Dissertation summary: Study on the isolation, chemical transformation and biological activities of triterpenoids from Centella asiatica (L.) Urban (Apiaceae)

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The aims of the dissertation are: Isolation and purification of the main chemical constituents of the plant Centella asiatica (L.) Urban collected from various regions in Vietnam; synthesis of new derivatives of asiatic acid and madecassic acid obtained from C. asiatica.

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Nội dung Text: Dissertation summary: Study on the isolation, chemical transformation and biological activities of triterpenoids from Centella asiatica (L.) Urban (Apiaceae)

VIETNAM ACADEMY OF SCIENCE AND MINISTERY OF TECHNOLOGY EDUCATION AND GRADUATE UNIVERSITY OF SCIENCE AND TRAINING TECHNOLOGY VO THI QUYNH NHU STUDY ON THE ISOLATION, CHEMICAL TRANSFORMATION AND BIOLOGICAL ACTIVITIES OF TRITERPENOIDS FROM CENTELLA ASIATICA (L.) URBAN - (APIACEAE) Scientific Field: Organic Chemistry Classification Code: 62 44 01 14 DISSERTATION SUMMARY HA NOI - 2017
The dissertation was completed at: Institute of Chemistry Vietnam Academy of Science and Technology Scientific Supervisors: 1. Prof. Dr. Sc. Tran Van Sung Institute of Chemistry - Vietnam Academy of Science and Technology 2. Dr. Tran Van Loc Institute of Chemistry - Vietnam Academy of Science and Technology 1st Reviewer: ............................................................................................................. .............................................................................................................. .............................................................................................................. nd 2 Reviewer: ............................................................................................................ .............................................................................................................. .............................................................................................................. 3rd Reviewer: ............................................................................................................. .............................................................................................................. .............................................................................................................. The dissertation will be defended at Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay District, Ha Noi City. At ….. hour….. date….. month …..2017. The dissertation can be found in National Library of Vietnam and the library of Institute of Chemistry, Vietnam Academy of Science and Technology.
I. INTRODUCTION 1. Background Nowadays, it is necessary to search for interesting active compounds and develop them to new drugs. Besides active natural products, study on synthesis of active derivatives from compounds isolated from plant sources is one of the most effective ways to search for interesting active compounds. Centella asiatica (L.) Urban (Apiaceae) is a tropical medicinal plant native to South and Southeast Asian countries including Vietnam. This plant has been reported to own a wide range of biological activities such as wound healing, anti- inflammatory, antipsoriatic, antiulcer, hepatoprotective, anticonvusant, sedative, immunostimulant, cardioprotective, antidiabetic, cytotoxic, antitumor, antiviral, antibacterial, anti-insecticidal, antifungal, and antioxidant activities. It also has been used for the treatment of lepra and venous deficiencies. The plant Centella asiatica contains several classes of natural substances, but triterpenes are its major and most important constituents, which are regarded as marker components in terms of quality control. Among the triterpenoids of C. asiatica , four compounds with the ursane triterpene skeleton, asiatic acid, madecassic acid, asiaticoside and madecassoside, play the most important role. In view of the high content of these biological active compounds in C. asiatica, many chemical modifications of their structure have been carried out in order to find more active substances with potential pharmaceutical uses. For the above reasons, we propose the dissertation: “Study on the isolation, chemical transformation and biological activities of triterpenoids from Centella asiatica (L.) Urban (Apiaceae)”. 