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«Molecules 2015, 20, 7309-7324; doi:10.3390/molecules20047309 OPEN ACCESS molecules ISSN 1420-3049 Article Synthesis, ...»

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Molecules 2015, 20, 7309-7324; doi:10.3390/molecules20047309



ISSN 1420-3049



Synthesis, Anti-Tumor and Anti-Angiogenic Activity

Evaluations of Asiatic Acid Amino Acid Derivatives

Yue Jing, Gang Wang, Ying Ge, Minjie Xu and Zhunan Gong *

Center for New Drug Research and Development, College of Life Science,

Nanjing Normal University, Nanjing 210023, China; E-Mails: jingyueshadow@126.com (Y.J.);

jiyehanyan@126.com (G.W.); emilygy0131@126.com (Y.G.); xmj1011@163.com (M.X.) * Author to whom correspondence should be addressed; E-Mail: gongzhunan@njnu.edu.cn;

Tel./Fax: +86-25-85891736.

Academic Editor: Derek J. McPhee Received: 6 March 2015 / Accepted: 17 April 2015 / Published: 21 April 2015 Abstract: Fifteen semi-synthetic derivatives of asiatic acid (AA) have been synthesized and evaluated for their biological activities. The successful modification of these compounds at the C-2, C-3, C-23 and C-28 positions was confirmed using NMR, MS and IR spectra.

Further, their anti-tumor effects were evaluated in vitro using different cancer cell lines (HeLa, HepG2, B16F10, SGC7901, A549, MCF7 and PC3), while their anti-angiogenic activities were evaluated in vivo using a larval zebrafish model. Among the derivatives, compounds 4–10 showed more potent cytotoxic and anti-angiogenic effects than AA, while compounds 11–17 had significantly less effects. The new derivative 10 was also included in finished formulations to evaluate its stability using HPLC due to its potential topical use.

The derivative 10 had markedly better anti-tumor activities than both AA and other derivatives, with similar stability as its parent compound AA.

Keywords: asiatic acid; amino acid derivatives; anti-tumor activity; anti-angiogenic effect;

stability; HPLC analysis

1. Introduction Asiatic acid (AA, 2α,3β,23-trihydroxyurs-12-ene-28-oic acid, Figure 1), one of the active pentacyclic triterpenoids found in Centalla asiatica, can be easily prepared from hydrolysis of asiaticoside. Besides Molecules 2015, 20 7310 its traditional usage to treat skin defects [1], AA also has other biological effects including anti-tumor [2–6], anti-inflammation [7], hepatoprotective [8], anti-depression, and anti-Alzheimer’s disease [9,10] activities, like other triterpenes.

However, the efficacy of the original AA is relatively poor. Many attempts have been made to improve this. For example, 2-hydroxypropyl-β-cyclodextrin has been used as an adjuvant for enhancing the encapsulation and releasing characteristics of asiaticoside [11]. Poly(L-lactide) (PLLA) nanoparticles loaded with asiatic acid (AA) have also been successfully produced using the rapid expansion of a subcritical solution into an aqueous receiving solution containing a dispersing agent [12]. Moreover, many researchers have synthesized various AA derivatives by adding new groups to AA [13–15].

Increasing the solubility of a compound usually can improve its bioavailability. For example, conjugation of an amino acid to oleanolic acid has been to shown to improve its water solubility as well as its anti-melanoma activity [16]. It is reported that a hydrogen donor group at either the C-3 position and/or C-28 positions of ursolic acid is essential for its cytotoxic activity [10]. To this end, a series of AA derivatives were synthesized by substituting seven different amio acids at positions of C-28. Their cytotoxic activities were then evaluated in vitro using seven cancer cell lines (HeLa, HepG2, B16F10, SGC7901, A549, MCF7 and PC3). We then sought to evaluate the anti-angiogenic activity of the derivatives using Tg(fli1:EGFP) zebrafish. Results showed that acetylation of the C-2, C-3, and C-23 hydroxy groups in conjunction with a substituted amino acid ester group at C-28 (compounds 4–10), resulted in derivatives not only having stronger cell growth inhibitory activity, but also exhibiting more powerful anti-angiogenic effects than AA.

–  –  –

2. Results and Discussion

2.1. Chemistry As shown in Scheme 1. AA (1) was used as the starting material, and a series of amino acid derivatives were synthesized. Full acetylation of 1 afforded 2α,3β,23-O-triacetylasiatic acid (2) in good yield.

