Synthesis of an Ursolic Acid Saponin with N-Acetylglucosamine-containing Trisaccharide Residue

Chinese Journal of Chemistry, 2006, 24, 1421—1426 Full Paper

* E-mail: [email protected] Received December 29, 2005; revised March 1, 2006; accepted June 5, 2006. Project supported by the National Natural Science Foundation of China (No. 30400564).

© 2006 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Synthesis of an Ursolic Acid Saponin with N-Acetylglucosamine-containing Trisaccharide Residue

WANG, Peng(王鹏) LI, Chun-Xia(李春霞) WANG, Guang-Fa(王广法) LI, Ying-Xia*(李英霞)

Key Laboratory of Marine Drugs of the Ministry of Education of China, Institute of Marine Drug and Food, Ocean University of China, Qingdao, Shandong 266003, China

The focus of this work is the synthesis of an ursolic acid saponin with an N-acetylglucosamine-containing trisaccharide residue. Therefore, ursolic acid 3-yl α-L-arabinopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→6)-2- acetamido-2-deoxy-β-D-glucopyranoside (1) was concisely synthesized in convergent synthesis with 48.0% overall yield. The structure of saponin 1 was confirmed by 1H NMR, 13C NMR and mass spectra.

Keywords ursolic acid, saponin, N-acetylglucosamine, glycosylation, synthesis

Introduction

Ursolic acid, a pentacyclic triterpene isolated from many traditional medicinal plants, has been reported to possess a wide range of pharmacological activities, in-cluding anti-tumour,1-3 anti-inflammatory4 and anti- HIV.5 It is also one of the most promising chemopre-ventive agents for cancer.6 Attracted by the interesting bioactivities, we have been searching and synthesizing the derivatives of ursolic acid7 for more significant bio-activity. It is well known that the naturally occurring saponins with N-acetylglucosamine oligosaccharide residue are rare, with their numbers being less than 30 and structures highly conservative.8 One of their typical sugar chains, α-L-arabinopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→6)-2-acetamido-2-deoxy-β-D-glucopyranosyl, occurred in the calliandra saponins A—L from Calliandra anomala.9 It is more exciting to find that the saponin of oleanolic acid with this trisaccharide chain shows significant activity against the A2780 and M109 lung cancer cell lines with an IC50 of 0.8 and 1.0 µg/mL, respectively, and it is the most active saponin of oleano-lic acid from nature reported up to now,10 indicating that the N-acetylglucosamine-containing trisaccharide resi-due boosts drastically the bioactivity of oleanolic acid. As a part of our continuing synthesis of ursolic acid saponins7, we have introduced this sugar chain into ur-solic acid to synthesize saponin 1 (Figure 1).

Results and discussion

It is essential for the synthesis of saponins in concise and effective procedure and in high overall yield. Sun. et. al8 reported the synthesis of oleanolic acid saponin

containing 3-O-sugar chain in stepwise glycosylation. Previous experience has shown that the resulting saponin can be prepared in high overall yield in con-vergent glycosylation fashion.7 Herein we planned to adopt the approach of convergent glycosylation to the target molecules, and the synthetic access to saponin 1 was described in this paper.

Figure 1 Structure of saponin 1.

The key trisaccharide donor 13 was prepared as shown in Scheme 1. Starting from 1,3,4,6-tetra-O-ace-tyl-2-deoxy-2-phthalimido-β-D-glucopyranose (2),11 compound 3 was obtained as a pure β-anomer (J1,2＝8.8 Hz) by reacting 2 with p-MeO-C6H4OH under the pro-motion of BF3•Et2O in 81.9% yield. After removal of acetyl groups in 3, 6-OH was protected selectively by Tr group12 and then 3,4-OHs were protected by acetyl groups in situ to produce compound 5 quantitatively. Cleavage of Tr protecting group in 5 with FeCl3•6H2O

13 afforded the key glycosyl acceptor 6 in 69.5% yield. Phenyl 3,4-di-O-acetyl-2-O-levulinoyl-1-thio-α-L-arabi- nopyranoside (7) was used as a synthon for the middle arabinopyranose having a distinguishable 2-O-protec-tion.7 Thus, glycosylation of 6 with 7 under the promo-tion of NIS-TfOH led to disaccharide 8 in satisfactory yield (93.5%). Selective removal of the 2-O-levulinoyl

1422 Chin. J. Chem., 2006, Vol. 24, No. 10 WANG et al.


Scheme 1

Reagents and conditions: (a) p-MeO-C6H4OH, BF3•Et2O, CH2Cl2, 81.9%. (b) NaOMe, MeOH, quant. (c) i TrCl, DMAP, Py; ii Ac2O, Py; quantitative. (d) FeCl3•6H2O, 69.5%. (e) NIS, TfOH (0.3 eq.), 93.5%. (f) NH2NH2•HOAc, quant. (g) 10, TMSOTf (0.1 eq.), 97.9%. (h) CAN (5 eq.), 94.2%. (i) DBU, CCl3CN, r.t., 70.4%.

