Anti-tumor Activity of Derivatives of Protopanaxadiol Prepared with Acid Anhydrides

School of Pharmaceutical Science & Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming 650500, China

Abstract [Objectives] This paper aimed to prepare derivatives of protopanaxadiol from Panax notoginseng (Burk.) FH Chen with acid anhydrides and study their anti-tumor activity. [Methods] The 3-hydroxyl group of protopanaxadiol was subjected to structural modification and reacted with acid anhydrides to prepare derivatives, in order to improve the anti-tumor activity of protopanaxadiol. None of the five compounds designed and synthesized had been reported in the literature, and they were novel compounds. The anti-tumor activity of the derivatives was studies using MTS method. Taking cisplatin and paclitaxel as positive control drugs, the bioactivity of the compounds 1-5 on anti-tumor cell lines (HL-60 cells, SMMC-7721 cells, A-549 cells, MCF-7 cells and SW480 cells) in vitro was screened. [Results] The compound 5 showed inhibitory effect on HL-60 cells, SMMC-7721 cells and A-549 cells. [Conclusions] The acid anhydride esterification method is simple to operate and easy to control. This study has reference value for the structural modification and anti-tumor activity research of protopanaxadiol from P. notoginseng (Burk.) FH Chen.

Key words Protopanaxadiol, Derivative, Panax notoginseng (Burk.) FH Chen, Structural modification

1 Introduction

Panaxnotoginseng(Burk.) FH Chen is a precious Chinese medicinal material in China. The saponins contained inP.notoginseng(Burk.) FH Chen are the main active ingredients for curative effect ofP.notoginseng(Burk.) FH Chen[1-2]. However, notoginsenosides are complex, and more than 70 monomer saponins have been isolated and identified[3-4]. As notoginsenosides are difficult to isolate and separate, the industrialization of isolation of monomers is not easy. In contrast, the aglycon of notoginsenosides is the ideal starting material for structural modification. At present, the structural modification of aglycones is mainly aimed at protopanaxadiol and protopanaxatriol. The two differ in structure only by one hydroxyl group. A lot of pharmacological studies have confirmed that notoginsenosides have good pharmacological activity, including for cardio-cerebral vascular system[5-6], immune system[7], nervous system[8], anti-aging[9]and anti-tumor[10-13]. The anti-tumor activity of notoginsenosides ranks as protopanaxadiol > protopanaxatriol and aglycon > monoglycoside > diglycoside > triglycoside > tetraglycoside[14]. Therefore, in this study, protopanaxadiol was used as a raw material for structural transformation. The derivatives of protopanaxadiol were prepared using the acid anhydride esterification method, and the anti-tumor activity of the derivatives was explored by the MTS method.

2 Materials and methods

2.1MaininstrumentsandreagentsThe used apparatus and instruments mainly included 78-1 magnetic stirrer (Hangzhou Instrument Motor Factory), KQ-100 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), FA2004 electronic balance (Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.), HH-2 digital thermostatic water bath (Guohua Electric Co., Ltd.), BUCHI R-200 rotary evaporator (Switzerland), SHZ-D circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd.), TN counter torque balance (Shanghai Second Balance Instrument Factory), DF-101S collector type constant temperature heating magnetic stirrer (Gongyi Yuhua Instrument Co., Ltd.), Bruker AVIII-500 nuclear magnetic resonance spectrometer (Bruker Company, USA) and Agilent 6520 high resolution Q-TOF-ESI-MS (Agilent Technology Co., Ltd.).

Silica gel for column chromatography (Qingdao Ocean Chemical Plant); high-performance thin-layer chromatography plate (Merck). Chemical synthesis reagents were purchased from Shanghai Jingchun Industrial Co., Ltd. (Aladdin) and Shanghai Titan Technology Co., Ltd. (Adamas). Most of them were of GR grade, and the others were of AR grade.

2.2Methods

2.2.1Preparation of compound 1. Certain amounts of protopanaxadiol (50 mg) and DMAP (25 mg) were placed in a 25-mL two-neck flask, added with 5 mL of anhydrous pyridine at room temperature to dissolve sufficiently, and added with 15 drops of anhydrous propionic anhydride while stirring at room temperature. The reaction was checked with TLC every 30 min. When the raw materials were dissolved completely, stirring was stopped. A certain volume (80 mL) of distilled water was poured into a 100-mL beaker. The reaction solution was then poured into the beaker. Under the condition of stirring, 5% HCL solution was added to the beaker dropwise to adjust the pH into 5.0-5.5, and when a large amount of white precipitate appeared, the stirring was stopped. After standing for 24 h, the solution in the beaker was filtered under reduced pressure. The white precipitate was washed with distilled water until neutral. After drying the white precipitate, the sample was chromatographed on a 300-400 mesh silica gel (300-400 mesh) column. Petroleum ether and ethyl acetate (20∶1) were used for elution. Finally, compound 1 was obtained (Fig.1).