Treatment of 2 with COCl2 afforded the corresponding acyl chloride 3, which was used for the following reactions without further purification. Reaction of 3 with concentrated Et3N solution and amino acid methyl ester hydrochlorides furnished amides 4–10, which were hydrolyzed with aqueous NaOH to give asiatic amides 11–17 (Scheme 1).

–  –  –

2.2. Antitumor Activity of the Compounds Seven different kinds of cancer cell lines (HeLa, HepG2, B16F10, SGC7901, A549, MCF7 and PC3) were chosen to determine the cytotoxic activity of AA and its derivatives. The antiproliferative effects of the compounds were dertermined using Cell Counting Kit-8, in which WST-8(2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium mono- sodium salt) was used as a substrate.

2.2.1. IC50 Values of the Compounds.

–  –  –

C-3 and C-23 positions only showed less activities than AA; (2) compounds with only conjugated amino acids at C-28 also showed less activity than AA; (3) compounds with both acetylated hydroxy groups at C-2, C-3 and C-23 positions, and an amino acid ester group at C-28, had stronger activities than AA.

Meanwhile, these activities varied based on alkyl side chains on the C-28 amide chain.

–  –  –

2.2.2. Cell Viability Suppression Activity of the Compounds To compare the anti-tumor effect of the derivatives with their parent compound AA, tumor cells were exposed to different compounds at 10 μM for 72 h. We found that compounds 4–10 showed a stronger cytotoxic effect on cell viability than AA, while compounds 11–17 showed a much smaller cytotoxic effect. Notably, AA-PMe (10) presented the strongest anti-tumor acitivity among all the compounds to most cancer cell lines (Figure 2).

–  –  –

Figure 2. Cell viability suppression effect of AA derivatives on the A549, B16F10, HeLa, HepG2, SGC7901, MCF7, and PC3 cells.

Cells were treated with 10 μM compounds for 72 h and the cell viability was measured. * p 0.05, ** p 0.01, *** p 0.001.

2.3. Anti-Angiogenic Activity of the Compounds in Zebrafish The anti-angiogenic effect of AA and its derivatives was evaluated in Tg(fli1:eGFP) zebrafish by examining their effect on vessel formation in embryos. As shown in Figure 3A, the intersegmental blood vessels (ISVs) were the most easily observed angiogenic vessels in the embryos at 48 hpf.

Figure 3. Cont.Molecules 2015, 20 7314

Figure 3. The anti-angiogenic activity of AA derivatives in zebrafish.

Tg(fli1:eGFP) zebrafish embryos at 24 hpf were immersed in culture media containg 0.1% DMSO (control), 150 nM VRI (positive control) or 10 μM compounds. (A) Live fluorescence microscopy highlights EGFP expressing intersegmental blood vessels (ISVs) and the subintestinal vessel plexus (SIVs), and the later which appears as a smooth basket-like structure with 5–6 arcades. Scale bar, 50 μm. (B,C) Quantification of the ISV length and number of SIV branch points in 72 hpf zebrafish embryos in the vehicle control group and compounds treated groups. (D) Evaluation of the anti-angiogenic activity of the 16 compounds using EAP assay. * p 0.05, ** p 0.01.

Compounds 4–10 showed an obvious inhibition on ISV formation at 10 μM, but no obvious effect was observed for compounds 11–17 at the same concentration. At 72 hpf, the subintestinal vessel plexus (SIVs) developed as a smooth basket-like structure with approximately 5–6 arcades in the vehicle control group. The numbers of SIV branch points in compounds 4–10 were much fewer than those in 11–17 treated groups. Among these compounds, compound 10 exhibited the strongest inhibition on vessel formation (Figure 3A–D). Compounds 5 and 10 were found to lead to pericardial edema (Figure 3A), which might because circulation was hampered by reduction in vessel formation [17].

2.4. Stability of the Compounds

–  –  –

AA-PMe (10) were soluble and stable without significant differences between different media, temperatures and time (Figure 4). We also found that both AA and AA-PMe (10) were stable at 37 °C and −20 °C. These results provided a theoretical basis for cell biology studies.