Scheme 2

Reagents and conditions: (a) TMSOTf (0.2 eq.), －70 ℃; then r.t. for 30 min; 71.8%. (b) i BuOH, NH2CH2CH2NH2; ii Ac2O, Py, 66.8%. (c) MeONa, MeOH, quantitative.

group in the presence of hydrazine acetate in CH2Cl2- MeOH afforded disaccharide acceptor 9 quantitatively. To avoid the formation of acetyl-transfer product,8 the benzoyl-protected arabinopyranosyl trichloroacetimi-date (10) was used as the glycosyl donor for introduc-tion of terminal arabinopyranosyl residue. Herein, gly-cosylation of 10 with acceptor 9 with 2'-OH free under standard glycosylation conditions afforded trisaccharide 11. The anomeric p-methoxyphenol group was removed by ammonium cerium nitrate14 to afford hemiacetal 12

in 94.2% yields, which was then converted into the key trisacchride donor 13 in satisfactory yields. The large coupling constants of J1,2 (7.0 Hz for 12, 8.8 Hz for 13) in 1H NMR signals indicated that 12 and 13 were ob-tained as pure β-anomers, which was different from the usual α, β-mixture.14

The next task was to introduce the trisaccharide residue 13 into ursolic acid. Starting from trityl ursolic ester,12 compound 14 as the acceptor was glycosylated with 13 at －70 ℃, followed by deprotection of Tr

Ursolic acid saponin Chin. J. Chem., 2006 Vol. 24 No. 10 1423


group by warming reaction solution to room tempera-ture for another 30 min to provide 15 in satisfactory yield (71.8%). The Phth group in compound 15 was removed15 in the presence of ethylenediamine in BuOH at 90 ℃ for 12 h to afford, after acetylation (Ac2O, pyridine), precursor 16 for two steps in 66.8% yield. Final removal of all protective groups in 16 (Me-ONa/MeOH) yielded the target saponin 1. Therefore, saponin 1 was synthesized concisely in three steps and 48.0% overall yield starting from trityl ursolic ester. The structure of saponin 1 was confirmed by NMR and MS. The anti-tumor bioactivity of saponin 1 was under in-vestigation.

Experimental

General methods

Solvents were purified in the usual way. Thin layer chromatography was performed on precoated Merk sil-ica gel 60 F254 plates, and flash column chromatography was performed on silica gel (200—300 mesh, Qingdao, China). Optical rotations were determined with a Perkin-Elmer Model 241 MC polarimeter. 1 H NMR and 13C NMR spectra were taken on a JEOL JNM-ECP 600 spectrometer with tetramethylsilane as the internal standard, and chemical shifts are recorded in δ values. Mass spectra were recorded on a Q-TOF Global mass spectrometer.

p-Methoxylphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (3)

To a solution of compound 2 (3.0 g, 6.28 mmol) and p-MeO-C6H4OH (1.95 g, 15.7 mmol) in CH2Cl2 (40 mL) was added BF3•Et2O (1.6 mL, 47%) at 0 ℃. After stirred at r.t. for 7 h, the solution was diluted with CH2Cl2 (120 mL) and successively washed with water (2×50 mL), saturated aqueous NaHCO3 (2×50 mL) and brine (2×50 mL). The organic layer was dried over anhydrous Na2SO4 and then concentrated under vacuum. The residue was purified by silica gel column chroma-tography (4∶1 to 3∶1, V∶V, petroleum ether-EtOAc) to afford compound 3 (2.78 g, 81.9%) as a white solid with Rf 0.44 (1∶1, V∶V, petroleum ether-EtOAc). 1H NMR (CDCl3) δ: 7.74—7.88 (m, 4H, Phth), 6.73—6.86 (m, 4H, PhOCH3), 5.86 (d, J＝8.8 Hz, 1H, H-1), 5.89 (dd, J＝9.1, 10.6 Hz, 1H, H-3), 5.25 (dd, J＝9.1, 10.8 Hz, 1H, H-4), 4.58 (dd, J＝8.4, 10.6 Hz, 1H, H-2), 4.35 (dd, J＝5.2, 12.1 Hz, 1H, H-6-1), 4.18 (dd, J＝2.2, 12.1 Hz, 1H, H-6-2), 3.97 (ddd, J＝2.2, 4.8, 10.3 Hz, 1H, H-5), 3.73 (s, 3H, OCH3), 2.11, 2.05, 1.99 (3s, 9H, 3×Ac).

p-Methoxylphenyl 2-deoxy-2-phthalimido-β-D-glucopyranoside (4)

