Synthesis and Anti-Fungal Activity of Seven Oleanolic Acid Glycosides

Abstract

In order to develop potential anti-fungal agents, seven glycoconjugates composed of α-L-rhamnose, 6-deoxy-α-L-talose, β-D-galactose, α-D-mannose, β-D-xylose-(1→4)-6-deoxy-α-L-talose, β-D-galactose-(1→4)-α-L-rhamnose, β-D-galactose-(1→3)-β-D-xylose-(1→4)-6-deoxy-α-L-talose as the glycone and oleanolic acid as the aglycone were synthesized in an efficient and practical way using glycosyl trichloroacetimidates as donors. The structures of the new compounds were confirmed by MS, ¹H-NMR and ¹³C- NMR. Preliminary studies based on means of mycelium growth rate, indicated that all the compounds possess certain fungicidal activity against Sclerotinia sclerotiorum (Lib.) de Bary, Rhizoctonia solani Kuhn, Botrytis cinerea Pers and Phytophthora parasitica Dast.

1. Introduction

During the course of growth and development, plants synthesize triterpenoid saponins which act as preformed chemical barriers against fungal attack [1]. Aside from their important role in plant growth, these glycosylated plant secondary metabolites show various kinds of biological activity and have been used widely as anti-inflammatory, anti-tumor, anti-HIV, and antifungal agents [2]. Consequently, triterpenoid saponin structures have become the synthetic targets of many research groups [3,4]. One common feature shared by all saponins is the presence of a sugar chain at the C-3 of the aglycone moiety [5,6]. These chains vary from saponin to saponin but usually consist of glucose, arabinose, glucuronic acid, xylose or rhamnose [7].

Recently Yadava et. al. reported a new triterpenoid saponin isolated from the seeds of L. scariola, which had the structure of 3-O-[β-D-galactopyranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-O-α-L-rhamnopyranosyl]-oleanolic acid (Figure 1, I) [8]. Interestingly, this triterpenoid saponin exhibited broad spectrum antibacterial and antifungal activities against Staphylococcus aureus, Escherichia coli, Penicillium digitatum and Aspergillus niger [8]. In a project for the discovery of novel environmentally friendly antifungal agents from natural resources, we engaged in the study of the synthesis and anti-fungal activity of glycoconjugate derivatives 1-7. We report herein the preliminary results of the study.

Figure 1. Structure of the triterpenoid saponin (I) and target compounds 1–7.

Figure 1. Structure of the triterpenoid saponin (I) and target compounds 1–7.

2. Results and Discussion

2.1. Chemistry

As shown in Figure 2, we envisioned that the target compounds 1-7 could be synthesized using nine suitably protected building blocks 9-16.

Figure 2. The building blocks 9-16 used for the synthesis of target compounds 1-7.

Figure 2. The building blocks 9-16 used for the synthesis of target compounds 1-7.

In our work, the Schmidt method [9] was used in the glycosylation, and benzyl was chosen as the protective group for the COOH group to avoid difficulties in the final deprotection.Since the synthons 9 [10], 12 [11], 13 [12] and 14 [13] were easily prepared according to the reported procedures, our attention was focused on the synthesis of 4-O-allyl-2,3-di-O-benzoyl-α-L-rhamnopyranosyl trichloroacetimidate (10), 4-O-allyl-2,3-di-O-benzoyl-6-deoxy-α-L-talopyranosyl trichloroacetimidate (11), 2,3,4,6-tetra–O-benzoyl-β-D-galactopyranose-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl tri-chloroacetimidate (15)and benzyl oleanolate 3-O-2,3-di-O-benzoyl-α-L-talopyranoside (16) (Scheme 1).

Scheme 1. Synthetic routes to the compounds 10, 11, 15 and 16.

Scheme 1. Synthetic routes to the compounds 10, 11, 15 and 16.

Among these compounds, donor 10 was prepared from the known p-methoxyphenyl 2,3-O-isopropylidene-α-L-rhamnopyranoside (17) [14]. Allylation of 17 with allyl bromide provided the corresponding p-methoxyphenyl 4-O-allyl-2,3-O-isopropylidene-α-L-rhamnopyranoside 18 quantitatively; then removal of the isopropylidene group with 70% HOAc followed by benzoylation gave 19 in high yield (88%); finally, cleavage of the p-methoxyphenyl glycoside in 19 with ceric ammonium nitrate (CAN) followed by trichloroacetimidation afforded the corresponding glycosyl donor 10 in 72% yield. Meanwhile, the donor 11 was prepared in a similar way, i.e., allylation of 20 [15] provided the corresponding p-methoxyphenyl 4-O-allyl-2,3-O-isopropylidene-6-deoxy-α-L-talo-pyranoside 21 quantitatively; and removal of the isopropylidenyl group followed by benzoylation gave 22 in high yield (80%); finally, cleavage of the p-methoxyphenyl glycoside in 22 with ceric ammonium nitrate (CAN) followed by trichloroacetimidation afforded the corresponding glycosyl donor 11 in 70% yield. Condensation of donor 11 with C-3-OH acceptor 9 [10] in the presence of TMSOTf gave the α-linked 6-deoxy-taloside 23, whose ¹H-NMR spectrum showed characteristic signals of a doublet at δ 5.42 ppm (J_1,2 = 3.6 Hz) for the H-1 of 6-deoxytalose, a multiplet at δ 5.88 ppm for CH₂=CH-CH₂O, and seven singlets at δ 1.12, 1.01, 0.92, 0.91, 0.89, 0.83, 0.60 ppm for the CH₃ groups of oleanolic acid. Deallylation of 23 with PdCl₂ gave the desired acceptor 16, and the ¹H- NMR showed that the characteristic allyl signals had disappeared.