2. Objectives of the dissertation The aims of the dissertation are: 1. Isolation and purification of the main chemical constituents of the plant Centella asiatica (L.) Urban collected from various regions in Vietnam . 2. Synthesis of new derivatives of asiatic acid and madecassic acid obtained from C. asiatica. 1
3. Determination of the biological activities of the synthesized derivatives to find new biologically active substances, and exploration of their structure - activity relationship. 3. New contributions of the dissertation  Quantitative determination of two main triterpenoids, asiatic acid and madecassic acid in three Centella asiatica samples collected in Son Tay (Hanoi), Nam Dinh province and Ho Chi Minh city was performed using column chromatography and HPLC. It was found that the content of both triterpenes in the Son Tay sample was the highest with values of 0.72% and 0.89% (of dry material) for asiatic acid and madecassic acid, respectively. This sample was chosen for extraction of these two triterpenes for chemical transformation.  The liver-protective activity of asiatic acid in the paracetamol-induced hepatotoxicity model in mice was evaluated. Asiatic acid showed a significant liver-protective activity (P
4. The layout of the dissertation The dissertation consists of 136 pages and contains 124 references. The layout of the dissertation includes the following sections: Preface (1 pages), Chapter 1: Overview (32 pages), Chapter 2: Objects and methods (3 pages), Chapter 3: Experimentals and results (38 pages), Chapter 4: Results and discussion (46 pages), Conclusions (1 pages), Recommendations (1 page), Publications (1 page), References (9 pages), and Appendix. 3
II. DISSERTATION CONTENTS PREFACE This part discusses the background, the scientific and practical significance, and the objectives of the research project. Chapter 1. Literature review 1.1. Chemistry and biological activities of Centella asiatica 1.2. Extraction and purification of the triterpenes and triterpene glycosides from Centella asiatica 1.3. Biological activities of Centella asiatica 1.4. Biological activities of asiatic acid and madecassic acid and their derivatives 1.5. Chemical transformations of asiatic acid 1.6. Researches on these topics in Vietnam Chapter 2. Methods 2.1. Isolation and purification methods Compounds were isolated and purified by using a combination of various chromatographic methods including thin-layer chromatography (TLC), column chromatography (CC) on different stationary phases such as Silica gel, YMC RP- 18, and Sephadex LH-20. 2.2. Methods for the determination of the chemical structures The chemical structures of isolated compounds were elucidated by a combination of physical parameters (melting point), modern spectroscopic methods (IR, UV, CD, MS, 1D-NMR, and 2D-NMR) with chemical conversion, and by comparing with literature data. 2.3. Chemical transformation methods 4
2.4. Cytotoxicity assay The in vitro cytotoxicity of the isolated compounds and their derivatives against HepG2, KB and LU human cancer cell lines were evaluated by MTT method. Cell viability was measured and IC50 values were calculated based on the Rawdata software. 2.5. Determination of the liver-protective activity 2.6. Molecular docking method Chapter 3. EXPERIMENTALS 3.1. Plant materials Samples of the plant Centella asiatica were collected in Son Tay, Hanoi City, and Nam Dinh province in April 2010. The sample from Ho Chi Minh City was collected in May 2010. All samples were identified by master of biology Nguyen The Anh, Institute of Chemistry, VAST, 18 Hoang Quoc Viet, Hanoi. 3.2. Isolation and purification of chemical constituents of Centella asiatica collected in Ho Chi Minh City 5
