Design, Synthesis and Evaluation of N13-Substituted Evodiamine Derivatives against Human Cancer Cell Lines

Attempting to improve the anticancer activity and solubility of evodiamine in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) solutions, thirty-eight N13-substituted evodiamine derivatives were designed, synthesized and tested for antitumor activities against six kinds of human cancer cell lines, namely prostate cancer (DU-145 and PC-3), lung cancer (H460), breast cancer (MCF-7), colon cancer (HCT-5) and glioblastoma (SF-268). The solubility of these compounds in SGF and SIF solutions was evaluated, and apoptosis induced by 2-2, 2-3, 2-16 and 3-2 was determined. The results showed: (1) among all compounds examined, 2-16 showed the highest antitumor activity and a broader spectrum of activity, with IC50 values ranging from 1–2 µM; (2) their solubility was obviously improved; (3) 2-3, 2-16 and 3-2 had a significant impact inducing apoptosis in some cancer cell lines. The preliminary structure-activity relationships of these derivatives were discussed.


Introduction
Evodiamine (1, Figure 1) is a quinolone alkaloid isolated from the fruit of Evodia rutaecarpa (Chinese name: Wu-Chu- Yu), which is one of the most popular and multi-purpose Traditional Chinese Medicines widely used for treating diverse human disorders [1] and is also very attractive as a component of healthy foods [2,3]. It was demonstrated by Hu [4] and Linag [1] that evodiamine possesses a wide range of biological activities related to anti-inflammatory, anti-obesity and antitumor properties. In particular, both the strong cytotoxicity of evodiamine against human cancer cells and the underlining mechanisms of growth inhibition, apoptosis induction and suppression of invasion and metastasis have attracted much attention [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Moreover, it was demonstrated that evodiamine sensitized chemoresistant breast cancer cells to adriamycin without obvious cytotoxicity against normal human peripheral blood cells [4,23], which indicated the potential of evodiamine for clinical application. In 2010, Sheng and his co-workers identified N13-substituted evodiamine derivatives to be potent topoisomerase I inhibitors by structure-based virtual screening and lead optimization [13]. Recently, the same group has constructed a library of new evodiamine derivatives bearing various substitutions or modified scaffold with substantially increased antitumor activity, which is attributable to the inhibition effect on topoisomerase I and II [16]. Despite its promosing anticancer potential, evodiamine is still unsuitable for clinical application because of its poor physicochemical properties.  13 14 Evodiamine (1) In this study, we were interested in using evodiamine as a lead compound to develop anticancer drug candidates, because of its: (1) known broad-spectrum antitumor activities; (2) significant difference of toxicity between cancer cells and normal peripheral blood mononuclear cells; (3) good drug-like molecule features as judged by both the criteria of Lipinski's "rule of five" and the "verb rule"; (4) good synthetic accessibility for its diversity of derivatives. In our continuous search for novel improved antitumor agents [24][25][26][27][28][29], we report herein the design, synthesis and testing of novel N13-substituted evodiamine derivatives as antitumor agents.

Synthesis of N13-Substituted Evodiamine Derivatives
The synthetic pathways to the target compounds are outlined in Schemes 1-4. The substitution reactions of evodiamine with alkyl halides or p-toluenesulfonic acid ester at the N13-position of evodiamine were completed smoothly in dried N,N-dimethylformaide (DMF) as reported in reference [13] with slight modifications.

Solubility of Evodiamine Derivatives in SGF and SIF Main Text Paragraph
We aimed to improve the aqueous solubility of evodiamine, subsequently, to promote its pharmacological or pharmacokinetic peculiarity to enhance its therapeutic effects. The solubilities of N13-position evodiamine derivatives in SGF and SIF solutions were evaluated and the results are shown in Table 1. It is interesting that the solubilities of these derivatives were obviously improved when compared to evodiamine. For example, the solubility of evodiamine itself was only 7.5-15 μg/mL and 24.8-49.5 µg/mL in SGF and SIF, respectively, while the solubilities in SGF and SIF for all evodiamine derivatives were nearly two fold that of evodiamine itself.

