Chem Biol Drug Des 2014 Research Letter

Synthesis and Antitumor Activity of Natural Compound Aloe Emodin Derivatives Naraganahalli R.Thimmegowda1,2,†, Chanmi Park1,†, Bettaswamigowda Shwetha1, Krisada Sakchaisri1, Kangdong Liu1,3, Joonsung Hwang1, Sangku Lee1, Sook J. Jeong1, Nak K. Soung1, Jae H. Jang4, In-Ja Ryoo4, Jong S. Ahn4, Raymond L. Erikson5 and Bo Y. Kim1,* 1

World Class Institute (WCI), Incurable Diseases Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Cheongwon 363-883, Korea 2 Department of Chemistry, Govt.S K S J T Institute, Bangalore, Karnataka 560001, India 3 Basic Medical College, Zhengzhou University, ZhengZhou 450001, China 4 Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Cheongwon 363-883, Korea 5 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA *Corresponding author: Bo Y. Kim, [email protected] † The first two are equal contributors to this work. In this study, we have synthesized novel water soluble derivatives of natural compound aloe emodin 4(a–j) by coupling with various amino acid esters and substituted aromatic amines, in an attempt to improve the anticancer activity and to explore the structure–activity relationships. The structures of the compounds were determined by 1H NMR and mass spectroscopy. Cell growth inhibition assays revealed that the aloe emodin derivatives 4d, 4f, and 4i effectively decreased the growth of HepG2 (human liver cancer cells) and NCI-H460 (human lung cancer cells) and some of the derivatives exhibited comparable antitumor activity against HeLa (Human epithelial carcinoma cells) and PC3 (prostate cancer cells) cell lines compared to that of the parent aloe emodin at low micromolar concentrations. Key words: aloe emodin, aloe emodin derivatives, antitumor activity, NCI-H460 cells, Hep G2 cells, structure activity relationship Abbreviations: CH2Cl2, dichloro methane; DMA, N,N-dimethyl acetamide; DMSO, dimethyl sulfoxide; KI, potassium iodide; mTORC2, mammalian target of rapamycin complex 2; NMR, nuclear magnetic resonance; TLC, thin-layer chromatography. ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12448

Received 8 July 2014, revised 1 September 2014 and accepted for publication 7 October 2014

Despite the intensive efforts and substantial advances that have occurred through focusing on improving treatments, cancer is still a leading cause of death worldwide. Several plant-derived compounds are currently successfully employed in cancer treatment. Natural compounds and their synthetic and semisynthetic analogs have served as a major route to new pharmaceuticals. Aloe emodin is a hydroxyanthraquinone naturally present in the leaves of Aloe vera (1–3). Aloe emodin and its derivatives were found to be potential anticancer agents (1,4–8). Our research group previously reported the inhibitory activity of natural compound aloe emodin against prostate cancer growth by targeting mTORC2 (4). In an effort to further develop this promising compound toward a potent anticancer agent, we have focused on the synthesis of novel water soluble aloe emodin derivatives to improve the potency and to examine the structure–activity relationships (SAR) for aloe emodin.

Experimental Section Chemistry All of the solvents and reagents used were obtained commercially and used as such unless noted otherwise. Dichloro methane solvent was freshly distilled over LiOH. Aloe emodin (>95% purity) was purchased from Sigma-Aldrich (St Louis, MO, USA). Moisture or air-sensitive reactions were conducted under nitrogen atmosphere in oven-dried glass apparatus. Reaction progress was monitored by thin-layer chromatography (TLC) on precoated silica gel plates (Kieselgel 60F254; Merck, White House Station, NJ, USA) and visualized by UV254 light or by treatment with phosphomolybdic acid and 10% ethanolic H2SO4. The solvents were removed under reduced pressure using standard rotary evaporators. Purification of products carried out by silica gel (particle size 40–63 lm; Merck) flash column chromatography. All NMR spectra were recorded on a Varian, Unity 300 and Inova 400 MHz spectrometer in Methanol-d4 and DMSO-d6 NMR solvents. The data are given as follows: chemical shift (d) in ppm. ESI-MS analyses were performed on a Finnigan MAT, Navigator.