Centella asiatica 1200g RMHCM n-Hexane residue n- Hexane, 3×5L extract, 29g CH2Cl2 ,3×4L residue CH2Cl2 extract, 33g n- BuOH, 3×3L n-BuOH extract, residue 50g Silica gel CH2Cl2/MeOH 95:5-70:30 madecassic stigmasterol β-Sitosterol asiatic acid (1) acid (10) (44) Madecassoside (14) (43) 185mg stigmasterol glucoside 168mg and β-sitosterol glucoside (144) Figure 3.2. Isolation scheme of extracts from Centella asiatica Spectral data of isolated compounds are given in 3.2.1.2. 3.3. Study the on the extraction, hydrolysis and separation of asiatic acid and madecassic acid from Centella asiatica The Centella asiatica sample was dried at room temperature and extracted three times with a mixture of EtOH/H2O 80:20 (each time 2h) at 80οC. After filtration and evaporation of the solvent, the residue was dissolved in 200 mL H2O/MeOH 80:20 and the solution was extracted with n-hexane for the elimination of fats and essential oil. Then it was hydrolyzed with 20% aqueous NaOH solution at 80 οC in 2h, cooled down to room temperature, then extracted with EtOAc. The aqueous layer was acidified with HCl to pH = 3-4 to obtained a 6
precipitation. The precipitation was filtered, washed with H2O and dried. This product was subjected to column chromatography and to HPLC for the determination of the content of asiatic acid and madecassic acid. 3.4. Synthesis of asiatic acid and madecassic acid derivatives 3.4.1. Synthesis of asiatic acid derivatives Isolation of asiatic acid (1) Dried and ground whole plant of Centella asiatica (1800 g) was extracted with MeOH three times (3x5 L) at 80 oC for 2 hours. The filtrate was concentrated under reduced pressure to yield a residue (330 g), which was suspended with H2O (0.8 L) and partitioned by n-hexane and EtOAc to obtain the corresponding residues of 40 g and 50 g. The aqueous phase was then concentrated to dryness and redissolved in MeOH, followed by treatment with 10% NaOH at 80 oC for 2 hours. Afterward, the solution was cooled to 10 oC with ice-water bath and acidified with 5% HCl to pH ≈ 4. The precipitated solid was collected and subjected to column chromatography on silica gel eluting with a gradient solvent of DCM/MeOH (95/5 → 90/10) to furnish asiatic acid (1) (6.8 g, 0.37% w/w). The chemical characterisation was based on the ESI-MS, 1H, 13C-NMR analysis and comparison with literature (Jing Yue et el. 2015). Synthesis of 2,3,23-triacetyl asiatic acid (145) A stirred solution of asiatic acid (1.46 g, 3 mmol) in pyridine (10 mL) was treated with acetic anhydride (10 mL) at room temperature for 12 h. The reaction mixture was then diluted with EtOAc (40 mL), washed successively with H2O, 1N HCl and saturated aqueous solution of NaHCO3. The solvent was evaporated under reduced pressure and the residue was chromatographed on silica gel column (n-hexane/EtOAc, 2/1) to yield 145 (1.47 g, 80%) as a white solid, M.p. = 160- 163 oC. The structure of 145 was confirmed by analysis of its ESI-MS, 1H, 13C- NMR spectral data and comparison with literature (Jing Yue et el. 2015). General procedure for the synthesis of 2,3,23-triacetylasiatic acid-28-amides (147-156) Oxalyl chloride (3.0 mmol, 6 equiv.) in DCM (2 mL) was added to a solution of 145 (307 mg, 0.5 mmol, 1 equiv.) in anhydrous dichloromethane (DCM, 15 7