On the other hand, we have also developed a novel strategy for the synthesis of 2,3,4,6-tetra–O-benzoyl-β-D-galactopyranose-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl trichloroacetimidate15 from 2,3,4,6-tetra–O-benzoyl-β-D-galactopyranosyl trichloroacetimidate 12 and 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose 24 [16]. 2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranose-(1→3)-1,2-O-isopropylidene-β-D-xylose 28 was conveniently prepared from 25 in 70% overall yield, via selective removal of the 5,6-O-isopropylidene group followed by NaIO₄ oxidation and NaBH₄ reduction in a similar way as reported in [17]. Subsequently, hydrolysis of 28 was carried out in an aqueous solution of sulfuric acid (4%) under heating at reflux, and the reaction was accompanied by ring expansion [18] to provide 2,3,4,6-tetra–O-benzoyl-β-D-galactopyranose-(1→3)-β-D-xylose 29, which was benzoylated with benzoyl chloride in pyridine. Regioselective removal of the 1-O-benzoyl group in 2 M MeOH-NH₃ followed by trichloroacetimidation with trichloroacetonitrile [9] afforded building block 15 in 68% yield (3 steps). Finally, condensation of the donor 10 with the acceptor 9 in the presence of TMSOTf gave benzyl oleanolate 3-O-4-O-allyl-2,3-di-O-benzoyl-α-L-rhamnopyranoside 30 in 88% yield (Scheme 2). The structure was confirmed by its ¹H-NMR spectrum, showing characteristic signals at δ 4.97 ppm (J_1,2 = 1.7 Hz) for the H-1 of rhamnose, δ 5.81 ppm for CH₂=CH-CH₂O, and δ 1.12, 1.02, 0.92, 0.92, 0.89, 0.88, 0.61 ppm for CH₃ of oleanolic acid, the ¹³C-NMR spectrum showed peaks at δ 99.6 ppm for anomeric C-1. Deallylation of 30 gave the desired acceptor 31 in 94% yields. The other five oleanolic acid glycosides 34, 36, 38, 40 and 42 were prepared from condensation of the donors and the acceptors 12 and 9, 13 and 9, 14 and 16, 12 and 31, 15 and 16 respectively, giving 86%~90% yields.

Scheme 2. Synthesis of the target compounds 1-7.

Scheme 2. Synthesis of the target compounds 1-7.

The 28-O-benzyl groups in 31, 16,34, 36, 38, 40, 42 were removed with Pd-C under H₂ atmosphere, and then the O-benzoyl groups were cleaved with MeOH-MeONa [19], furnishing the target compounds 1-7 in satisfactory yields, the structure of the target compounds were established by ¹H-NMR and ¹³C-NMR spectroscopy. For example, the ¹H-NMR spectrum of 7 showed characteristic signals such as δ 5.26, 5.25, 4.72 ppm for three H-1, and δ 1.28, 1.00, 0.99, 0.95, 0.90, 0.83, 0.78 for the CH₃ groups of oleanolic acid, the ¹³C-NMR spectrum showed peaks at δ 106.3, 105.9, 104.9 ppm for three anomeric C-1s.

2.2. Bioassay of Fungicidal Activities

Fungicidal activities of the target compounds against Sclerotinia sclerotiorum (Lib.) de Bary, Rhizoctonia solani Kuhn, Botrytis cinerea Pers and Phytophthora parasitica Dast were evaluated using the mycelium growth rate test [20]. The diameter of the mycelia was measured and the inhibition rate was calculated according to formula (1):

(1)

where I is the inhibition rate, is the average diameter of mycelia in the blank test, and is the average diameter of mycelia in the presence of compounds 1-7: The inhibition rates of compounds 1-7 against the four fungi at 50 µg/mL are given in Table 1. Compounds 1-7 exhibited more fungicidal activity against R. solani than the other fungi, compounds 1 and 2 are more active against B. cinerea and Phytophthora parasitica Dast than the other compounds.

Table 1. Inhibition Rate of Compounds 1-7 against four Fungi.

Compd no.	Inhibition rate (%)
	S. sclerotiorum	R. solani	B. cinerea	Phytophthora parasitica Dast
1	71.90	96.05	75.41	79.21
2	67.35	93.24	77.29	83.54
3	78.27	95.29	68.42	67.24
4	65.16	96.17	74.59	63.55
5	73.47	93.86	71.73	52.57
6	71.90	95.93	71.44	69.72
7	71.10	88.48	67.17	70.06

Table 1. Inhibition Rate of Compounds 1-7 against four Fungi.

Compd no.	Inhibition rate (%)
	S. sclerotiorum	R. solani	B. cinerea	Phytophthora parasitica Dast
1	71.90	96.05	75.41	79.21
2	67.35	93.24	77.29	83.54
3	78.27	95.29	68.42	67.24
4	65.16	96.17	74.59	63.55
5	73.47	93.86	71.73	52.57
6	71.90	95.93	71.44	69.72
7	71.10	88.48	67.17	70.06

3. Experimental

3.1. General methods

Solvents were purified in the usual way. All commercially available reagents were used as received. All reactions were monitored by TLC analysis and TLC was performed on silica gel HF with detection by charring with 30% (v/v) H₂SO₄ in CH₃OH or by UV detection. Column chromatography was conducted by elution of a column (8 × 100, 16 × 240, 18 × 300, 35 × 400mm) of silica gel (200-300 mesh) with EtOAc-PE (b.p. 60-90 °C) as the eluent. Air and moisture sensitive reactions were performed under dry N₂ atmosphere. Optical rotations were recorded using a Perkin-Elmer 241 polarimeter. NMR spectra were recorded on a Varian XL-300 spectrometer with TMS as the internal standard. Elemental analysis was performed on a Yanaco CHN Corder MF-3 automatic elemental analyzer. Mass spectra were recorded with a VG PLATFORM mass spectrometer using the electronspray ionization (ESI) mode. Solutions were concentrated at a temperature <60 °C under diminished pressure.