Compound 3 (2.78 g, 5.14 mmol) was dissolved in MeOH-CH2Cl2 (1∶1, 70 mL), and then NaOMe in MeOH (50%) was added to adjust pH 8—9. After stirred at r.t. for 30 min, the solution was neutralized with ion-exchange resin (H＋), and then filtered and

concentrated to provide 4 (2.13 g, 100%) as a white solid. 1H NMR (CD3OD) δ: 7.90—7.82 (m, 4H, Phth), 6.72—6.83 (m, 4H, PhOCH3), 5.65 (d, J＝8.5 Hz, 1H, H-1), 4.30 (dd, J＝8.4, 10.7 Hz, 1H, H-3), 4.21 (dd, J＝8.4, 10.6 Hz, 1H, H-2), 3.94 (dd, J＝2.2, 12.1 Hz, 1H, H-6-1), 3.78 (dd, J＝5.5, 12.2 Hz, 1H, H-6-2), 3.68 (s, 3H, OCH3), 3.54 (ddd, J＝2.2, 5.5, 9.9 Hz, 1H, H-5), 3.49 (dd, 1H, J＝9.9, 8.5 Hz, H-4).

p-Methoxylphenyl 3,4-di-O-acetyl-6-O-trityl-2-deoxy- 2-phthalimido-β-D-glucopyranoside (5)

Compound 4 (2.12 g, 5.14 mmol) was dissolved in anhydrous pyridine (40 mL), and then TrCl (7.16 g, 25.70 mmol) and DMAP (0.18 g, 1.03 mmol) were added. After stirred at 80 ℃ for 3 h, the mixture was cooled to r.t., and then Ac2O (10 mL) was added. The mixture was stirred continuously at r.t. for overnight, and then diluted with EtOAc (200 mL) and successively washed with water (3×50 mL), saturated aqueous Na-HCO3 (3×50 mL) and brine (3×50 mL). The organic layer was dried over anhydrous Na2SO4 and then con-centrated under vacuum. The residue was purified by silica gel column chromatography (4∶1, V∶V, petro-leum ether-EtOAc) to produce compound 5 (3.81 g, 100%) as a white solid with Rf 0.37 (1∶1, V∶V, petro-leum ether-EtOAc). 1H NMR (CDCl3) δ: 7.74—7.87 (m, 4H, Phth), 7.23—7.87 (m, 15H, 3×Ph), 6.74—7.00 (m, 4H, PhOCH3), 5.84 (d, J＝8.5 Hz, 1H, H-1), 5.79 (dd, J＝9.2, 10.6 Hz, 1H, H-3), 5.23 (dd, J＝9.2, 10.3 Hz, 1H, H-4), 4.61 (dd, J＝8.4, 10.6 Hz, 1H, H-2), 3.81 (ddd, J＝2.2, 5.5, 10.6 Hz, 1H, H-5), 3.73 (s, 3H, OCH3), 3.26 (dd, J＝2.2, 10.6 Hz, 1H, H-6-2), 3.21 (dd, J＝5.5, 10.3 Hz, 1H, H-6-2), 1.87, 1.75 (s each, 3H each, 2×Ac).

p-Methoxylphenyl 3,4-di-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (6)

To a solution of 5 (1.70 g, 2.29 mmol) in dried CH2Cl2 (30 mL) was added FeCl3•6H2O (1.25 g, 4.58 mmol). After stirred at r.t. for 5 h, the solution was di-luted with CH2Cl2 (100 mL) and successively washed with water (3×50 mL) and brine (3×50 mL). The or-ganic layer was dried over anhydrous Na2SO4 and then concentrated in vacuo. The residue was purified by sil-ica gel column chromatography (1∶1, V∶V, petroleum ether-EtOAc) to yield compound 6 (0.79 g, 69.5%) as a white solid with Rf 0.23 (1∶1, V∶V, petroleum ether- EtOAc). 1H NMR (CDCl3) δ: 7.87—7.74 (m, 4H, Phth), 6.74—6.85 (m, 4H, PhOCH3), 5.92, (d, J＝8.4 Hz, 1H, H-1), 5.90 (dd, J＝8.8, 10.6 Hz, 1H, H-3), 5.19 (t, J＝9.9 Hz, 1H, H-4), 4.55 (dd, J＝8.5, 10.3 Hz, 1H, H-2), 3.77—3.82 (m, 2H, H-5, H-6-1), 3.72 (s, 3H, OCH3), 3.66—3.69 (m, 1H, H-6-2), 2.32 (dd, J＝5.9, 8.1 Hz, 1H, OH), 2.08, 1.90 (s each, 3H each, 2×Ac); 13C NMR (CDCl3) δ: 170.1, 155.6, 150.3, 114.4—134.3 (Ph), 97.1 (C-1), 74.2, 70.5, 69.1, 61.2, 55.5, 54.5, 20.6, 20.4; ESI-MS calcd for C25H29NO10 517.2, found 517.2 [M＋NH4]