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Synthesis of 3-(bromomethyl)-1,8dihydroxyanthracene-9,10-dione (2) The compound 2 was prepared by a procedure as reported (9). A suspension of aloe emodin (1 g, 3.7 mmol) in 48% hydrobromic acid was refluxed for 5 h. The reaction was monitored by TLC. Upon completion, the reaction was allowed to cool to room temperature, and the solid was filtered, washed with water, and finally recrystallised from acetic acid. 0.85 g (yield: 69%) of the desired product was obtained as a bright yellow solid. Rf = 0.86 (EtOAc–hexane 1:1); 1H NMR (300 MHz, DMSO-d6) d: 4.82 (2H, s), 7.39–7.48 (2H, m), 7.73–7.86 (3H, m), 11.93 (2H, s).

General procedure for the synthesis of aloe emodin derivatives 3(a–j) and 4(a–j) The corresponding amine (five equiv) dissolved in N,Ndimethyl acetamide solvent (10 volumes), cesium carbonate (three equiv), and catalytic amount of potassium iodide were added to it, and the resulting mixture was stirred for 30 min at room temperature, and then, compound 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (1.2 equiv) was added to it, and the resulting reaction mixtures were stirred under nitrogen atmosphere for 5–10 h at room temperature. The reaction was monitored by TLC, and upon completion, the product was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, then filtered, and concentrated. The resulting residue was purified by silica gel flash chromatography using different ratios of ethyl acetate/hexane and dichloromethane/methanol mobile phase. The corresponding purified compounds 3(a–j) were dissolved in dichloromethane cooled to 0 °C then added hydrogen chloride solution (2 M in diethyl ether), and it was stirred for 30 min at 0 °C and then at room temperature for 5–10 h. The reaction was monitored by TLC, and upon completion, the resulting precipitate was collected by filtration and dried under vacuum (Scheme 1).

OH O

OH

OH O

Synthesis of (S)-methyl 2-(((4,5-dihydroxy-9,10-dioxo9,10-dihydroanthracen -2-yl)methyl) amino)-3(1H-indol-3-yl)propanoate hydrochloride (4a). The compound 4a was synthesized from 3-(bromomethyl)-1,8dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), L-tryptophan methyl ester hydrochloride (95.6 m, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield: 32% (12 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 471.3 [M + H]+. 1H NMR (300 MHz, Methanol-d4) d: 3.45–3.49 (2H, m), 3.77 (3H, s), 4.30–4.41 (3H, m), 6.95–7.02 (2H, m), 7.21–7.26 (2H, m), 7.33–7.41 (3H, m), 7.75–7.84 (3H, m). Synthesis of (S)-diethyl 2-(((4,5-dihydroxy-9,10-dioxo9,10-dihydroanthracen -2-yl)methyl) amino) pentanedioate hydrochloride (4b). The compound 4b was synthesized from 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), L-glutamic acid diethyl ester hydrochloride (90 mg, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield:43% (16 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 456.3 [M + H]+. 1H NMR (300 MHz, Methanol-d4) d: 1.23–1.28 (3H, t), 1.330–1.38 (3H, m), 2.22–2.41 (2H, m), 2.53–2.66 (2H,), 4.12–4.26 (3H, m), 4.31–4.45 (4H, m), 7.34–7.37 (1H, dd), 7.48–7.49 (1H, d), 7.75–7.82(2H, m), 7.93–7.93 (1H, d). Synthesis of (S)-methyl 2-(((4,5-dihydroxy-9,10-dioxo9,10-dihydroanthracen -2-yl)methyl) amino)-3-phenylpropanoate hydrochloride (4c). The compound 4c was synthesized from 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), L-phenylalanine methyl ester hydrochloride (80.9 mg, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen

OH

OH

OH

ii

i OH O 1 (Aloe emodin)

O

H N

Br O 2

R

O 3(a-j) iii OH

O

OH H N

O 4 (a-j)

R HCl

Scheme 1: Synthetic route to aloe emodin derivatives 4(a–j). Reagents and conditions: (i) 48% HBr, 100 °C, 5 h; (ii) R-NH2, Cs2CO3, KI, N,N-dimethyl acetamide, rt, 5–10 h; (iii) CH2Cl2, HCl in ether, 0 °C to rt, 5–10 h.