3.2. Chemical synthesis

p-Methoxyphenyl 4-O-allyl-2,3-O-isopropylidene-α-L-rhamnopyranoside (18). Sodium hydride (2.3 g, 47.4 mmol) and allyl bromide (3.6 mL, 41.1 mmol) were successively added to a soln. of compound 17 [14] (9.8 g, 31.6 mmol) in N,N-dimethylformamide (50 mL) which was cooled in an ice-salt bath. Then the reaction mixture was slowly allowed to reach room temperature and stirred for 20 min at the end of which time TLC (4:1 petroleum ether-EtOAc) indicated that the reaction was complete. The reaction mixture was diluted with EtOAc (100 mL), washed with ice-water, and dried (Na₂SO₄). The soln was concentrated, and the residue was subjected to column chromatography (8:1 petroleum ether-EtOAc) to give the desired product 18 (10.5 g, 95%) as a foamy solid. R_f = 0.68 (4:1 petroleum ether-EtOAc); -61.4 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 7.00-6.81 (m, 4 H, Bz-H), 5.93 (m, 1 H, CH₂=CH-CH₂O), 5.59 (s, 1 H, H-1), 5.32-5.16 (m, 2 H), 4.40-4.32 (m, 3 H), 4.14 (m, 1 H), 3.83-3.77 (m, 4 H, H-5, OCH₃), 3.21 (m, 1 H), 1.56 (s, 3 H, CH₃), 1.40 (s, 3 H, CH₃), 1.23 (d, 3 H, J = 6.3 Hz, H-6). Anal. Calcd. for C₁₉H₂₆O₆: C, 65.13; H, 7.48; found: C, 65.29; H, 7.63.

p-Methoxyphenyl 4-O-allyl-2,3-di-O-benzoyl-α-L-rhamnopyranoside (19). Compound 18 (7.8 g, 22.3 mmol) was dissolved in 70% HOAc (200 mL) and stirred for 2 h at 75°C, at the end of which time TLC (2:1 petroleum ether-EtOAc) indicated the completion of the reaction. The mixture was concentrated under reduced pressure and then coevaporated with toluene (2 × 40 mL). To a soln of the residue (7.3 g, 23.5 mmol) in pyridine (60 mL) was added benzoyl chloride (8.2 mL, 70.5 mmol) dropwise. After stirring for 8 h at rt, TLC (3:1 petroleum ether-EtOAc) indicated that the reaction was complete. Methanol (1 mL) was added to quench the reaction and then water (100 mL) was added to the reaction mixture. The aq. soln. was extracted with EtOAc (3 × 200 mL), the extract was washed with 1 M HCl and saturated aq. sodium bicarbonate, dried (Na₂SO₄) and concentrated. The residue was passed through a short silica-gel column with 6:1 petroleum ether-EtOAc as the eluent to give 19 (10.2 g, 88% for two steps) as a foamy solid. R_f = 0.42 (4:1 petroleum ether-EtOAc); +21.1 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.07-7.34 (m, 10 H, Bz-H), 7.08-6.83 (m, 4 H, MeOC₆H₄), 5.87-5.75 (m, 3 H), 5.52 (d, 1 H, J = 1.8 Hz, H-1), 5.17 (m, 1 H), 5.08 (m, 1 H), 4.20-4.08 (m, 3 H), 3.78-3.71 (m, 4 H, H-5, OCH₃), 1.41 (d, J = 6.2 Hz, 3 H, H-6); Anal. Calcd. for C₃₀H₃₀O₈: C, 69.49; H, 5.83; found: C, 69.55; H, 5.58;

4-O-Allyl-2,3-di-O-benzoyl-α-L-rhamnopyranosyl trichloroacetimidate (10). To a soln. of 19 (10.0 g, 19.3 mmol) in 80% MeCN (200 mL) was added ceric ammonium nitrate (42.3 g, 77.2 mmol). The mixture was stirred for 20 min at 35 °C, at the end of which time TLC (4:1 petroleum ether-EtOAc) indicated that the reaction was complete. The solvents were evaporated in vacuo at 50 °C to give a residue, which was dissolved in CH₂Cl₂, and washed with water. The organic phase was dried (Na₂SO₄) and concentrated. Purification by silica gel chromatography with 5:1 petroleum ether-EtOAc as the eluent afforded a foamy residue. The residue was dried under high vacuum for 2 h, then was dissolved in dry CH₂Cl₂ (50 mL), trichloroacetonitrile (2.5 mL, 24.3 mmol) and 1,8-diaza-bicyclo[5.4.0] undecene (DBU) (0.3 mL, 30 mmol) were added. The mixture was aged under the nitrogen atmosphere until completion (TLC, 4:1 petroleum ether-EtOAc). Concentration of the reaction mixture and purification of the residue by column chromatography (5:1 petroleum ether-EtOAc) gave 10 (7.6 g, 72% for two steps) as a white foamy solid. R_f = 0.67 (4:1 petroleum ether-EtOAc); +39.3 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.74 (s, 1 H, C=NH), 8.06-7.34 (m, 10 H, Bz-H ), 6.39 (d, 1 H, J = 1.9 Hz, H-1), 5.85-5.71 (m, 3 H), 5.21-5.07 (m, 2 H), 4.22-4.15 (m, 3 H), 3.77 (dd, 1 H, J = 9.6, 9.6 Hz, H-4), 1.48 (d, J = 6.2 Hz, 3 H, H-6). Anal. Calcd. for C₂₅H₂₄Cl₃NO₇: C, 53.93; H, 4.34; N, 2.52; found: C, 53.79; H, 4.23; N, 2.29.