＋; ESI-MS calcd for C25H25NNaO10 522.2, found 522.2 [M＋Na]＋.



p-Methoxylphenyl 2-O-lev-3,4-di-O-acetyl-α-L-arabinopyranosyl-(1→6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (8)

To a mixture of compound 6 (0.90 g, 1.80 mmol), 7 (1.15 g, 2.70 mmol) and powdered 0.4 nm molecular sieves (300 mg) in dried CH2Cl2 (20 mL) at －20 ℃ were added NIS (0.81 g, 3.60 mmol) and TfOH (47 µL, 0.54 mmol). After stirred at －20 ℃ for 2 h, the reac-tion mixture was quenched with Et3N. The solid was then filtered off. The filtrate was concentrated under vacuum to produce an yellow syrup, which was purified by column chromatography (1∶1, V∶V, petroleum ether- EtOAc) to afford compound 8 (1.37 g, 93.5%) as a white solid with Rf 0.30 (1∶1, V∶V, petroleum ether- EtOAc). 1H NMR (CDCl3) δ: 6.79—7.86 (m, 8H, Ph, NPhth), 5.88 (d, J＝8.7 Hz, 1H, H-1), 5.85 (dd, J＝9.1, 10.5 Hz, 1H, H-3), 5.23 (brs. 1H, H-4'), 5.21 (dd, J＝7.4, 9.7 Hz, 1H, H-2'), 5.06 (dd, J＝9.2, 10.6 Hz, 1H, H-4), 5.02 (dd, J＝3.2, 9.7 Hz, 1H, H-3'), 4.50—4.54 (m, 2H, H-1', H-2), 4.01—4.05 (m, 2H, H-5'-1, H-5), 3.92 (dd, J＝1.8, 11.4 Hz, 1H, H-6-1), 3.72—3.75 (m, 4H, Ph-OCH3, H-6-2), 3.57 (d, J＝12.8 Hz, 1H, H-5'-2), 2.43—2.70 (m, 4H, Lev), 2.15, 2.14, 2.08, 2.05, 1.88 (s each, 3H each, Ac×5); 13C NMR (CDCl3) δ: 206.1 (CH3COCH2CH2COO), 171.4, 170.3, 170.1, 169.7, 155.5, 150.6, 134.4, 131.2, 126.7, 118.2, 114.6, 100.7 (C-1'), 97.0 (C-1), 73.8 (C-5), 70.6 (C-3), 70.0 (C-3'), 69.4 (C-4), 69.0 (C-2'), 67.8 (C-4'), 67.5 (C-6), 55.6 (PhOCH3), 54.5 (C-2), 37.6, 29.6, 27.7, 20.9, 20.7, 20.6, 20.4; ESI-MS calcd for C39H43NNaO18 836.2, found 836.3 [M＋Na]＋.

p-Methoxylphenyl 3,4-di-O-acetyl-α-L-arabinopyranosyl-(1→6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido- β-D-glucopyranoside (9)

To a solution of 8 (1.07 g, 1.31 mmol) in CH2Cl2- MeOH (1∶1, 10 mL) was added NH2NH2• HOAc (0.24 g, 2.62 mmol). After stirred at r.t. for 3 h, the mixture was concentrated in vacuo to give a yellow syrup, which was purified by column chromatography (1∶1, V∶V, petroleum ether-EtOAc) to yield compound 9 (0.93 g, 100%) as a white solid with Rf 0.22 (1∶2, V∶V, petro-leum ether-EtOAc). 1H NMR (CDCl3) δ: 6.74—7.76 (m, 8H, Phth, Ph), 5.90 (d, J＝8.7 Hz, 1H, H-1), 5.88 (dd, J＝9.2, 10.6 Hz, 1H, H-3), 5.36 (t, J＝9.6 Hz, 1H, H-4), 5.26 (s-like, 1H, H-4'), 4.90 (dd, J＝3.7, 10.1 Hz, 1H, H-3'), 4.58 (dd, J＝8.3, 10.6 Hz, 1H, H-2), 4.25 (d, J＝7.8 Hz, 1H, H-1'), 4.14 (dd, J＝1.4, 12.4 Hz, 1H, H-6-1), 4.00 (dd, J＝2.3, 13.3 Hz, 1H, H-5'-1), 3.96—3.99 (m, 1H, H-5), 3.88 (dd, J＝7.3, 10.1 Hz, 1H, H-2'), 3.73 (s, 3H, PhOCH3), 3.66 (dd, J＝5.5, 11.0 Hz, 1H, H-6-2), 3.60 (d, J＝12.8 Hz, 1H, H-5'-2), 2.78 (brs, 1H, OH), 2.14, 2.09, 2.06, 1.90 (s each, 3H each, 4×Ac); 13C NMR (CDCl3) δ: 170.3, 170.1, 155.6, 150.4, 134.4, 131.3, 123.7, 118.6, 114.4, 103.8, 97.2, 73.0, 72.1, 70.6, 69.0, 68.9, 64.3, 55.5, 54.5, 20.9, 20.8, 20.7, 20.4; ESI-MS calcd for C34H37NNaO16 738.2, found 738.3 [M＋Na]＋.