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Aloe Emodin Derivatives

chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield: 40% (14 mg), Yellow solid. ESI-MS revealed quasi-molecular ion peak at m/z 432.3 [M + H]+.1H NMR (300 MHz, Methanol-d4) d: 3.17–3.25 (1H, q), 3.39–3.47 (1H, q), 3.72 (3H, s), 4.36–4.40 (3H, m), 7.25– 7.39 (6H, m), 7.44 (1H, s), 7.75–7.82 (2H, m), 7.89 (1H, s). Synthesis of (S)-methyl 2-(((4,5-dihydroxy-9,10-dioxo9,10-dihydroanthracen -2-yl)methyl) amino)-3-hydroxypropanoate hydrochloride (4d). The compound 4d was synthesized from 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), L-serine methyl ester hydrochloride (58.4 mg, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield: 29% (9 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 372.3 [M + H]+. 1H NMR (300 MHz, Methanol-d4) d: 3.89 (3H, s), 4.09–4.11 (2H, d), 4.25–4.27 (1H, t), 4.42 (2H, s), 7.35–7.38 (1H, dd), 7.49– 7.50 (1H, d), 7.76–7.85 (2H, m), 7.96–7.96 (1H, d). Synthesis of (S)-methyl 2-(((4,5-dihydroxy-9,10-dioxo9,10-dihydroanthracen -2-yl)methyl) amino)-2-phenylacetate hydrochloride (4e). The compound 4e was synthesized from 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), L-phenylglycine methyl ester hydrochloride (75.6 mg, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedure 4.1.2. Yield: 53% (18 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 418.3 [M + H]+. 1H NMR (300 MHz, Methanol-d4) d: 3.83 (3H, s), 4.217–4.26 (1H, d), 4.36–4.40 (1H, d), 5.34 (1H, s), 7.35–7.42 (2H, dd), 7.54 (5H, s), 7.76–7.83 (2H, m), 7.88–7.89 (1H, d). Synthesis of ethyl 3-(((4,5-dihydroxy-9,10-dioxo-9,10dihydroanthracen-2-yl)methyl)amino) propanoate hydrochloride (4f). The compound 4f was synthesized from 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), b-alanine ethyl ester hydrochloride (57.6 mg, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield: 30% (9 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 370.3[M + H]+. 1H NMR (400 MHz, Methanol-d4) d: 1.26–1.29 (3H, t), 2.81–2.84 (2H, t), 3.38–3.41 (2H, t), 4.18–4.23 (2H, q), 4.38 (2H, s), 7.36–7.38 (1H, dd), 7.49–7.48 (1H, d), 7.77–7.84 (2H, m), 7.94–7.94 (1H, d). Chem Biol Drug Des 2014

Synthesis of 1,8-dihydroxy-3-(((2-(5-hydroxy1H-indol-3-yl)ethyl)amino) methyl)anthracene -9,10dione hydrochloride (4g). The compound 4g was synthesized from 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), serotonin hydrochloride (79.8 mg, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield: 55% (19 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 429.3[M + H]+. 1H NMR (300 MHz, Methanol-d4) d: 3.09–3.14 (2H, q), 3.35–3.40 (2H, t), 4.31 (2H, s), 6.56–6.59 (1H d), 6.80–6.80 (1H, d), 7.09–7.12 (2H, m), 7.34–7.39 (2H, m), 7.75–7.81 (3H, m). Synthesis of 1,8-dihydroxy-3-(((2-(pyridin-2-yl)ethyl) amino)methyl) anthracene-9,10-dione hydrochloride (4h). The compound 4h was synthesized from 3(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), 2-(2-Aminoethyl)pyridine (45 lL, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield: 32% (10 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 375.3[M + H]+. 1H NMR (300 MHz, Methanol-d4) d: 3.44–3.54 (2H, m), 3.61–3.66 (2H, m), 4.45 (2H, s), 7.36–7.39 (1H, dd), 7.55–7.55 (1H, d), 7.77–7.98 (5H, m), 8.40–8.46 (1H, m), 8.77–8.79(1H, d). Synthesis of 1,8-dihydroxy-3-(((2-(pyridin-3-yl)ethyl) amino)methyl) anthracene-9,10-dione hydrochloride (4i). The compound 4i was synthesized from 3(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), 3-(2-Aminoethyl)pyridine (45 lL, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedure 4.1.2. Yield: 36% (11 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 375.3[M + H]+. 1H NMR (300 MHz, Methanol-d4) d: 3.34–3.40 (2H, m), 3.52–3.57 (2H, t), 4.43 (2H, s), 7.35–7.38 (1H, dd), 7.54–7.54 (1H, d), 7.76–7.84 (2H, m), 7.96–7.97 (1H, d), 8.08–8.13 (1H, t), 8.64–8.66 (1H, d), 8.82 (1H, d), 8.94 (1H, s). Synthesis of 1,8-dihydroxy-3-(((4-methoxybenzyl) amino)methyl)anthracene-9,10-dione hydrochloride (4j). The compound 4j was synthesized from 3-(bromomethyl)-1,8-dihydroxyanthracene-9,10-dione (2) (25 mg, 0.075 mmol), 4-Methoxy benzyl amine (49 lL, 0.375 mmol), cesium carbonate (73.3 mg, 0.225 mmol), and hydrogen chloride solution (2 M in diethyl ether) as reactants using general procedures. Yield: 59% (19 mg), Yellow solid. ESI-MS revealed quasimolecular ion peak at m/z 390.3[M + H]+. 1H NMR 3