–  –  –

3. Experimental Section

3.1. General All reagents were obtained from Aladdin (Shanghai, China) and used without further purification.

Thin-layer chromatography was shown with silica gel 60 GF254 (200–300 mesh). Infrared (IR) spectra were recorded by a Cary5000 instrument (Varian, Palo Alto, CA, USA). Nuclear magnetic resonance (1H-NMR) spectra were measured by an Avance 400 spectrometer (Bruker, Ettlingen, Germany) with DMSO-d6 or CDCl3 as solvents and tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) were recorded in ppm, and coupling constant (J) in Hz. Mass spectra were recorded by a 1290/6460 LC-MS spectrometer (Agilent, Santa Clara, CA, USA). Melting points were determined using an RY-1 digital melting point apparatus (Baytree Packaging Machinery and Material Co., Limited, Shanghai, China). Asiatic acid was purchased from Guangxi Changzhou Natural Products Development Co., Ltd.

(Nanning, China).

3.2. Synthesis and Characterization Data Figure 1 shows the chemical structures of the AA derivatives, which were synthesized by modification of AA (1) at the C-2, C-3, C-23 and C-28 positions.

3.2.1. Asiatic Acid (1)

–  –  –

37.75, 36.78, 32.63, 30.66, 27.96, 24.27, 23.79, 23.44, 21.56, 17.90, 17.51, 17.43, 17.36, 14.23;

ESI-MS: 489.7 ([M+H]+).

3.2.2. 2α,3β,23-Triacetoxyurs-12-en-28-oic Acid (2) Asiatic acid (1, 3 g, 6 mmol) was dissolved in pyridine (30 mL) and stirred for 0.5 h, then Ac2O (6.125 g, 60 mmol) was slowly added into the solution followed by about 1 h stirring and cooling. DMAP (0.03 g) was added to the mixture which was then stirred for 3 h at room temperature (RT). After slowly dropped and quickly stirred into ice to end the reaction, the mixture was extracted with CH2Cl2, and then acidified with aq. HCl. The org. layer was washed with sat. NaHCO3, and brine in sequence, dried (Na2SO4), and concentrated under reduced pressure. The crude product was purified by CC (petroleum ether(PE)/EtOAc 3:1) to give 2 (3.3 g, 92.8%). White solid; M.p. 93–95°; IR (KBr): 3380, 2920, 1740, 1230; 1H-NMR (DMSO-d6) δ 11.97 (s, 1H, COOH), 5.13 (t, J = 3.6 Hz, 1H, H-12), 5.09–5.01 (m, 1H, H-2), 4.94 (d, J = 10.3 Hz, 1H, H-3), 3.81 (d, J = 11.7 Hz, 1H, H-23), 3.50 (d, J = 11.8 Hz, 1H, H-23), 2.12 (d, J = 11.2 Hz, 1H, H-18), 2.01 (s, 3H), 1.98 (s, 3H), 1.92 (s, 3H), 1.05 (d, J = 7.7 Hz, 6H), 0.92 (s, 3H), 0.82 (d, J = 8.8 Hz, 6H), 0.76 (s, 3H); 13C-NMR (DMSO-d6) δ 178.69, 170.35 (COO), 170.17 (COO), 170.10 (COO), 138.71, 124.64, 74.72 (C-3), 69.50 (C-2), 65.36 (C-23), 52.83, 47.54, 47.31, 47.27, 43.68, 42.12, 41.98, 38.94, 38.91, 37.75, 36.73, 32.56, 30.66, 27.92, 24.23, 23.50, 23.36, 21.53, 21.15, 20.97, 20.94, 17.94, 17.39, 17.29, 17.03, 14.00; ESI-MS: 637.2 ([M+Na]+).

3.2.3. N-(2α,3β,23-Acetoxyurs-12-en-28-oyl)acyl Chloride (3) A mixture of 2 dissolved in CH2Cl2 and COCl2 was refluxed for 24 h at RT and the excess reagent was removed under reduced pressure. The residue was extracted with cyclohexane three times (50 mL each time) to give acyl chloride 3.