p-Methoxylphenyl 2,3,4-tri-O-benzoyl-α-L-arabinopyranosyl-(1→2)-3,4-di-O-acetyl-α-L-arabinopyranosyl- (1→6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (11)

To a mixture of compound 9 (0.18 g, 0.25 mmol), 10 (0.23 g, 0.38 mmol) and powdered molecular sieves (0.4 nm, 300 mg) in dried CH2Cl2 (8 mL) at －40 ℃ was added TMSOTf (4.4 µL, 0.025 mmol) under Ar protec-tion. After stirred at －40 ℃ for 45 min, the mixture was quenched with Et3N. The solid was then filtered off and the filtrate was concentrated under vacuum to afford an yellow syrup, which was purified by column chro-matography (2∶ 1→1∶ 1, V∶V, petroleum ether- EtOAc) to yield compound 11 (0.28 g, 97.9%) with Rf 0.50 (1∶1, V∶V, petroleum ether-EtOAc). 1H NMR (CDCl3) δ: 6.84—8.01 (m, 23H, Ph), 5.99 (d, J＝8.7 Hz, 1H, H-1), 5.90 (dd, J＝9.2, 10.5 Hz, 1H, H-3), 5.61 (dd, J＝6.9, 9.2 Hz, 1H, H-2"), 5.59—5.60 (m, 1H, H-4"), 5.14—5.15 (m, 1H, H-4'), 5.09—5.12 (m, 2H, H-3", H-4), 4.97 (d, J＝6.9 Hz, 1H, H-1"), 4.92 (dd, J＝3.7, 8.7 Hz, 1H, H-3'), 4.60 (d, J＝6.0 Hz, 1H, H-1'), 4.55 (dd, J＝8.3, 10.6 Hz, 1H, H-2), 4.32 (dd, J＝3.7, 13.3 Hz, 1H, H-5"-1), 4.10—4.15 (m, 1H, H-5), 3.89—3.95 (m, 3H, H-2', H-5'-1, H-6-1), 3.82—3.86 (m, 2H, H-5"-2, H-6-2), 3.56 (dd, J＝2.8, 13.7 Hz, 1H, H-5'-2), 2.07, 1.95, 1.89, 1.76 (s each, 3H each, 4×Ac); 13C NMR (CDCl3) δ: 170.3, 170.2, 170.0, 169.6, 165.6, 165.3, 165.0, 155.5, 150.2, 134.4, 133.3, 133.2, 133.2, 131.3, 128.3—129.9 (Ph), 123.7, 117.6, 114.8, 101.9, 100.9, 96.1, 76.3, 73.7, 71.1, 70.8, 70.7, 69.6, 68.2, 67.8, 67.2, 62.3, 55.5, 54.6, 20.7, 20.7, 20.5, 20.3; ESI-MS calcd for C60H57NNaO23 1182.3, found 1182.3 [M＋Na]＋.

2,3,4-Tri-O-benzoyl-α-L-arabinopyranosyl-(1→2)-3,4-di-O-acetyl-α-L-arabinopyranosyl-(1→6)-3,4-di-O- acetyl-2-deoxy-2-phthalimido-β-D-glucopyranose (12)

To a solution of 11 (1.02 g, 0.88 mmol) in CH3CN- H2O (4∶1, 25 mL) was added ammonium cerium ni-trate (2.41 g, 4.40 mmol). After stirred at r.t. for 1 h, the mixture was diluted with EtOAc (100 mL) and succes-sively washed with water (3×30 mL) and brine (3×30 mL). The organic layer was dried over anhydrous Na2SO4 and then concentrated in vacuo to yield a yel-low syrup, which was purified by column chromatog-raphy (1∶1, V∶V, petroleum ether-EtOAc) to yield compound 12 (0.87 g, 94.2%) as a white solid with Rf 0.30 (1∶2, V∶V, petroleum ether-EtOAc). 1H NMR (CDCl3) δ: 7.36—8.05 (m, 19H, 3×Bz, Phth), 5.82 (dd, J＝8.8, 10.6 Hz, 1H, H-3), 5.73—5.74 (m, 1H, H-4"), 5.69 (dd, J＝6.2, 8.0 Hz, 1H, H-2"), 5.64 (dd, J＝3.7, 8.1 Hz, 1H, H-3"), 5.16 (s-like, 1H, H-4'), 5.08 (d, J＝4.8 Hz, 1H, H-1"), 4.92 (dd, J＝9.1, 10.2 Hz, 1H, H-4), 4.86 (dd, J＝3.3, 9.5 Hz, 1H, H-3'), 4.60 (d, J＝5.1 Hz, 1H, H-1'), 4.57 (d, J＝7.0 Hz, 1H, H-1), 4.20 (dd, J＝8.4, 10.6 Hz, 1H, H-2), 4.05—4.07 (m, 1H, H-5), 3.98— 4.03 (m, 3H, H-5'-1, H-5"-1, H-6-1), 3.92—3.95 (m, 2H, H-5"-2, H-6-2), 3.68 (dd, J＝8.8, 11.7 Hz, 1H, H-2'),