mL). After being stirred at room temperature for 24 h, the solvent and excess of oxalyl chloride were removed under reduced pressure. The residue was redissolved in anhydrous dichloromethane (20 mL) followed by an addition of triethylamine (3.0 mmol, 6 equiv.) and a solution of monoamine (0.75 mmol, 1.5 equiv.) or diamine compounds (1.2 mmol, 4.0 equiv.) in DCM (2 mL). After stirring for further 20 h, the reaction mixture was quenched with DCM (30 mL) and washed with HCl 5%. The organic phase was dried over Na 2SO4 and evaporated to dryness. The pure products 147-156 were obtained by purification by silica gel column chromatography using n-hexane/EtOAc as eluent. The products were characterised by their ESI-MS, 1H, 13C-NMR spectral data. General procedure for the synthesis of the amides 157-161 To a stirred solution of the corresponding amides (147-150, 156) (1.0 equiv.) and DMAP (0.01 mol%) in dry CH2Cl2 (20 mL), acetyl chloride (2 equiv.) was added. The resulting solution was stirred for further 2 h, then quenched with aqueous solution of NaHCO3 10%. The organic phase was washed with H2O, brine solution and dried over Na2SO4. After removal of the solvent, the residue was subjected to column chromatography on silica gel eluting with n- hexane/EtOAc to yield the pure acetylated products. The products were characterised based on ESI-MS, 1H, 13C-NMR analysis. General procedure for the synthesis of the compounds 162-166 The amides 147-149, 154, 156 (1 equiv.) in MeOH (15 mL) were treated with 4N aqueous KOH (2 mL) at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, acidified with HCl 5% to pH≈4 and extracted with DCM (3x20 mL). The combined organic phase was washed with aqueous NaHCO3 10% and dried over Na2SO4. The pure products 162-166 were obtained by purification on silica gel column (CH2Cl2/MeOH, 95/5) and characterised by the analysis of their IR, MS, 1H, 13C-NMR spectra. Synthesis of compound 167 To a solution of 145 (307 mg, 0.5 mmol, 1 equiv.) in anhydrous dichloromethane (DCM, 15 mL), oxalyl chloride (3.0 mmol, 6 equiv.) in DCM (2 mL) was added. After being stirred at room temperature for 24 h, the solvent and 8
excess of oxalyl chloride were removed under reduced pressure. The residue was redissolved in dichloromethane (20 mL) followed by an addition of 30% aqueous ammonia solution (2 mL). After stirring for further 20 h, the solvent was evaporated under reduced pressure. Pure 167 (270 mg, 88%) was obtained by purification on silica gel column (n-hexane/EtOAc; 3/1). Compound 167 was characterised by MS, 1H, 13C-NMR spectra. Synthesis of compound 168 2,3,23-Triacetyl asiatic acid-28-amides 167 (1 equiv.) in MeOH (10 mL) were treated with 4N KOH (2 mL) at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, acidified with 5% HCl to pH≈4 and extracted with DCM (3x20 mL). The combined organic phase was washed with 10% NaHCO3 and dried over Na2SO4. The pure product 168 was obtained by column chromatography purification on silica gel (CH2Cl2/MeOH, 95/5). The chemical chacterization was based on MS, 1H, 13C-NMR analysis. Synthesis of compound 169 Acetyl chloride (2 equiv.) was added to a stirred solution of 167 (307 mg, 0.5 mmol) and DMAP (6 mg; 0.05 mmol) in dry CH2Cl2 (10 mL). The resulting solution was stirred further 2 h, then quenched with 10% aqueous NaHCO3. The organic phase was washed with H2O, brine solution and dried over Na2SO4. After removal of the solvent, the residue was subjected to column chromatography on silica gel, eluting with n-hexane/ EtOAc to yield pure 169 (252 mg, 85%). The chemical chacterization was based on ESI-MS, 1H, 13C-NMR analysis. Synthesis of compound 170 Compound 169 (595 mg, 1 mmol) in MeOH (10 mL) were treated with 4N KOH (2 mL) at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, acidified with 5% HCl to pH≈4 and extracted with DCM (3x20 mL). The combined organic phase was washed with 10% NaHCO3 and dried over Na2SO4. The pure product 170 was obtained in 74% (368 mg) by column chromatography purification on silica gel (CH2Cl2/MeOH, 95/5). The chemical chacterization was based on ESI-MS, 1H, 13C-NMR analysis. Synthesis of compound 171 9