Activity of Evodiamine Derivatives against Human Cancer Cell Lines
To evaluate the antiproliferative effects of the evodiamine derivatives on human cancer cells, the derivatives were used to treat a variety of human cancer cell lines derived from prostate cancer (DU-145 and PC-3), lung cancer (NCl-H460), breast cancer (MCF-7), colon cancer (HCT-15) and glioblastoma (SF-268) and the growth inhibition effect was tested by MTT cytotoxicity assays using evodiamine as a reference. The results are listed in Table 1.
Moreover, we tried to make a dimer using a linker between N13-position, as shown in Table 1, which exhibited moderate (compound 3-1) to good (compound 3-2) antitumor activity against all the tested tumor cell lines. It should be very intriguing that the evodiamine derivative with a benzoylmethyl group (Table 1, compound 2-8) was inactive against the six tested tumor cell lines, although it was reported that one with a benzoyl group at N13-position showed higher antitumor activity and broad spectrum activity against A549, MDA-MB-435 and HCT116 [13].

Analysis of Anticancer Activities by Spatial Three-Dimensional Structures
In order to elucidate the effect of the spatial structure of evodiamine derivatives on anticancer activity, three-dimensional models of evodiamine, N13-(2-methoxyethyl)evodiamine, N13-(2butoxyethyl)evodiamine, and N13-(4-methoxybubyl)evodiamine were obtained using ChemBioDraw Ultra 11.0 [30] ( Figure 2).  Comparing the three-dimensional models of these four compounds, we could not find obvious differences in respect of the spatial three-dimensional structures of the evodiamine moiety, that is, the spatial three-dimensional structure of evodiamine was not changed due to the introduction of alkoxy groups. Therefore, it could be speculated that the reason leading to the difference in the anticancer activities of these four compounds was the differences in the structures of substituents.

Apoptosis of Human Cancer Cell Lines Induced by Evodiamine Derivatives
The significant difference of antitumor potency resulting from such subtle change in the structures encouraged us to further explore their antitumor mechanism. Hence, compounds 2-2, 2-3, 2-16 and 3-2 were chosen for testing their effect on inducing apoptosis in breast MCF-7, lung NCl-H460 and colon HCT-15 cancer cell lines.
Apoptosis was determined by flow cytometry analysis using the Annexin V-FITC/PI Apoptosis Kit (Becton Dickinson, Franklin Lakes, NJ, USA) and the results are presented in Figure 3 and in Table 2. As shown in Figure 3, compounds 2-3 and 3-2 strongly induced early apoptosis in MCF-7 with the percentages increasing from 3.21% (medium control) to 84.58% and 55.62%, respectively, while 2-16 and 3-2 obviously induced late apoposis in HCT-15 with the percentages increasing from 3.55% (medium control) to 59.01% and 65.67%, respectively. These two compounds also induced late apoptosis in NCl-H460 with a moderate intensity with the percentages increasing to 40%-50%.

General
Evodiamine was purchased from Aladdin Industrial Inc. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker Avance 300 spectrometer (Bruker Company, Saarbrücken, Germany) using CDCl 3 or DMSO-d 6 as solvent, chemical shifts were given in ppm (δ). Elemental analyses were performed with a Vario EL cube instrument (Elementar Company, Hanau, Germany). The mass spectra were recorded on a LCQ DECA XP LC-MS mass spectrometer (Thermo Company, Waltham, MA, USA) and high resolution mass spectra were recorded on a MAT95XP High Resolution Mass Spectrometer (Thermo Company). X-ray data were collected on a Smart 1000 CCD Single Crystal Diffractometer (Bruker).

Solubility of N13-Substituted Evodiamine Derivatives in SGF and SIF Solutions
SGF and SIF solutions were prepared as described in the United States Pharmacopoeia [31], and the solubilities of N13-substituted evodiamine derivatives in SGF and SIF solutions were determined by the reported method [32][33][34][35] with slight modifications.

Inhibitory Effects of N13-Substituted Evodiamine Derivatives toward Six Cultured Human Cancer Cell Lines
The growth inhibition effects of the thirty N13-substituted evodiamine derivates on human prostate cancer cell lines (DU-145 and PC-3), lung cancer cell line (H460), breast cancer cell line (MCF-7), colon cancer cell line (HCT-5) and glioblastoma cell lines (SF-268) were determined by MTT assay with evodiamine as positive control.