Thimmegowda et al.

(300 MHz, Methanol-d4) d: 3.80 (3H, s), 4.26–4.32 (4H, d), 7.01 (2H, s), 7.43 (4H, s), 7.80 (3H, s).

Cell culture and cell growth inhibition assay PC3 and NCI-H460 cancer cells were purchased from ‘American Type Culture Collection’ (ATCC, Manassas, VA, USA). Cyto XTM obtained from ‘LPS solution,’ penicillin/streptomycin was from ‘cellgro,’ and 10% fetal bovine serum was purchased from ‘Gibco,’ United States. PC3 (prostate cancer) cells were propagated in F-12K Nutrient mixture (Gibco, Grand Island, NY, USA). HeLa and HepG2 cells were maintained in DMEM/high glucose (Hyclone, Logan, UT, USA). NCI-H460 cells were maintained in RPMI-1640 medium (Hyclone). All media contain 100 U penicillin/100 lL/mL streptomycin (Cellgro, Manassas, VA, USA) and 10% fetal bovine serum (Gibco, USA) at 37 °C in a humidified incubator with 5% CO2. All the cells were maintained within 15 days.

substituted aromatic amines in the presence of cesium carbonate, potassium iodide and N,N-dimethyl acetamide by nucleophilic substitution reaction afforded the corresponding alkylamino derivatives 3(a–j). Compounds 3(a–j) were purified by silica gel flash column chromatography using different ratios of ethyl acetate/hexane and dichloromethane/methanol as mobile phase. Finally, the compounds 3(a–j) were converted to their corresponding hydrochloride salts 4(a–j) (Table 1) by treating with 2 M hydrogen chloride solution in diethyl ether. The structures of the compounds were determined by H NMR and mass spectroscopy (Supporting Information).

Biological activities The cell growth inhibition of the target compounds 4(a–j) were evaluated over HepG2 (human liver cancer cells), NCIH460 (human lung cancer cells), PC3 (prostate cancer cells), and HeLa cells (Human epithelial carcinoma cells) by MTS assay using Cyto XTM in comparison with aloe emodin (Table 2).

MTS assay Human prostate cancer PC3 cells, human epithelioid cervical carcinoma HeLa cells, and human hepatocellular carcinoma HepG2 cells were grown in DMEM medium. Human large lung cancer NCI-H460 cells were cultivated in RPMI1640 media. All medium contained 100 U penicillin/ (100 lL/mL) streptomycin (cellgro) and 10% fetal bovine serum (Gibco, USA) at 37 °C in a humidified incubator with 5% CO2. Same condition and materials were applied after experimental process.

As shown in Table 2, some of the aloe emodin derivatives exhibited better antitumor activity than the aloe emodin. For instance, compounds 4d, 4f, and 4i showed more potent inhibitory effect against HepG2 cells, with the IC50 values of 4.789, 11.069 and 15.593 lM, respectively, compared to aloe emodin (IC50 = 26.168 lM). Compounds 4d, 4f, 4h, and 4i were found to have good inhibitory effect, with IC50 values of 19.054, 18.476, 14.922 and 15.865 lM, respectively, while aloe emodin was less effective (IC50 = 33.721 lM) against NCI-H460 cells. Some of the aloe emodin derivatives showed moderate inhibitory effect against HeLa and PC3 cells.