Fig.1Syntheticroutesofcompounds1-5

2.2.2Preparation of compound 2. Certain amounts of protopanaxadiol (30 mg) and DMAP (15 mg) were placed in a 25-mL two-neck flask, added with 5 mL of anhydrous pyridine at room temperature to dissolve sufficiently, and added with 0.8 mL of butyric anhydride while stirring at room temperature. The reaction was checked with TLC every 30 min. When the raw materials were dissolved completely, stirring was stopped. The subsequent treatment was the same as compound 1. After drying the white precipitate, the sample was separated with silica-gel (300-400 mesh) column chromatography. Petroleum ether and ethyl acetate (15∶1) were used for elution. Finally, compound 2 was obtained (Fig.1).

2.2.3Preparation of Compound 3. Certain amounts of protopanaxadiol (50 mg) and DMAP (25 mg) were placed in a 25-mL two-neck flask, added with 5 mL of anhydrous pyridine at room temperature to dissolve sufficiently, and added with 10 drops of isobutyric anhydride while stirring at room temperature. The reaction was checked with TLC every 30 min. When the raw materials were dissolved completely, stirring was stopped. The subsequent treatment was the same as compound 1. After drying the white precipitate, the sample was separated with silica-gel (300-400 mesh) column chromatography. Petroleum ether and ethyl acetate (25∶1) were used for elution. Finally, compound 3 was obtained (Fig.1).

2.2.4Preparation of Compound 4. Certain amounts of protopanaxadiol (50 mg) and DMAP (25 mg) were placed in a 25-mL two-neck flask, added with 5 mL of anhydrous pyridine at room temperature to dissolve sufficiently, and added with 50 mg of succinic anhydride while stirring at room temperature. The reaction was checked with TLC every 30 min. When the raw materials were dissolved completely, stirring was stopped. The subsequent treatment was the same as compound 1. After drying the white precipitate, the sample was separated with silica-gel (300-400 mesh) column chromatography. Petroleum ether and acetone (7∶3) were used for elution. Finally, compound 4 was obtained (Fig.1).

2.2.5Preparation of Compound 5. Certain amounts of protopanaxadiol (50 mg) and DMAP (25 mg) were placed in a 25-mL two-neck flask, added with 5 mL of anhydrous pyridine at room temperature to dissolve sufficiently, and added with 180 mg of phthalic anhydride while stirring at room temperature. The reaction was checked with TLC every 30 min. When the raw materials were dissolved completely, stirring was stopped. The subsequent treatment was the same as compound 1. After drying the white precipitate, the sample was separated with silica-gel (300-400 mesh) column chromatography. Petroleum ether and acetone (3∶1) were used for elution. Finally, compound 5 was obtained (Fig.1).

2.2.6Screening of anti-tumor activity by MTS method[15-16]. The anti-tumor activity of the five samples was detected by the Center for Drug Screening and Research of Kunming Institute of Botany, Chinese Academy of Sciences. The bioactivity of the five compounds for anti-tumor cell lines, HL-60 cells, SMMC-7721 cells, A-549 cells, MCF-7 cells and SW480 cells was screenedinvitroby MTS method, with cisplatin and paclitaxel as positive controls.

3 Results

3.1SpectraldataCompound 1, white grease, yield 76%;1H NMR (400 MHz, CDCl3) δ: 0.81 (3H, s, H-19); 0.81 (3H, s, H-30); 0.85 (3H, s, H-28); 0.93 (3H, s, H-18); 0.98 (3H, s, H-29); 1.06 (3H, s, H-26); 1.56 (3H, s, H-27); 1.62 (3H, s, H-21); 4.46 (1H, dd,J=4.0 Hz, 11.0 Hz, H-3); 5.05 (1H, t,J=6.0 Hz, H-24);13C NMR (CDCl3,100 MHz) δ: 39.69 (t, C-1), 28.31 (t, C-2), 81.15 (d, C-3), 38.44 (s, C-4), 55.83 (d, C-5), 21.88 (t , C-6), 34.47 (t, C-7), 42.05 (s, C-8), 48.68 (d, C-9), 37.91 (s, C-10), 31.63 (t, C-11) , 73.46 (d, C-12), 45.45 (d, C-13), 52.71 (s, C-14), 31.08 (t, C-15), 27.13 (t, C-16), 49.96 (d, C-17), 16.47 (q, C-18), 16.22 (q, C-19), 76.22 (s, C-20), 23.00 (q, C-21), 37.02 (t, C-22), 23.52 (t, C-23), 124.91 (d, C-24), 131.02 (s, C-25), 25.69 (q, C-26), 21.07 (q, C-27), 29.66 (q, C-28), 17.60 (q, C-29), 18.07 (q, C-30), 174.03 (s, C-1), 27.93 (t, C-2), 9.27 (q, C-3), 174.04 (s, C-4), 27.83 (t, C-5), 8.78 (q, C-6); ESI-MS (m/z): 572.86[M+H]+. It was determined to be 3,12-propionate-protopanaxadiol.

