided the best activities for NCI-H460 (IC 50 = 6.5 μM) and K562 (IC 50 = 6.1 μM, which is better than that of lycorine, IC 50 = 7.5 μM) while the bulky aliphatic substituent derivative 9b demonstrated the best potency against HCT116 (IC 50 = 6.4 μM) and MCF-7 (IC 50 = 5.2 μM).

General
All solvents were treated by standard techniques before use. Lycorine hydrochloride (purity > 98%) was purchased from Baoji Guokang Biotechnology Co. Ltd (Baoji, China). Cell culture medium, fetal bovine serum, and trypsin were purchased from Thermo Scientific (Waltham, MA, USA). All reagents were purchased from the commercial suppliers and were used without further purification. 1 H-and 13 C-NMR spectra were recorded on a Bruker AVANCE-400 (400 MHz) NMR spectrometer (Billerica, MA, USA) ( 1 H, 400 MHz; 13 C, 100 MHz) in CDCl 3 , CD 3 OD using TMS as an internal standard. Chemical shifts are reported in parts per million δ in ppm and coupling constants are provided in hertz (Hz). ESI-MS spectra were recorded on a Shimadzu LCMS-2020 (Shimadzu, Kyoto, Japan). HRMS were recorded on a Varian 7.0 T high resolution ESI-FTICR mass spectrometer (Palo Alto, CA, USA). Silica gel TLC (GF254) was used to monitor the reactions, which were visualized under UV light at 254 nm and 365 nm. Flash chromatography was performed using silica gel (200-300 mesh).

Cell Lines
The cell lines used in this study were purchased from the Shanghai Cell Bank, Chinese Academy of Medical Sciences (Shanghai, China). All cell lines were maintained in a humidified atmosphere containing 5% CO 2 at 37 °C. All the cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were seeded into 96 well plates in 100 μL at plating density of 2 × 10 3 cells per well. After incubation for 24 h, the cells were allowed to attach. The cells were treated with different concentrations of compounds and then incubated for 72 h under the same conditions. Compounds were dissolved in DMSO at stock concentrations of 20 mM.

In Vitro Growth Inhibition Assay
The concentrations which were used to calculate the IC 50 values were: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 μM. The stock solutions were diluted with complete medium before use. Untreated cells were used as controls. The cells were then treated for 72 h with varying concentrations of compounds. 20 μL of 3-(4,5 dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT, Sigma, St. Louis, MO, USA) solution (5 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C. After the removal of the medium, MTT formazan was dissolved in 150 μL DMSO and monitored using a microplate reader at a wavelength of 570 nm. All experiments were performed in triplicate wells and were repeated at least three times.

General Procedure for the Preparation of 9
Compound 8 was dissolved in amine (5 mL, piperidine, diethylamine, pyrrole, n-butylamine, isopropylamine for 9a-e, respectively), and the solution was heated to reflux overnight. The amine was removed under reduced pressure and the crude residue was purified using silica gel chromatography to give the products.