Ursolic acid saponin Chin. J. Chem., 2006 Vol. 24 No. 10 1425


3.59 (dd, J＝1.1, 12.8 Hz, 1H, H-5'-2), 2.03, 2.00, 1.96, 1.87 (s each, 3H each, 4×Ac); 13C NMR (CDCl3) δ: 170.1, 170.1, 170.0, 169.8, 167.6, 165.6, 165.4, 123.6—134.7 (Ph), 102.2, 100.0, 92.4, 74.3, 73.9, 72.5, 70.7, 70.0, 69.8, 68.6, 68.2, 67.9, 63.4, 55.9, 54.5, 20.9, 20.8, 20.7, 20.5; ESI-MS calcd for C53H54N2O22 1071.3, found 1071.3 [M＋NH4]

＋; ESI-MS calcd for C53H51N- NaO22 1076.3, found 1076.3 [M＋Na]＋.

2,3,4-Tri-O-benzoyl-α-L-arabinopyranosyl-(1→2)-3,4-di-O-acetyl-α-L-arabinopyranosyl-(1→6)-3,4-di-O- acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl- trichloroacetimidate (13)

To a solution of 12 (0.85 g, 0.81 mmol) in dried CH2Cl2 (15 mL) were added DBU (60 µL, 0.40 mmol) and CCl3CN (0.98 mL, 9.72 mmol) at 0 ℃. After stirred at r.t. for 1 h, the mixture was concentrated in vacuo to afford a yellow syrup, which was purified by silica gel column chromatography (2∶1, V∶V, petro-leum ether-EtOAc) to afford 13 (0.68 g, 70.4%) as a foam solid with Rf 0.50 (1 ∶ 2, V∶V, petroleum ether-EtOAc). 1H NMR (CDCl3) δ: 8.91 (s, 1H, NH), 7.27—8.06 (m, 19H, Ph), 6.70 (d, J＝8.8 Hz, 1H, H-1), 5.93 (dd, J＝9.1, 10.6 Hz, 1H, H-3), 5.68 (dd, J＝6.5, 8.5 Hz, 1H, H-2"), 5.63—5.64 (m, 1H, H-4"), 5.51 (dd, J＝3.3, 8.8 Hz, 1H, H-3"), 5.21 (t, J＝9.1 Hz, 1H, H-4), 5.12 (d, J＝6.6 Hz, 1H, H-1"), 5.11 (s, 1H, H-4'), 4.94 (dd, J＝3.3, 7.7 Hz, 1H, H-3'), 4.63 (d, J＝5.5 Hz, 1H, H-1'), 4.59 (dd, J＝8.8, 10.6 Hz, 1H, H-2), 4.34 (dd, J＝3.7, 12.8 Hz, 1H, H-5"-1), 4.15—4.17 (m, 1H, H-5), 3.93—4.01 (m, 4H, H-5"-2, H-6-1, H-6-2, H-5'-1), 3.78 (dd, J＝5.9, 11.0 Hz, 1H, H-2'), 3.57 (dd, J＝2.2, 12.5 Hz, 1H, H-5΄-2), 2.07 (s, 3H, Ac), 1.90 (s, 9H, 3×Ac).

Ursolic acid-3-yl 2,3,4-tri-O-benzoyl-α-L-arabinopy- ransyl-(1→2)-3,4-di-O-acetyl-α-L-arabinopyranosyl- (1→6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-β-D- glucopyranoside (15)

To a mixture of compound 14 (0.24 g, 0.35 mmol), 13 (0.50 g, 0.42 mmol) and powdered molecular sieve (0.4 nm, 300 mg) in dried CH2Cl2 (8 mL) at －70 ℃ was added TMSOTf (12.2 µL, 0.070 mmol) under Ar protection. After stirred at －70 ℃ for 45 min and then at r.t. for 30 min, the mixture was quenched with Et3N. The solid was then filtered off and the filtrate was concentrated under vacuum to afford a yellow syrup, which was purified by silica gel (200—300 mesh) col-umn chromatography (2∶1, V∶V , petroleum ether-EtOAc) to yield compound 15 (0.38 g, 71.8%) as a white solid with Rf 0.22 (1∶1, V∶V, petroleum ether-EtOAc). 20