Compound 2, yield 67%;1H NMR (400 MHz, CDCl3) δ: 0.81 (3H, s, H-19); 0.84 (3H, s, H-30); 0.91 (3H, s, H-28); 0.91 (3H, s, H-18); 0.98 (3H, s, H-29); 1.06 (3H, s, H-26); 1.56 (3H, s, H-27); 1.62 (3H, s, H-21); 4.46 (1H, dd,J=4.2 Hz, 11.3 Hz, H-3 ); 5.07 (1H, t,J=6.0 Hz, H-24);13C NMR (CDCl3, 100 MHz) δ: 42.02 (t, C-1), 31.66 (t, C-2), 80.08 (d, C-3), 38.25 (s, C-4), 55.79 (d, C-5), 18.06 (t, C-6), 36.62 (t, C-7), 39.66 (s, C-8), 48.45 (d, C-9), 37.83 (s, C-10), 31.30 (t, C-11), 73.45 (d, C-12), 45.39 (d, C-13), 52.70 (s, C-14), 27.13 (t, C-15), 23.51 (t, C-16), 49.95 (d, C-17), 16.21 (q, C-18), 15.39 (q, C-19), 76.16 (s, C-20), 23.09 (q, C-21), 38.40 (t, C-22), 18.53 (t, C-23), 124.87 (d, C-24), 131.02 (s, C-25), 25.68 (q, C-26), 17.57 (q, C-27), 27.90 (q, C-28), 16.47 (q, C-29), 17.57 (q, C-30), 173.26 (s, C-1′), 34.43 (t, C-2′), 21.84 (q, C-3′), 13.68 (q, C-4′), 172.20 (s, C-1″), 34.34 (t, C-2‴), 21.74 (t, C-3″),13.50 (q.C-4″); ESI-MS (m/z): 600.91[M+H]+. It was determined to be 3,12-butyrate-protopanaxadiol.

Compound 3, yield 70%;1H NMR (400 MHz, CDCl3) δ: 0.85 (3H, s, H-19); 0.87 (3H, s, H-30); 0.89 (3H, s, H-28); 1.03 (3H, s, H-18); 1.10 (3H, s, H-29); 1.18 (3H, s, H-26); 1.61 (3H, s, H-27); 1.67 (3H, s, H-21); 4.49 (1H, dd,J=5.2 Hz, 11.0 Hz, H-3); 5.10 (1H, t,J=6.0 Hz, H-24);13C NMR (CDCl3, 100 MHz) δ: 42.05 (t, C-1), 27.92 (t, C-2), 79.89 (d, C-3), 38.39 (s, C-4), 55.82 (d, C-5), 18.38 (t, C-6), 34.47 (t, C-7), 39.71 (s, C-8), 48.34 (d, C-9), 37.03 (s, C-10), 31.72 (t, C-11), 73.46 (d, C-12), 45.40 (d, C-13), 52.80 (s, C-14), 27.18 (t, C-15), 23.45 (t, C-16), 49.99 (d, C-17), 16.24 (q, C-18), 15.45 (q, C-19), 76.21 (s, C-20), 23.17 (q, C -21), 38.01 (t, C-22), 21.88 (t, C-23), 124.990 (d, C-24), 131.13 (s, C-25), 25.70 (q, C-26), 17.43 (q, C-27), 28.17 (q, C-28), 16.50 (q, C-29), 17.60 (q, C-30), 176.67 (s, C-1′), 34.39 (t, C-2′), 19.17 (q, C-3′), 18.04 (q, C-4′), 175.96 (s, C-1″), 34.24 (t, C-2″), 18.88 (t, C-3″), 17.71 (q.C-4″); ESI-MS (m/z): 600.91[M+H]+. It was determined to be 3,12- isobutyrate-protopanaxadiol.