To a stirred solution of asiatic acid (1) (2.44 g, 5 mmol) and TsOH.H2O (10 mg, 0.05 mmol) in DMF (20 ml), 2,2-dimethoxypropane (580 mg, 5.5 mmol) was added dropwise and kept for further 5 h at room temperature. The reaction mixture was neutralized with NaOH 5% to pH≈8 and extracted with ethyl acetate (2x100 mL). The combined organic phase was washed with H2O (3x50 ml), dried over Na2SO4 and evaporated under reduced pressure. Compound 171 was obtained in 88% (2.32 g) yield by column chromatography purification on silica gel (CH2Cl2/MeOH, 20/1). The characterisation of compound 171 was based on the ESI-MS, 1H, 13C-NMR spectra analysis and comparison with the literature (Jeong B.S. et al. 2007 and 2006). Synthesis of compound 172 To a stirred solution of 171 (264 mg, 0.5 mmol) and triethylamine (1 mmol) in dry CH2Cl2 (10 mL) at room temperature, succinic anhydride (60 mg, 0.6 mmol) was added. After 12 h, the reaction mixture was diluted with ethyl acetate (20 mL), washed with 1N HCl and followed by an aqueous solution of 10% NaHCO3 The solvent was removed and the residue was chromatographed on a silica gel column (n-hexane/EtOAc, 2/1) to yield pure product 172 (232 mg, 74%). Compound 172 was characterised by ESI-MS, 1H, 13C-NMR. Synthesis of compound 173 To a stirred solution of compound 171 (264 mg, 0.50 mmol) in pyridine (5 ml), acetic anhydride (76 mg, 0.74 mmol) was added. After keeping further 12 h, ethyl acetate (40 mL) was added. The organic phase was washed with 1N HCl, aqueous solution of NaHCO3, and brine solution. Removal of the solvent and subsequent purification on a silica gel column (n-hexane/EtOAc, 2/1) afforded the pure product 173 (234 mg, 82%). The chemical chacterization was based on ESI- MS, 1H, 13C-NMR analysis. Synthesis of compound 174 Compound 173 (570 mg, 1 mmol) in MeOH (10 ml) was treated with 1N HCl (2 ml) at room temperature for 4 h. The solvent was removed and the residue was dissolved in H2O (15 ml), followed by extraction with CH2Cl2 (3x30 mL). The combined organic phase was washed with brine solution and dried over 10
Na2SO4. Product 174 (477 mg, 90%) was obtained by column chromatography purification on silica gel (n-hexane/EtOAc, 7/3). The characterisation was based on the ESI-MS, 1H, 13C-NMR analysis. 3.4.2. Synthesis of madecassic acid derivatives Isolation of madecassic acid (10) from Centella asiatica: Dried and ground Centella asiatica plant (1.800 g) was extracted with MeOH three times (3x5 L) at 80 oC for 2 hours. The filtrate was concentrated under reduced pressure to yield a raw material (330 g), which was suspended with H2O (0.8 L) and partitioned by n-hexane and EtOAc to obtain the corresponding residues of 40 and 50 gram, respectively. The aqueous solution was concentrated to dryness and re-dissolved in MeOH, followed by treatment with 10% NaOH at 80 oC for 2 hours. The solution was cooled down to 10 oC and acidified with 5% HCl to pH ≈ 4. The precipitate was collected and subjected to column chromatography on silica gel, eluted with a gradient solvent of DCM/MeOH (95/5 → 90/10) to furnish madecassic acid (10) (6.8 g, 0.34% w/w) as a white powder. 6β-hydroxy-2α,3β,23-triacetoxy-urs-12-ene-28-oic acid (175) To a stirred solution of 10 (502 mg, 0.996 mmol) in pyridine (10 mL), acetic anhydride (300 mg, 2.921 mmol) was added. After stirring 18 hours at room temperature, ethyl acetate (60 mL) was added. The solution was washed with 1N HCl, brine solution and dried over Na2SO4. Solvent was evaporated and the residue was chromatographed on SiO2 column (n-hexane/EtOAc; 2/1) to give 175 (533.4 mg, 85%) as a white powder. Compound 175 was then reacted with oxalyl chloride in dichloro-methane (DCM) at room temperature to afford compound 176 as the key intermediate. General procedure for the synthesis of compounds 177 - 185 Oxalyl chloride (6 mmol) was added to a solution of compound 175 (1 mmol) in dry DCM (20 mL) and stirred overnight at room temperature. The solvent and excess of oxalyl chloride were evaporated. The residue was redissolved in DCM (20 mL), followed by addition of TEA (6 mmol) and the corresponding amines (1.5 mmol). After being stirred 20 hours at room temperature, the solution was washed with 5% HCl, brine solution and dried over 11