D[ ]α ＋92.2 (c 0.20, CHCl3); 1H NMR

(DMSO) δ: 7.00—7.95 (m, 19H, 3×Bz, Phth), 5.70—5.76 (m, 2H, H-3', H-3'''), 5.67 (d-like, H-4'''), 5.48 (dd, J＝7.3, 9.2 Hz, 1H, H-2'''), 5.27 (d, J＝7.8 Hz, 1H, H-5'''), 5.25 (d, J＝7.3 Hz, 1H, H-1'), 5.06 (t-like, 1H, H-12), 4.97 (d-like, 1H, H-4"), 4.90 (t, J＝9.7 Hz, 1H, H-4'), 4.77 (dd, J＝3.7, 8.7 Hz, 1H, H-3"), 4.73 (d, J＝6.4 Hz, 1H, H-1"), 4.33 (d, J＝11.0 Hz, 1H, H-5'''-1), 4.25 (d, J＝12.8 Hz, 1H, H-5'''-2), 4.09 (dd, J＝8.7,

11.0 Hz, 1H, H-2΄), 3.91—3.94 (m, 1H, H-5'), 3.80—3.83 (m, 4H, H-2", H-5"-1, H-5-2", H-6'-1), 3.65 (d, J＝11.9 Hz, 1H, H-6'-2), 3.27 (dd, J＝4.6, 11.5 Hz, 1H, H-3), 2.25, 2.05, 1.81, 1.78 (s each, 3H each, 4×Ac), 2.08 (d, J＝11.9 Hz, 1H, H-18), 1.02, 0.74, 0.66, 0.36, 0.23 (s each, 3H each, 5×Me), 0.82 (d, J＝6.4 Hz, 3H), 0.53 (d, J＝13.3 Hz, 1H, H-5); 13C NMR (DMSO) δ: 178.2 (C-28), 164.4— 169.6 (Bz, Ac), 143.5, 142.0, 140.5, 137.8 (C-13), 136.9 135.0, 134.3, 133.6, 133.5, 130.6, 130.3, 128.1—129.3, 127.5, 126.6, 125.5, 124.6 (C-12), 123.5, 123.2, 101.8 (C-1"), 100.5 (C-1'''), 99.0 (C-1'), 87.9 (C-3), 75.8 (C-2"), 73.1 (C-5'), 71.3 (C-3'''), 70.9 (C-2'''), 70.3 (C-3"), 69.9 (C-3'), 69.2 (C-4'), 69.0 (C-4'''), 66.9 (C-4"), 66.8 (C-5"), 63.2 (C-5'''), 62.0 (C-6'), 54.3 (C-2'), 54.2 (C-5), 52.1 (C-18), 39.9, 38.9, 38.4, 37., 37.5, 37.2, 36.2, 36.0, 32.3, 30.0, 27.6, 27.4, 26.7, 25.4, 24.0, 23.8, 23.7, 22.8, 22.5, 21.0, 20.9, 20.1, 17.3, 17.0, 16.7, 16.0, 14.9; ESI-MS calcd for C83H96NO24 1490.6322, found 1490.6393 [M－H]－.

Ursolic acid-3-yl 2,3,4-tri-O-acetyl-α-L-arabinopyranosyl-(1→2)-3,4-di-O-acetyl-α-L-arabinopyranosyl- (1→6)-3,4-di-O-acetyl-2-deoxy-2-acetamido-β-D-glucopyranoside (16)

To a mixture of 15 (0.35 g, 0.23 mmol) in BuOH (20 mL) was added NH2CH2CH2NH2 (10 mL). After stirred during 80—90 ℃ overnight, the mixture was concen-trated to afford a yellow syrup, which was coevaporated with toluene twice and then dissolved in 1∶1 pyri-dine-Ac2O (10 mL). The resulting mixture was stirred at r.t. overnight and then concentrated in vacuo to yield an yellow solid, which was purified by silica gel column chromatography (50∶1, V∶V, CHCl3-CH3OH) to pro-vide compound 16 (0.19 g, 66.8%) as a white solid with Rf 0.40 (20∶1, V∶V, CHCl3-CH3OH). 20

D[ ]α ＋8.9 (c 0.20, CHCl3);