PC3 cells (3 9 103), HepG2 cells (3 9 103), HeLa cells (2 9 103), and NCI-H460 cells (2 9 103) were seeded in triplicates in 96-well plates. After 16 h, cells were refreshed with new media containing different concentrations (1, 2.5, 5 and 10 lM) of aloe emodin derivatives and aloe emodin. Finally, after incubation 24, 48, 72 h, 10 mL of the Cyto XTM (LPS solution, Daejeon, Republic of Korea) was added to each well and cells were then incubated for another 2 h at 37 °C in a 5% CO2 incubator. Absorbance was measured at 450 nm.

There was a difference in the cell growth inhibition of aloe emodin derivatives. Compounds having L-serine methyl ester, b-alanine ethyl ester, and 3-(2-Aminoethyl)pyridine substituents were more potent than aloe emodin. The result suggests that some of the aloe emodin derivatives can effectively decrease the growth of different cancer cell lines at lower micromolar concentrations compared to that of the parent aloe emodin.

Conclusions Results and Discussion Chemistry The semisynthesis of natural compound aloe emodin derivatives was carried out according to the general pathway illustrated in the reaction Scheme 1. The intermediate compound 3-(bromomethyl)-1,8-dihydroxyanthracene9,10-dione (2) was obtained by converting hydroxymethyl group at position 3 of aloe emodin to corresponding methyl bromide using 48% hydrobromic acid under reflux condition (9). Treatment of compound 2 with various amino acid methyl esters, amino acid ethyl esters and 4

In summary, we have synthesized 10 aloe emodin derivatives and evaluated their antitumor activities against four different cancer cell lines. Biological evaluation revealed that the compounds 4d, 4f, and 4i exhibited better antitumor activity against HepG2 and NCI-H460 tumor cell lines than aloe emodin, and some of the derivatives exhibited comparable antitumor activity against HeLa and PC3 cells. The structure activity relationship study showed L-serine methyl ester, b-alanine ethyl ester, and 3-(2-Aminoethyl)pyridine substituents are important for their increased antitumor activity. Hence, the present study provides a new insight of this Chem Biol Drug Des 2014

Aloe Emodin Derivatives Table 1: Structure of aloe emodin and its derivatives Compound

Amines (R-NH2)

Aloe emodin derivatives

4a

OH O H2N

HCl

NH O

4b

O

H2N

O O

O

OCH3

OH

CH2 HCl

O

H N

CH3

H2N

HCl NH

O

OH O

L-Glutamic acid diethyl ester HCl 4c

H N

OCH3

L-Tryptophan methyl ester HCl

O

OH

O

O

OH O

O

O

CH3 HCl

CH3

OH

HCl

O

H N

OCH3

L-Phenylalanine methyl ester HCl

HCl

O

4d

H2N

OH

O

OCH3

OH O

HCl

4e

O

OH O O

H N

CH3 HCl

O

OH O

O

β-Alanine ethyl ester HCl OH H2N

OCH3

H N O OH O

O

CH3

HCl

O OH

OH H N

HCl NH

HCl

OH

O

4g

OCH3

OH

OCH3

L-phenylglycine methyl ester HCl O

O

HCl

OH

HCl

H2N

H2N

OCH3

OH H N

L-Serine methyl ester HCl

4f

O

O

HCl NH

Serotonin hydrochloride 4h

H2N

OH O

N

2-(2-Aminoethyl)pyridine 4i

H2N

N

3-(2-Aminoethyl)pyridine

Chem Biol Drug Des 2014

OH H N

N HCl

O

OH O

OH H N

O

N

HCl

5

Thimmegowda et al. Table 1: continued Compound

Amines (R-NH2)

4j

Aloe emodin derivatives OH O

OCH3

OH

H2N

OCH3

H N

HCl

O

4-Methoxy benzyl amine 1

OH O

OH OH

O

Aloe emodin

Table 2: Cell growth inhibition of aloe emodin and its derivatives against four different tumor cell lines IC50 (lM)a Compound