Na2SO4. The residue was chromatographed over SiO2 column (n-hexane/EtOAc) to afford products 177 - 185. General procedure for the hydrolysis of 2α,3β,23-triacetoxy groups (synthesis of 186-191) Compounds 177-183 (1 mmol) in MeOH (10 mL) were treated with 4N aqueous NaOH solution (2 mL) at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, acidified with HCl 5% to pH≈4 and extracted with DCM (3x50 mL). The combined organic phase was washed with water and dried over Na2SO4. The pure products 186-191 were obtained by purification on silica gel column (CH2Cl2/MeOH; 95/5) and characterised by analysis of their IR, ESI-MS, 1H, 13C-NMR spectra. 3.5. Biological assay 3.6. Study on the biological activities of synthetized derivatives 3.6.1. Assay for in vitro cytotoxicity Our goal in this work was to evaluate the cytotoxicity of the synthesized compounds against three human cancer cell lines: KB (melanoma cancer), Hep G2 (liver cancer) and Lu (lung cancer) using the MTT method (Mosmann T. 1983, Cos P.et al. 2006, Scudiero D.A et al. 1998). Ellipticine was used as positive control (detailed description is in Experimental part). The results are given in Table 4.3, 4.4. 3.6.2. Determaination of the liver-protective activity on mice Chapter 4. RESULTS AND DISCUSSION 4.1. Isolation of asiatic acid and madecassic acid 4.1.1. Chemical composition of Centella assiatica (L.) urban collected in Ho Chi Minh City During the screening for biological active compounds from the medicinal plant Centella asiatica, a sample from Ho Chi Minh City has been studied. From the n-BuOH extract of the plant six compounds have been isolated: stigmasterol, β-sitosterol, asiatic acid, madecassic acid, mixture of stigmasterol glucoside and 12
β-sitosterol glucoside (1:1) and madecassoside. Their structures were determined by IR, ESI-MS and NMR (1D and 2D) spectroscopy as well as by comparison with the literature data. 4.1.2. Quantitative determination of the main triterpenic acids in Centella asiatica samples from North and South of Vietnam by HPLC method Table 4.1: The total content in percent of asiatic acid and madecassic acid in three Centella asiatica samples determind by HPLC method RMST 4.25 g RMND 4.20 g RMHCM 1.67 g Constituent % % in dry % % in dry % % in dry Area material Area material Area material Asiatic acid 33.81 0.718 30.49 0.615 28.92 0.241 Madecassic 41.93 0.891 43.02 0.865 28.61 0.239 acid 4.2. Conditions for the separation of asiatic acid and madecassic acid from Centella asiatica Conditions for the extraction and hydrolysis to obtain high amount of asiatic acid and madecassic acid were investigated. The optimal conditions were found as follows: Extraction conditions: -Solvent: EtOH/H2O 80:20 (v/v) -Temperature: 80οC -Time: 2 hours Hydrolysis conditions: -Concentration of the aqueous NaOH solution: 20% (w/w) -Time: 2 hours -Temperature: reflux (80οC). -Neutralization after hydrolysis with 5% HCl solution. 4.3. Synthesis of asiatic acid and madecassic acid derivatives 4.3.1. Synthesis of asiatic acid derivatives 13
The synthetic procedures are presented in Scheme 4.1 and 4.2. Scheme 4.1 was designed to obtained different amides of asiatic acid (1) by the reaction of asiatic acid triacetate (145), which was obtained when asiatic acid reacted with acetic anhydride in pyridine for twelve hours at room temperature. The yield was about 80% after silica gel column chromatography. Structure of the triacetate 145 was confirmed by its positive ESI–MS with the ion peaks at m/z 655 [M+1- CH3COOH]+, 495 [M+1- 2xCH3COOH]+, its IR spectrum [1746.26 and 1237.00 (acetate), 1698.88 (-COOH)]. The 1H and 13C-NMR spectra of 145 clearly showed beside the corresponding signals in asiatic acid (1) signals of three acetyl groups at δH (ppm) 1.92, 1.96, 2.12 (all s, 3H) and δC (ppm) 170.83, 170.47, 170.37. The signals of H-2β, H-3α and H-23 were shifted to lower field and appeared at δH (ppm) 3.51, 3.78 (each 1H, d, J = 11.5 Hz, 2x H-23), 5.01 (1H, d, J = 10.5 Hz, H- 3α) and 5.09 (1H, dt, J = 4.5, 10.5 Hz, H-2β) in comparison to the corresponding signals [δH (ppm) 3.29 and 3.52 (each 1H, d, J = 11.0 Hz, H-23), 3.38 (1H, d, J = 9.5 Hz, H-3α), 3.72 (1H, dt, J = 4.5, 9.5 Hz, H-2β)] of compound 1. The spectral data of compound 145 are in agreement with the literature (Zhang L. et al. 2009). The triacetate 145 was then converted to the chloride 146 by the reaction with oxalyl chloride at room temperature. Compound 156 reacted then with different amines in the presence of triethylamine to afford the corresponding amides 147- 156 with the yields of 75-92% after column chromatography. The structures of the compounds 147-156 were evidenced by the ESI-MS spectra with the corresponding pseudomolecular ion peaks [M+H]+ or [M-H]- as well as by the additional signals for the amide side chains in their 1H and 13C-NMR spectra. The signal of the 28-COOH group in compound 145 (δC 183.68) was high-field shifted to δC: 177.38-180.00 ppm due to the amidation. The hydroxyl and amino groups in the amide side chains of compound 147-150, 156 was acetylated to obtain the corresponding tetraacetyl derivatives 157-160 and the penta-acetate 161 with high yield (75-80%) after column chromatography. The structures of the derivatives 157-161 were confirmed by their ESI-MS spectra and by the signals of the additional acetyl groups in their 1H and 13C-NMR spectra. There were typical changes in the 1H-NMR spectra. The signal of H-3’ in compound 157 appeared 14
clearly as a triplet at δH 4.10 ppm (J = 6.0 Hz) in comparison with its parent compound 150 as multiplet at δH 3.53 -3.63 ppm, overlapped with H-23a, H-2’a. The –NH signal of 157 is also high-field shifted to δH 6.05 (t, J = 5.0 Hz) in comparison with δH 6.18 (t, J = 5.5 Hz) in 150. In compounds 158-160 the signals of the H-7’, H-9’, H-10’ appeared down-field shifted at δH 3.22 ppm (2H, m) instead at about δH 3.12 ppm (2H, m) in the corresponding compounds 147-149. The new –NH amide proton signals appeared as broad singlets at δH 5.61, 5.96, 5.71, respectively. In compound 161, the signal of H-2’ was at δH 4.25 (4H, brs) instead at δH 3.72 (4H, brs) in compound 156. Deacetylation of compounds 147- 149 and 154, 156 in methanolic potassium hydroxide at room temperature afforded compounds 162-166 with the yields from 72-80% after SiO2 column chromatography. Beside the corresponding pseudomolecular ion peaks in the ESI- MS spectra, their 1H and 13C-NMR spectra contained no signals for the acetyl groups. Accordingly, the resonance signals of H-2β, H-3α and H-23 are shifted to high-field and appeared at the similar positions to those in the spectrum of compound 1. In another option, compound 146 was reacted with 30% aqueous ammoniac solution at room temperature to afford the triacetyl amide 167 which was deacetylated to afford compound 168. In an attempt to get the N-acetyl derivative of compound 167 by reaction with acetyl chloride/ dimethylaminopyridine (DMAP) at room temperature, the nitril 169 was obtained instead of the N-acetyl derivative. The nitril 169 was formed due to dehydration of the amide group in the presence of acetyl chloride and DMAP. Hydrolysis of compound 169 in methanolic potassium hydroxide afforded the nitril 170. The 1H-NMR spectrum of compound 167 contained the signals of the –NH2 amide protons at δH 5.48 and 5.83 ppm (each 1H, brs). The 1H and 13C-NMR spectroscopic data of compounds 167, 168 are in good agreement with the literature (Zhang L. et al. 2009). The presence of a nitril group in compound 169 and 170 was confirmed by the carbon signals in their 13C-NMR at δC (ppm) 122.94 and 126.41, respectively. Further evidences were shown by the absorption at 2228.8 cm-1 in the IR spectrum, as 15