p-Methoxyphenyl 4-O-allyl-2,3-O-isopropylidene-6-deoxy-α-L-talopyranoside (21). Compound 20 (4.9 g, 15.8 mmol) was allylated under the same conditions as used for the preparation of 18 from 17, giving 21 (5.1 g, 92%) as a foamy solid; R_f = 0.73 (4:1 petroleum ether-EtOAc); -49.1 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 7.01-6.80 (m, 4 H, Bz-H), 5.93 (m, 1 H, CH₂=CH-CH₂O), 5.56 (d, 1 H, J = 1.5 Hz, H-1), 5.29-5.17 (m, 2 H), 4.49 (m, 1 H), 4.34-4.25 (m, 2 H), 4.09-4.00 (m, 2 H), 3.77 (s, 3 H, OCH₃), 3.60 (m, 1 H), 1.59 (s, 3 H, CH₃), 1.40 (s, 3 H, CH₃), 1.31 (d, 3 H, J = 6.6 Hz, H-6). Anal. Calcd. for C₁₉H₂₆O₆: C, 65.13; H, 7.48; found: C, 65.25; H, 7.29.

p-Methoxyphenyl 4-O-allyl-2,3-di-O-benzoyl-6-deoxy-α-L-talopyranoside (22). Sequential de-O-isopropylidenation and then benzoylation of compound 21 (7.8 g, 22.3 mmol) under the same conditions as those used for the preparation of 19 from 18, gave 22 (9.3 g, 80%) as a foamy solid; R_f = 0.67 (3:1 petroleum ether-EtOAc); -7.7 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.25-7.25 (m, 10 H, Bz-H), 7.07-6.82 (m, 4 H, MeOC₆H₄), 5.93 (m, 1 H, CH₂=CH-CH₂O), 5.77 (dd, 1 H, J = 3.49, 3.34 Hz, H-3), 5.68-5.67 (m, 2 H), 5.29-5.12 (m, 2 H), 4.33-4.27 (m, 2 H), 4.07 (m, 1 H), 3.83-3.76 (m, 4 H, CH₂=CH-CH₂O, OCH₃), 1.37 (d, J = 6.5 Hz, 3 H, H-6); Anal. Calcd. for C₃₀H₃₀O₈: C, 69.49; H, 5.83; found: C, 69.63; H, 5.66.

4-O-allyl-2,3-di-O-benzoyl-6-deoxy-α-L-talopyranosyl trichloroacetimidate (11). Compound 22 (5.0 g, 9.7 mmol) was trichloroacetimidated under the same conditions as used for the preparation of 10 from 19, giving 11 (3.7 g, 70% for two steps) as a foamy solid. R_f = 0.70 (4:1 petroleum ether-EtOAc); +6.14 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.74 (s, 1 H, C=NH), 8.24-7.32 (m, 10 H, Bz-H), 6.48 (d, J = 1.4 Hz, 1 H, H-1), 5.90 (m, 1 H), 5.67-5.62 (m, 2 H), 5.18-5.09 (m, 2 H), 4.31-4.07 (m, 2 H), 3.87 (m, 1 H), 3.70 (m, 1 H), 1.44 (d, J = 6.5 Hz, 3 H, H-6). Anal. Calcd. for C₂₅H₂₄Cl₃NO₇: C, 53.93; H, 4.34; N, 2.52; found: C, 53.87; H, 4.15; N, 2.78.

Benzyl oleanolate 3-O-4-O-allyl-2,3-di-O-benzoyl-6-deoxy-α-L-talopyranoside (23). Compound 11 (4.3 g, 7.8 mmol), 9 [10] (3.6 g, 6.4 mmol) and 4 Å molecular sieves (1.0 g) were added to anhydrous redistilled CH₂Cl₂ (60 mL). TMSOTf (130 μL, 0.7 mmol) was added dropwise at -10 °C under nitrogen protection. The reaction mixture was allowed to raise to rt and stirred for 2 h, and then quenched with Et₃N (2 drops). Filtration of the reaction mixture, concentration of the filtrate, followed by purification of the residue by column chromatography (5:1 petroleum ether-EtOAc) provided 23 (5.2 g, 85%). R_f= 0.47 (8:1 petroleum ether-EtOAc). +39.3 (c 0.5, CHCl₃), ¹H-NMR (CDCl₃): δ 8.23-7.30 (m, 15 H, Ar-H), 5.88 (m, 1 H, CH₂=CH-CH₂O), 5.53 (dd, 1 H, J = 3.4, 3.5 Hz, H-3'), 5.42 (m, 1 H), 5.29 (br s, 1 H, H-12), 5.23-5.02 (m, 5 H), 4.32-4.23 (m, 2 H), 4.03 (m, 1 H), 3.76 (s, 1 H, CH₂=CH-CH₂O), 3.17 (dd, 1 H, J = 5.1, 10.7 Hz, H-3), 2.90 (dd, 1 H, J = 3.8, 13.7 Hz, H-18), 1.35 (d, 3 H, J = 6.5 Hz, H-6'), 1.12, 1.01, 0.92, 0.91, 0.89, 0.83, 0.60 (s, 7 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 177.4, 166.3, 165.6 (3 C=O), 143.7, 136.4, 135.0, 133.1, 133.0, 130.3, 130.1, 129.7, 129.7, 128.4, 128.4, 128.4, 128.4, 128.4, 128.2, 128.2, 128.0, 128.0, 128.0, 127.9, 122.5, 116.7, 100.6 (C-1'), 89.3, 76.4, 74.3, 70.1, 69.0, 66.5, 65.9, 55.4, 47.6, 46.7, 45.9, 41.7, 41.4, 39.3, 39.0, 38.4, 36.7, 33.9, 33.1, 32.7, 32.4, 30.7, 28.3, 27.6, 25.8, 25.2, 23.6, 23.4, 23.1, 18.3, 16.9, 16.5, 16.5, 15.3; Anal. Calcd. for C₆₀H₇₆O₉: C, 76.56; H, 8.14; found: C, 76.65; H, 8.31.