3.2.4. N-(2α,3β,23-Acetoxyurs-12-en-28-oyl)-L-phenylalanine Methyl Ester (4) L-Phenylalanine methyl ester hydrochloride (6 mmol) in CH2Cl2 (200 mL) was added to 3, and then Et3N was added (3 mL). The mixture was then stirred for 4 h at RT, washed with water, dried (Na2SO4), and concentrated under reduced pressure. The crude product was purified by CC (petroleum ether(PE)/EtOAc 3:1) to give 4 (2.7 g, 62%). White solid; M.p. 120–123°; IR (KBr): 3401, 2923, 1739, 1228; 1H-NMR (CDCl3) δ 6.35 (d, J = 6.2 Hz, 1H, N-H), 5.25 (t, J = 3.3 Hz, 1H, H-12), 5.19–5.10 (m, 1H, H-2), 5.07 (d, J = 10.3 Hz, 1H, H-3), 4.76–4.65 (m, 1H, H-2’), 3.84 (d, J = 11.8 Hz, 1H, H-23), 3.68 (s, 3H), 3.57 (d, J = 11.8 Hz, 1H, H-23), 2.08 (d, J = 10.9 Hz, 1H, H-18), 2.07 (s, 3H), 2.02 (s, 3H), 1.97 (s, 3H), 1.05 (d, J = 3.4 Hz, 6H), 0.94 (s, 3H), 0.87 (s, 3H), 0.83 (d, J = 6.4 Hz, 3H), 0.60 (s, 3H);

C-NMR (CDCl3) δ 177.29, 172.13 (CON), 170.81 (COO), 170.42 (COO), 170.35 (COO), 138.36, 136.21 (Ar-C), 129.35 (Ar-C), 128.44 (Ar-C), 127.00 (Ar-C), 125.86, 74.77 (C-3), 69.88 (C-2), 65.22 (C-23), 53.53, 53.45, 52.14, 47.66, 47.56, 47.43, 43.72, 42.19, 41.88, 39.55, 39.02, 38.09, 37.71, 37.19, 32.27, 30.81, 27.64, 25.27, 24.68, 23.34, 23.22, 21.17, 21.07, 20.87, 20.78, 17.80, 17.07, 17.03, 16.41, 13.90; ESI-MS: 798.3 ([M+Na]+).

Molecules 2015, 20 7317

3.2.5. N-(2α,3β,23-Acetoxyurs-12-en-28-oyl)glycine Methyl Ester (5)

As described for the preparation of 4, treatment of 2 (5.8 mmol) with L-glycine methyl ester

hydrochloride (6 mmol) afforded 5 (3.4 g, 85%). Yield: 85%; White solid; M.p. 174–177°; IR (KBr):

2921, 2865, 1741, 1367, 1228; 1H-NMR (DMSO-d6) δ 7.68 (t, J = 5.6 Hz, 1H, N-H), 5.18 (t, J = 3.5 Hz, 1H, H-12), 5.09–5.01 (m, 1H, H-2), 4.94 (d, J = 10.3 Hz, 1H, H-3), 3.79 (d, J = 17.0 Hz, 1H, H-2’) 3.64 (d, J = 11.6 Hz, 1H, H-23), 3.59 (s, 3H), 3.50 (d, J = 11.8 Hz, 1H, H-23), 2.16 (d, J = 10.9 Hz, 1H, H-18), 2.00 (s, 3H), 1.98 (s, 3H), 1.92 (s, 3H), 1.04 (d, J = 9.0 Hz, 6H), 0.93 (s, 3H), 0.83 (d, J = 3.5 Hz, 6H), 0.67 (s, 3H); 13C-NMR (DMSO-d6) δ 177.14, 170.93 (CON), 170.35 (COO), 170.17 (COO), 170.11 (COO), 138.68, 124.73, 74.74 (C-3), 69.51 (C-2), 65.37 (C-23), 52.30, 51.92, 47.56, 47.35, 46.98, 43.70, 42.03, 41.98, 41.35, 39.52, 39.19, 38.91, 37.73, 37.16, 32.57, 30.87, 27.66, 23.98, 23.51, 23.37, 21.58, 21.15, 20.97, 20.94, 17.93, 17.52, 17.03, 16.97, 14.00; ESI-MS: 686.3 ([M+H]+); 708.3 ([M+Na]+).

3.2.6. N-(2α,3β,23-Acetoxyurs-12-en-28-oyl)-L-alanine Methyl Ester (6)

As described for the preparation of 4, treatment of 2 (5.8 mmol) with L-alanine methyl ester hydrochloride (6 mmol) afforded 6 (2.4 g, 60%). Pale yellow solid. Pale yellow solid; M.p. 220–223°;

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