well as by a pseudomolecular ion peaks [M+Na]+ at m/z 618 and 492 in the (+) ESI –MS spectra, respectively. Scheme 4.1. Synthesis of derivatives of asiatic acid Reagents and conditions: a) (CH3CO)2O, pyridine, rt., 12 h, 80%, b) oxalyl chloride, DCM, rt., 24 h, c) RNH2, DCM, rt, 24 h, (75-92%), d) AcCl, DMAP, DCM, 2h, 75-80%, e) KOH, MeOH, rt, 16 h, 72-80%, f) acetyl chloride, DMAP, rt, 85%. As shown in scheme 4.2, some changes in the A-ring of asiatic acid (1) were carried out to evaluate their effect on the cytotoxicity of the obtained derivatives. Compound 1 was reacted with 2,2-dimethoxy propane in toluenesulfonic acid (TsOH) at room temperature to afford compound 171 (yield 88%). Compound 171 showed identical NMR spectra with those in the literature (Jeong B. S. et al. 2007). The (-)-ESI-MS of 171 contained a pseudomolecular ion peak at m/z 527 16
[M-H]- (100). Its 1H and 13C-NMR contained additional signals of an isopropylidenedioxy group at δH 1.42, 1.48 (each 3H,s); δC 100.72 ppm. Through ketal formation the signals of C-2 and C-23 were shifted to lower field and appeared at δC 83.03 and 73.66 instead of beeing at δC 69.68 and 66.31 as in asiatic acid (1) while the C-3 signal is shifted to higher field and appeared at δC 66.15 in comparison with δC 78.17 ppm in compound 1. The H-23a signal was down-field shifted to δH 3.50 instead at δ 3.29 in asiatic acid (1). For obtaining a succinate derivative, which is assumed to be biologically active, compound 171 was reacted with succinic anhydride in DMAP and TEA at room temperature to yield the succinate 172. Besides, compound 171 was acetylated to the acetyl derivative 173, which yielded the 2-acetyl-asiatic acid (174) on hydrolysis. The structures of compounds 171-174 were confirmed by their spectroscopical data. The (-)-ESI-MS of compound 172 showed a pseudomolecular ion peak at m/z 627 [M-H]- (100). Its 1H and 13C-NMR spectra exhibited the corresponding signals at δH 5.03 (1H, dt, J = 4.5, 9.5 Hz, H-2) and 3.54 (1H, d, J = 9.5 Hz, H-3) in comparison with δH 3.73 (1H, dt, J = 4.5, 10.5 Hz, H-2) and 3.40 (1H, d, J = 10.5 Hz, H-3) in compound 171. Carboxyl signals of the succinic residue appeared at δC 171.71 (COO-) and 178.04 (COOH) in the 13C-NMR spectrum of 172. The (-)- ESI-MS spectrum of compound 173 contained a pseudomolecular ion peak at m/z 569 [M-H]- (100). The 2α-O-acetyl group of 173 was evidenced by the signals at δH 4.99 (dt, J = 4.5, 10.5, H-2β) and 2.01 (3H, s, CH3CO) and δC 78.89 (C-2), 170.62 (CH3COO-). The (+)-ESI-MS spectrum of compound 174 showed a pseudomolecular ion peak at m/z 531 [M+H]+ (100). Compared to the 1H and 13C- NMR spectra of compound 173 the resonance signals for the isopropylidene group disappeared in the spectra of compound 174. 17
Scheme 4.2: Synthesis of derivatives of asiatic acid Reagents and conditions: a) 2,2-dimethoxypropane, TsOH.H2O, rt., 7 h, 88%,b) succinic anhydride, DMAP, TEA, rt. 12 h, 74%, c) (CH3CO)2O, pyridine, rt. 12 h, 82%, d) 5% HCl, rt. 4h, 90%. 4.3.2. Synthesis of madecassic acid derivatives Among the chemical constituents of C. asiatica, madecassic acid (10) and its oligoglucoside madecassoside are two of the main active components. It was reported that madecassoside and asiaticoside are responsible for burn wound healing of C. asiatica extract, and madecassoside is more effective than asiaticoside. Madecassic acid lowered glucose level and increased plasma insulin level in a dose-dependent manner if supplied to diabetic mice for six weeks. Madecassic acid and madecassoside inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. Madecassoside possesses excellent anti-rheumatoid arthritis property after oral administration, ameliorates bleomycine-induced pulmonary fibrosis in mice through promoting the generation of hepatocyte growth factor via PPAR-y in colon. Madecassic acid was reported to have protective property against hypoxia- induced oxidative stress in retinal microvascular endothelial cells and to be anti- colitis in mice by oral admimistration. Because of the high content of madecassic acid in C. asiatica, it is interesting to synthesize its derivatives and study their biological activities. 18