Benzyl oleanolate 3-O-2,3-di-O-benzoyl-6-deoxy-α-L-talopyranoside (16). To a soln of compound 23 (5.0 g, 5.2 mmol) in MeOH-CH₂Cl₂ = 1/1 (50 mL) was added PdCl₂ (304 mg, 1.0 mmol). The mixture was stirred for 12 h, at the end of which time TLC (8:1 petroleum ether-EtOAc) indicated that the reaction was complete. The reaction mixture was diluted with dichloromethane (100 mL), washed with water and satd aq Na₂CO₃. The organic layer was concentrated, and the residue was passed through a short silica gel column with 8:1 petroleum ether-EtOAc as the eluent to give 16 (4.4 g, 92%). R_f= 0.32 (8:1 petroleum ether-EtOAc). +45.0 (c 0.5, CHCl₃), ¹H-NMR (CDCl₃): δ 8.07-7.26 (m, 15 H, Ar-H), 5.49-5.47 (m, 2 H), 5.29 (br s, 1 H, H-12), 5.07 (m, 3 H), 4.32-3.96 (m, 2 H, H-4', H-5'), 3.20 (dd, 1 H, J = 5.8, 9.8 Hz, H-3), 2.90 (dd, 1 H, J = 4.4, 13.9 Hz, H-18), 2.55 (d, 1 H, J = 11.1 Hz, OH), 1.34 (d, 3 H, J = 6.5 Hz, H-6'), 1.12, 1.02, 0.92, 0.92, 0.89, 0.85, 0.61 (s, 7 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 177.4, 165.5, 165.5 (3 C=O), 143.7, 136.5, 133.6, 133.2, 129.8, 129.8, 129.7, 129.7, 129.5, 128.7, 128.7, 128.4, 128.4, 128.3, 128.0, 128.0, 127.9, 127.9, 126.8, 122.5, 100.4 (C-1'), 89.8, 70.6, 70.2, 68.9, 66.7, 65.9, 55.4, 47.6, 46.8, 45.9, 41.7, 41.4, 39.3, 39.0, 38.4, 36.7, 33.8, 33.1, 32.7, 32.4, 30.7, 28.3, 27.6, 25.9, 25.3, 23.6, 23.4, 23.1, 18.3, 16.9, 16.5, 16.2, 15.3; Anal. Calcd. for C₅₇H₇₂O₉: C, 75.97; H, 8.05; found: C, 75.83; H, 8.19.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranose-(1→3)-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose(25). Compound 12 [11] (3.87 g, 5.2 mmol) and 24 [16] (1.24 g, 4.8 mmol) were coupled under the same conditions as that used for the preparation of 23 from 11 and 9, giving 25 (3.5 g, 88%) as a foamy solid. R_f = 0.16 (4:1 petroleum ether-EtOAc); -61.4 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.09-7.25 (m, 20 H, Bz-H), 5.99 (dd, 1 H, J = 0.8, 3.3 Hz), 5.76 (dd, 1 H, J = 7.9, 10.5 Hz, H-2'), 5.62 (dd, 1 H, J = 3.4, 10.4 Hz, H-3'), 5.50 (d, 1 H, J = 3.6 Hz), 4.95 (d, 1 H, J = 7.9 Hz, H-1'), 4.67 (dd, 1 H, J = 6.3, 11.1 Hz), 4.50-4.25 (m, 6 H), 4.16-4.03 (m, 2 H), 1.43, 1.42, 1.34, 1.12 (s, 4 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 166.0, 165.5, 165.5, 164.9 (4 C=O), 133.6, 133.5, 133.3, 133.3, 129.9, 129.9, 129.9, 129.9, 129.9, 129.8, 129.6, 129.6, 129.4, 129.1, 129.0, 128.7, 128.7, 128.6, 128.6, 128.5, 128.5, 128.3, 128.3, 111.9, 108.6, 104.9, 100.6 (2 × C-1), 82.9, 81.8, 80.5, 77.2, 73.1, 71.8, 71.5, 69.9, 68.0, 66.3, 61.9, 26.7, 26.6, 25.9, 25.3; Anal. Calcd. for C₄₆H₄₆O₁₅: C, 65.86; H, 5.53; found: C, 65.72; H, 5.75.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranose-(1→3)-1,2-O-isopropylidene-α-D-glucofuranose (26). The compound 25 (3.0 g) was dissolved in 60% HOAc (100 mL) and stirred for 6 h at 25°C, at the end of which time TLC (2:1 petroleum ether-EtOAc) indicated the completion of the reaction. The mixture was concentrated under reduced pressure and then co evaporated with toluene (2 × 40 mL). The residue was passed through a short silica-gel column with 3:1 petroleum ether-EtOAc as the eluent to give 26 (2.4 g, 83%) as a foamy solid. R_f = 0.68 (1:1 petroleum ether-EtOAc); +98.2 (c 1.0, CHCl₃); ¹H-NMR (CDCl₃): δ 8.08-7.23 (m, 20 H, Bz-H), 6.01 (d, 1 H, J = 2.5 Hz), 5.79 (dd, 1 H, J = 8.0, 10.5 Hz, H-2'), 5.61 (dd, 1 H, J = 3.4, 10.5 Hz, H-3'), 5.53 (d, 1 H, J = 3.7 Hz), 4.98 (d, 1 H, J = 7.9 Hz, H-1'), 4.57 (d, 2 H, J = 6.1 Hz), 4.50-4.29 (m, 3 H), 4.23 (d, 1 H, J = 3.7 Hz), 4.19-4.07 (m, 3 H), 3.92-3.85 (m, 1 H), 3.69 (dd, 1 H, J = 5.7, 11.5 Hz, H-18), 1.42, 1.06 (s, 2 × 3 H, CH₃); ¹³C- NMR (CDCl₃): δ 166.1, 165.5, 165.5, 164.8 (4 C=O), 133.8, 133.6, 133.4, 133.4, 133.3, 130.0, 129.9, 129.8, 129.8, 129.8, 129.6, 129.1, 129.0, 129.0, 128.8, 128.8, 128.7, 128.7, 128.6, 128.6, 128.5, 128.3, 128.3, 112.2, 105.2, 101.9 (2 × C-1), 83.6, 83.2, 80.0, 77.2, 72.4, 71.3, 69.5, 68.7, 68.0, 64.4, 62.2, 26.7, 26.2; Anal. Calcd. for C₄₃H₄₂O₁₅: C, 64.66; H, 5.30; found: C, 64.49; H, 5.38.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranose-(1→3)-5-aldehyde-1,2-O-isopropylidene-α-D-gluco-furanose(27). To a vigorously stirred suspension of silicagel-supported NaIO₄ reagent which was prepared as the reported method [17] (2.0 g) in CH₂Cl₂ (5 mL) was added a soln of the compound 26 (0.8 g, 1 mmol) in CH₂Cl₂ (5 mL). The mixture was stirred at rt for 25 min, and TLC (2:1 petroleum ether-EtOAc) indicated that the reaction was complete. The mixture was filtered, and the silica gel was thoroughly washed with CHCl₃. Purification by silica gel chromatography with 2:1 petroleum ether-EtOAc as the eluent afforded 27 (0.7 g, 88%) as a foamy solid. R_f = 0.41 (2:1 petroleum ether-EtOAc); +70.2 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 9.68 (d, 1 H, J = 1.5 Hz, CHO), 8.08-7.25 (m, 20 H, Bz-H), 5.97 (dd, 1 H, J = 0.9, 3.4 Hz), 5.74-5.59 (m, 3 H), 4.89 (d, 1 H, J = 7.8 Hz, H-1'), 4.70-4.54 (m, 3 H), 4.48-4.30 (m, 3 H), 1.44, 1.18 (s, 2 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 197.9, 166.0, 165.7, 165.5, 164.8 (5 C=O), 133.7, 133.6, 133.3, 133.3, 130.3, 130.0, 130.0, 129.9, 129.8, 129.8, 129.8, 129.6, 129.6, 129.4, 129.2, 129.1, 129.0, 128.9, 128.8, 128.7, 128.7, 128.6, 128.5, 128.3, 112.8, 105.7, 100.6 (2 × C-1), 83.9, 83.0, 82.9, 77.2, 71.8, 71.5, 69.6, 67.9, 61.9, 26.6, 26.1; Anal. Calcd. for C₄₃H₄₀O₁₄: C, 66.15; H, 5.16; found: C, 66.34; H, 5.25.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranose-(1→3)-1,2-O-isopropylidene-β-D-xylose (28). To a soln of 27 (1.4 g, 1.8 mmol) in 7:3 EtOAc-H₂O (50 mL) at 0 ^oC was added NaBH₄ (109 mg, 2.7 mmol). The mixture was stirred at 0°C for 15 min, and TLC (3:1 petroleum ether-EtOAc) indicated that the reaction was complete. The aq. soln. was extracted with EtOAc (3 × 100 mL), the extract was washed with 1 M HCl and saturated aq sodium bicarbonate, dried (Na₂SO₄) and concentrated. Purification by silica gel chromatography with 5:1 petroleum ether-EtOAc as the eluent afforded 28 (1.3 g, 96%) as a foamy solid. R_f = 0.29 (3:2 petroleum ether-EtOAc); +184.2 (c 1.0, CHCl₃); ¹H-NMR (CDCl₃): δ 8.08-7.26 (m, 20 H, Bz-H), 6.00 (dd, 1 H, J = 0.8, 3.3 Hz), 5.80-5.55 (m, 3 H), 4.96 (d, 1 H, J = 7.9 Hz, H-1'), 4.70-4.30 (m, 6 H), 4.16-3.91 (m, 2 H), 2.56 (dd, 1 H, J = 6.7, 6.7 Hz), 1.44, 1.12 (s, 2 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 166.1, 165.5, 165.5, 164.9 (4 C=O), 133.8, 133.6, 133.4, , 133.4, 130.1, 130.1, 130.0, 130.0, 129.8, 129.8, 129.7, 129.6, 129.2, 129.0, 128.9, 128.8, 128.7, 128.7, 128.7, 128.6, 128.5, 128.5, 128.3, 112.1, 104.9, 101.3 (2 × C-1), 83.6, 82.8, 79.8, 77.2, 72.1, 71.4, 69.6, 68.0, 62.2, 59.9, 26.9, 26.1; Anal. Calcd. for C₄₂H₄₀O₁₄: C, 65.62; H, 5.24; found: C, 65.35; H, 5.37.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranose-(1→3)-β-D-xylose (29). Compound 28 (1.22 g, 1.6 mmol) was dissolved in 4% aq H₂SO₄ (100 mL) and then refluxed for 4 h. TLC (1:1 petroleum ether-EtOAc) indicated that the reaction was complete. The resulting soln. was cooled down to room temperature and extracted three times with EtOAc. The extract was washed with saturated aq. sodium bicarbonate, dried (Na₂SO₄) and concentrated. Purification by silica gel chromatography with 2:1 petroleum ether-EtOAc as the eluent afforded 29 (0.9 g, 85%) as a foamy solid. R_f = 0.31 (1:1 petroleum ether-EtOAc); +331.6 (c 1.0, CHCl₃); ¹H-NMR (CDCl₃): δ 8.10-7.25 (m, 20 H, Bz-H), 6.01 (d, 1 H, J = 3.3 Hz), 5.85 (m, 1 H), 5.65 (m, 1 H), 5.07-5.02 (m, 2 H), 4.58-4.40 (m, 3 H), 3.79-3.74 (m, 3 H), 3.50 (m, 1 H), 3.27 (m, 1 H); ¹³C-NMR (CDCl₃): δ 166.1, 165.6, 165.5, 165.5 (4 C=O), 133.7, 133.5, 133.4, 133.4, 130.0, 130.0, 129.8, 129.7, 129.1, 129.0, 128.8, 128.8, 128.7, 128.6, 128.5, 128.5, 128.4, 128.3, 102.8, 102.6 (2 × C-1), 97.3, 92.4, 88.2, 85.7, 77.2, 73.4, 72.0, 71.5, 70.7, 70.0, 69.9, 68.2, 68.1, 62.4, 62.0; Anal. Calcd. for C₃₉H₃₆O₁₄: C, 64.28; H, 4.98; found: C, 64.39; H, 4.83.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranose-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl trichloro-acetimidate (15). Compound 29 (3.5 g, 4.8 mmol) was benzoylated under the same conditions as used for the preparation of 19. Then the resultant residue was dissolved in 2 M MeOH-NH₃ (200 mL) and stirred at 35 °C at the end of which time TLC (3:1 petroleum ether-EtOAc) indicated that the reaction was complete. The solvents were evaporated in vacuo at 50 °C to give a residue, which was dissolved in CH₂Cl₂, and washed with water. The organic phase was dried (Na₂SO₄) and concentrated. Purification by silica gel chromatography with 5:1 petroleum ether-EtOAc as the eluent afforded a foamy residue. The residue was trichloroacetimidated under the same conditions as used for the preparation of 10 from 19, giving 15 (3.5 g, 68% for three steps) as a white foamy solid. R_f = 0.42 (3:1 petroleum ether-EtOAc); +18.4 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.52 (s, 1 H, C=NH), 8.18-7.08 (m, 30 H, Bz-H ), 6.54 (d, 1 H, J = 3.5 Hz), 5.87 (d, 1 H, J = 3.3 Hz), 5.66 (dd, 1 H, J = 7.9, 10.4 Hz), 5.48-5.37 (m, 2 H), 5.27 (dd, 1 H, J = 3.5, 9.7 Hz), 5.14 (d, 1 H, J = 7.9 Hz, H-1'), 4.67 (dd, 1 H, J = 9.4, 9.4 Hz), 4.41-4.18 (m, 4 H), 3.95 (dd, 1 H, J = 10.9, 11.0 Hz). Anal. Calcd. for C₅₅H₄₄Cl₃NO₁₆: C, 61.09; H, 4.10; N, 1.30; found: C, 61.34; H, 4.27; N, 1.49.