HepG2

NCI-H460

PC3

HeLa

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j Aloe emodin

>25 >25 >25 4.789 >25 11.069 >25 >25 15.593 >25 26.168

>25 >25 >25 19.054 >25 18.476 >25 14.922 15.865 >25 33.721

>25 >25 >25 16.647 >25 16.624 >25 >25 24.272 >25 16.372

>25 >25 >25 >25 >25 15.729 >25 18.471 >25 >25 15.493

a

IC50 values were calculated from three independent experiments.

novel aloe emodin derivatives serving as a potential anticancer agents. From the above summary, it can be concluded that the presence of more number of oxygen atoms and hydroxyl group of aliphatic esters in compounds 4d and 4f may increase the solubility in water by hydrogen bonding and it may increase the cell permeability and and these groups may be responsible for increased anti tumor activity. But exact interaction is not known. Further studies are needed to develop still best anticancer drugs. Synthesis of novel aloe emodin derivatives by changing the substituents at different position of the aloe emodin nucleus to enhance the anticancer activity is under progress at our laboratory.

Acknowledgments This work was supported by the World Class Institute (WCI) Program (WCI 2009-002), Global R&D Center (GRDC) Program funded by the Ministry of ICT, Science and Future Planning (MISP), and also supported by KRIBB Research Initiative Program. 6

References 1. Pecere T., Gazzola M.V., Mucignat C., Parolin C., Vecchia F.D., Cavaggioni A., Basso G., Diaspro A., Salvato B., Carli M., Palu G. (2000) Aloe-emodin Is a new type of anticancer agent with selective activity against neuroectodermal tumors1. Cancer Res;60:2800–2804. 2. Reynolds T. (1985) The compounds in Aloe leaf exudates: a review. Bot J Linn Soc;90:157–177. 3. Fairbairn J.W. (1980) Natural anthraquinone drugs. Pharmacology;20:2–122. 4. Liu K., Park C., Li S., Lee K.W., Liu H., He L., Soung N.K., Ahn J.S., Bode A.M., Dong Z., Kim B.Y., Dong Z. (2012) Aloe-emodin suppresses prostate cancer by targeting the mTOR complex 2. Carcinogenesis;33:1406– 1411. 5. Chiu T.H., Lai W.W., Hsia T.C., Yang J.S., Lai T.Y., Wu P.P., Ma C.Y., Yeh C.C., Ho C.C., Lu H.F., Wood W.G., Chung J.G. (2009) Aloe-emodin induces cell death through S-phase arrest and caspase-dependent pathways in human tongue squamous cancer SCC-4 cells. Anticancer Res;29:4503–4511. 6. Lin M.L., Lu Y.C., Chung J.G., Li Y.C., Wang S.G., N G S.H., Wu C.Y., Su H.L., Chen S.S. (2010) Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway. Cancer Lett;291:46–58. 7. Cui X.R., Takahashi K., Shimamura T., Koyanagi J., Komada F., Saito S. (2008) Preparation of 1,8-di-O-alkylaloe-emodins and 15-amino-, 15-thiocyano-, and 15selenocyanochrysophanol derivatives from aloe-emodin and studying their cytotoxic effects. Chem Pharm Bull;56:497–503. 8. Abramson H.N., Banning J.W., Nachtman J.P., Roginski E.T., Sardessai M., Wormser H.C., Wu J.D., Nagia Z., Schroeder R.R., Bernardo M.M. (1986) Synthesis of anthraquinonyl glucosaminosides and studies on the influence of aglycone hydroxyl substitution on superoxide generation, DNA binding, and antimicrobial properties. J Med Chem;29:1709–1714. 9. Alexander J., Bhatia A.V., Clark G.W. III, Leutzow A., Mitscher L.A., Omoto S., Suzuki T. (1980) Novel ring Chem Biol Drug Des 2014

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hydroxylation of aloe-emodin and further elaboration to anthracycline synthons. J Org Chem;45:24–28.

Figure S1. Hep G2 cell growth inhibition by aloe emodin and its derivatives.

Supporting Information

Figure S2. H460 cell growth inhibition by aloe emodin and its derivatives.

Additional Supporting Information may be found in the online version of this article:

Figure S3. PC3 cell growth inhibition by aloe emodin and its derivatives.

Appendix S1. 1H NMR and Mass spectra of aloe emodin derivatives.

Figure S4. HeLa cell growth inhibition by aloe emodin and its derivatives.

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