1H NMR (CDCl3) δ: 11.22 (brs, 1H, COOH), 5.70 (d, J＝7.7 Hz, 1H, NHAc), 5.26—5.27 (m, 2H, H-3', H-4'''), 5.23 (t, J＝3.3 Hz, 1H, H-12), 5.12—5.17 (m, 2H, H-2''', H-4"), 4.99—5.03 (m, 3H, H-3", H-3''', H-4'), 4.67 (d, J＝7.3 Hz, 2H, H-1', H-1'''), 4.63 (d, J＝4.4 Hz, 1H, H-1"), 4.05 (dd, J＝2.9, 12.8 Hz, 1H, H-5'''-1), 3.99 (dd, J＝6.2, 11.8 Hz, 1H, H-5"-1), 3.77—3.88 (m, 3H, H-2', H-2", H-6'-1), 3.65—3.69 (m, 3H, H-5', H-6'-2, H-5'''-2), 3.52 (dd, J＝3.3, 12.1 Hz, 1H, H-5"-2), 314 (dd, J＝4.4, 11.3 Hz, 1H, H-3), 2.18 (d, J＝10.6 Hz, 1H, H-18), 2.14, 2.12, 2.10, 2.09, 2.03, 2.02, 2.01, 1.93 (s each, 3H each, 8×Ac), 1.06, 0.92, 0.91, 0.75, 0.74 (s each, 3H each, 5×Me), 0.94 (d, J＝6.2 Hz, 3H, H-30), 0.86 (d, J＝6.2 Hz, 3H, H-29); 13C NMR (CDCl3) δ: 183.3 (C-28), 169.3—171.1 (CH3CO), 137.9 (C-13), 125.8 (C-12), 103.0, 100.9, 100.4, 89.9 (C-3), 74.9, 72.9, 72.4, 70.3, 69.4, 69.1, 67.6, 67.2, 66.4, 63.4, 55.4, 55.3, 52.5, 47.9, 47.5, 41.8, 39.4, 39.0, 38.9, 38.8, 38.4, 36.7, 32.8, 30.6, 28.0, 27.9, 25.9, 24.0, 23.5, 23.4, 23.2, 21.2, 21.0, 20.8, 20.7 (×2), 18.1, 17.1, 17.0, 16.4, 15.4; ESI-MS calcd for C62H92NO23 1218.6060, found 1218.6085 [M＋H]＋.



Ursolic acid 3-yl α-L-arabinopyranosyl-(1→2)-α-L- arabinopyranosyl-(1→6)-2-deoxy-2-acetamido-β-D- glucopyranoside (1)

Compound 16 (0.106 g, 0.087 mmol) was dissolved in MeOH-CH2Cl2 (2∶1, 12 mL), and then NaOMe in MeOH (20 mg, 50%) was added. After stirred at r.t. for 4 h, the solution was neutralized with ion-exchange resin (H＋), and then filtered and concentrated. The residue was purified by column chromatography (20∶1→2∶1, V∶V, CHCl3-MeOH) to afford 1 as an amor-phous solid (0.080 g, 100%) with Rf 0.40 (2∶2∶0.1, V∶V∶V, CHCl3-CH3OH-H2O). 20

D[ ]α ＋12.4 (c 0.20, CH3OH); 1H NMR (CD3OD) δ: 5.21 (t, J＝3.3 Hz, 1H, H-12), 4.50 (d, J＝5.9 Hz, 1H), 4.48 (d, J＝7.0 Hz, 1H), 4.42 (d, J＝8.4 Hz, 1H), 4.05 (d, J＝10.6 Hz, 1H), 3.97 (dd, J＝2.9, 12.4 Hz, 1H), 3.43—3.85 (m, 13H), 3.16 (dd, J＝4.4, 11.4 Hz, 1H, H-3), 2.19 (d, J＝11.3 Hz, 1H, H-18), 1.94 (s, 3H, Ac), 1.11, 0.96, 0.94, 0.83, 0.75 (s each, 3H each, Me×5), 0.94 (d, J＝7.6 Hz, 3H, H-30), 0.87 (d, J＝6.2 Hz, 3H, H-29), 0.77 (d, J＝11.7 Hz, 1H, H-5); 13C NMR (CD3OD) δ: 173.4 (C-28), 139.7 (C-13), 126.8 (C-12), 105.8, 104.9, 103.4, 90.6, 80.4, 76.4, 75.7, 74.2, 73.4, 72.9, 72.0, 69.7, 69.5, 68.7, 67.2, 65.7, 57.7, 56.8, 54.8, 54.4, 43.3, 40.8, 40.5, 40.4, 39.9, 39.8, 38.2, 37.8, 34.3, 31.8, 29.2, 28.6, 27.0, 25.4, 24.4, 24.1, 23.1, 21.6, 19.3, 17.9, 17.7, 17.1; ESI-MS calcd for C48H77- NO16 924.5321, found 924.5333 [M＋H]＋.

References

1 Hsu, Y. L.; Kuo, P. L.; Lin, C. C. Life Sci. 2004, 75, 2303. 2 Es-saady, D.; Simon, A.; Ollier, M.; Maurizis, J. C.; Chulia,

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Okabe, H.; Cosentino, L. M.; Lee, K. H. J. Nat. Prod. 2000, 63, 1619.

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11 Baker, B. R.; Joseph, J. P.; Schaub, R. E.; William, J. H. J. Org. Chem. 1954, 19, 1786.

12 Yu, B.; Xie, J.; Deng, S.; Hui, Y. J. Am. Chem. Soc. 1999, 121, 12196.

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(E0512293 SONG, J. P.; LING, J.)

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