Benzyl oleanolate 3-O-4-O-allyl-2,3-di-O-benzoyl-α-L-rhamnopyranoside (30). Compound 10 (4.3 g, 7.8 mmol) and 9 [10] (3.6 g, 6.4 mmol) were coupled under the same conditions as used for the preparation of 23 from 11 and 9, giving 30 (5.4 g, 88%) as a foamy solid. R_f = 0.45 (8:1 petroleum ether-EtOAc); +73.7 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.06-7.31 (m, 15 H, Ar-H), 5.81 (m, 1 H, CH₂=CH-CH₂O), 5.66 (dd, 1 H, J = 3.2, 9.5 Hz, H-3'), 5.57 (dd, 1 H, J = 1.7, 3.2 Hz, H-2'), 5.29 (br s, 1 H, H-12), 5.20-5.03 (m, 4 H, PhCH2, CH₂=CH-CH₂O), 4.97 (d, 1 H, J_1,2 = 1.7 Hz, H-1'), 4.22-4.04 (m, 3 H), 3.67 (dd, 1 H, J = 9.5, 9.5 Hz, H-4'), 3.15 (dd, 1 H, J = 6.3, 9.8 Hz, H-3), 2.91 (dd, 1 H, J = 4.1, 13.4 Hz, H-18), 1.40 (d, 3 H, J = 6.2 Hz, H-6'), 1.12, 1.02, 0.92, 0.92, 0.89, 0.88, 0.61 (s, 7 × 3H, CH₃); ¹³C-NMR (CDCl₃): δ 177.4, 165.5, 165.3 (3 C=O), 143.7, 136.4, 134.5, 133.2, 132.9, 130.0, 130.0, 129.9, 129.7, 129.5, 129.5, 128.4, 128.4, 128.4, 128.4, 128.3, 128.0, 128.0, 127.9, 122.5, 117.2, 99.6 (C-1'), 89.7, 79.1, 77.2, 74.0, 72.5, 71.5, 67.8, 65.9, 55.4, 47.6, 45.9, 41.7, 41.4, 39.3, 39.0, 38.4, 36.7, 33.9, 33.1, 32.7, 32.4, 30.7, 28.3, 27.6, 25.9, 25.3, 23.6, 23.4, 23.1, 18.3, 18.0, 16.9, 16.5, 15.3; Anal. Calcd. for C₆₀H₇₆O₉: C, 76.56; H, 8.14; found: C, 76.73; H, 8.43.