Compound 4, yield 36%;1H NMR (400 MHz, CDCl3) δ: 0.85 (3H, s, H-19); 0.90 (3H, s, H-30); 0.95 (3H, s, H-28); 1.00 (3H, s, H-18); 1.11 (3H, s, H-29); 1.25 (3H, s, H-26); 1.60 (3H, s, H-27); 1.67 (3H, s, H-21); 4.49 (1H, dd,J=5.4 Hz, 10.6 Hz, H-3); 5.11 (1H, t,J=6.5 Hz, H-24);13C NMR (CDCl3,100 MHz) δ: 42.00 (t, C-1), 29.44 (t, C-2), 81.26 (d, C-3), 37.84 (s, C-4), 55.88 (d, C-5), 21.55 (t, C-6), 34.59 (t, C-7), 39.67 (s, C-8), 49.69 (d, C-9), 37.03 (s, C-10), 31.20 (t, C-11), 70.82 (d, C-12), 45.33 (d, C-13), 51.60 (s, C-14), 27.93 (t, C-15), 23.75 (t, C-16), 49.93 (d, C-17), 16.19 (q, C-18), 15.54 (q, C-19), 74.66 (s, C-20), 21.89 (q, C-21), 36.95 (t, C-22), 23.88 (t, C-23), 124.50 (d, C-24), 131.91 (s, C-25), 26.32 (q, C-26), 17.68 (q, C-27), 29.67 (q, C-28), 16.48 (q, C-29), 16.99 (q, C-30), 171.99 (s, C-1′), 34.59 (t, C-2′), 34.57 (q, C-3′); 176.71 (s, -COOH); ESI-MS (m/z): 460.41[M+H]+. It was determined to be 3-succinate-protopanaxadiol.

Compound 5, yield 62%;1H NMR (400 MHz, CDCl3) δ: 0.80 (3H, s, H-19); 0.88 (3H, s, H-30); 0.94 (3H, s, H-28); 1.02 (3H, s, H-18); 1.16 (3H, s, H-29); 1.25 (3H, s, H-26); 1.66 (3H, s, H-27); 1.71 (3H, s, H-21); 4.49 (1H, dd,J=5.0 Hz, 11.0 Hz, H-3); 5.17 (1H, t,J=7.0 Hz, H-24);13C NMR (CDCl3, 100 MHz) δ: 42.00 (t, C-1), 29.67 (t, C-2), 82.48 (d, C-3), 38.55 (s, C-4), 56.04 (d, C-5), 18.13 (t, C-6), 34.58 (t, C-7), 39.68 (s, C-8), 48.22 (d, C-9), 37.01 (s, C-10), 31.52 (t, C-11), 71.03 (d, C-12), 45.40 (d, C-13), 51.83 (s, C-14), 28.04 (t, C-15), 22.72 (t, C-16), 50.20 (d, C-17), 16.42 (q, C-18), 15.46 (q, C-19), 75.06 (s, C-20), 21.86 (q, C-21), 37.95 (t, C-22), 22.72 (t, C-23), 124.49 (d, C-24), 131.77 (s, C-25), 25.75 (q, C-26), 17.64 (q, C-27), 31.22 (q, C-28), 16.53 (q, C-29), 17.15 (q, C-30), 169.41 (s, C-1′), 129.88 (s, C-2′), 129.88 (s, C-3′), 127.95 (q, C-4′), 130.16 (d, C-5′), 131.77 (d, C-6″), 127.95 (t, C-7″); 172.36 (s, -COOH); ESI-MS (m/z): 608.85[M+H]+. It was determined to be 3-phthalate-protopanaxadiol.

3.2Resultsofanti-tumoractivityscreeningThe results show that compound 5 (3-phthaloyl-protopanaxadiol) showed cytotoxic activity on HL-60 CELLS, SMMC-7721 cells and A-549 cells, and theirIC50were 13.27, 27.32 and 35.92 μmol/L, respectively (Table 1).

Table1TheIC50valuesofcompounds1-5onhumancancercelllines

μmol/L

4 Discussions

Screening compounds isolated from plants one by one is the traditional method for finding lead compounds, and it is also the key to the research and development of modern new drugs. During the isolation and purification of monomer compounds, research is facing a series of problems, such as shortage of plant resources, difficulty in isolation and purification, waste of human and material resources and low probability of obtaining new compounds from plant species through traditional separation methods. Structural modification for compounds with high contents in plants can help solve the above problems efficiency, and also, new compounds with high yield and stable structure can be obtained.P.notoginseng(Burk.) FH Chen is a valuable traditional Chinese medicine with medicinal value. At present, the structure modification of protopanaxadiol, an active ingredient ofP.notoginseng(Burk.) FH Chen, is mainly concentrated in 3-OH. Due to the steric hindrance of the 3-OH, the reaction is slow in rate and incomplete. Considering that the reaction between acid anhydrides and protopanaxadiol is affected by reaction conditions and catalyst, so in this study, the basic catalyst DMAP commonly used in the esterification reaction was selected, and anhydrous pyridine was used as the reaction solvent to make the reaction more complete.

The preliminary investigation of the anti-tumor activity of protopanaxadiol derivativesinvitroby MTS method showed that the target product 5 has a significant cytostatic effect. This lays a solid foundation for the further research on anti-tumor activity of protopanaxadiol. Moreover, the synthetic route designed based on the acid anhydride esterification method is simple, and the reaction is easy to control. This study has a reference value for structural modification of aglycon components inP.notoginseng(Burk.) FH Chen.