Benzyl oleanolate 3-O-2,3-di-O-benzoyl-α-L-rhamnopyranoside (31). Compound 30 (5.0 g, 5.2 mmol) was deallylated under the same conditions as that used for the preparation of 16 from 23, giving 31 (4.5 g, 94%) as a foamy solid; R_f = 0.16 (8:1 petroleum ether-EtOAc); +29.5 (c 1.0, CHCl₃); ¹H- NMR (CDCl₃): δ 8.09-7.26 (m, 15 H, Ar-H), 5.57-5.48 (m, 2 H), 5.29 (br s, 1 H, H-12), 5.07 (dd, 2 H, J = 12.6, 17.1 Hz, PhCH₂), 5.00 (d, 1 H, J = 1.6 Hz, H-1'), 4.06-3.87 (m, 2 H), 3.18 (dd, 1 H, J = 2.9, 13.0 Hz, H-3), 2.90 (dd, 1 H, J = 4.4, 14.2 Hz, H-18), 2.49 (d, 1 H, J = 5.1 Hz, OH), 1.41 (d, 3 H, J = 6.1 Hz, H-6'), 1.12, 1.01, 0.92, 0.92, 0.89, 0.87, 0.61 (s, 7 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 177.4, 166.8, 165.6 (3 C=O), 143.6, 136.4, 133.3, 133.2, 129.7, 129.7, 129.7, 129.7, 129.5, 129.4, 128.4, 128.4, 128.3, 128.3, 127.9, 127.9, 127.9, 127.8, 126.8, 122.4, 99.7 (C-1'), 89.7, 73.4, 72.1, 71.3, 68.7, 65.9, 55.4, 47.5, 46.7, 45.8, 41.6, 41.4, 39.3, 38.9, 38.4, 36.7, 33.8, 33.0, 32.7, 32.3, 30.6, 28.3, 27.6, 25.8, 25.3, 23.6, 23.4, 23.0, 18.2, 17.5, 16.8, 16.5, 15.3; Anal. Calcd. for C₅₇H₇₂O₉: C, 75.97; H, 8.05; found: C, 75.81; H, 8.29.

Oleanolic acid 3-O-α-L-rhamnopyranoside (1). A suspension of 31 (1.3 g, 1.4 mmol) and 10% Pd-C (1.5 g) in EtOAc (30 mL) was refluxed and bubbled up with H₂ (20 mL/min). When TLC (2:1, petroleum-EtOAc) showed that the reaction had completed, Pd-C was removed through filtration and the filtrate was concentrated to dryness. The resulted amorphous solid was dissolved in dry CH₂Cl₂-MeOH (1:2, 30 mL), to which a newly prepared NaOMe/MeOH (1.0 mol/L, 20 mL) was added. The soln was stirred at rt for 2 h and then neutralized with Dowex H⁺ resin to pH 7 and filtered. The filtrate was concentrated and subjected to a flash column chromatography (CHCl₃-MeOH-H₂O 7:3:1, organic layer) to give 1 [13] (737 mg, 86% for two steps) as a white powder.

Oleanolic acid 3-O-6-deoxy-α-L-talopyranoside (2). Compound 2 was prepared from 16 by the same procedure as for 1. Yield: 81%; white powder, m.p. 288-290 °C, R_f = 0.29 (10:1:0.1 CHCl₃-MeOH-H₂O); +6.1 (c 0.5, MeOH); ¹H-NMR (pyridine-d₅): δ 5.47 (br s, 1 H, H-12), 5.31 (d, 1 H, J = 1.3 Hz, H-1'), 4.85 (dd, 1 H, J = 1.5, 3.0 Hz, H-2'), 4.25-4.21 (m, 2 H), 4.06 (d, 1 H, J = 1.4 Hz), 3.29 (dd, 1 H, J = 4.0, 13.7 Hz, H-3), 3.13 (dd, 1 H, J = 4.3, 11.5 Hz, H-18), 1.54 (d, 3 H, J = 6.5 Hz, H-6'), 1.28, 1.00, 0.99, 0.95, 0.90, 0.85, 0.80 (s, 7 × 3 H, CH₃); ¹³C-NMR (pyridine-d₅): δ 180.2, 144.9, 122.6, 104.9 (C-1'), 88.6, 74.3, 72.4, 67.8, 67.5, 55.7, 48.1, 46.7, 46.6, 42.2, 42.1, 39.8, 39.2, 38.6, 37.1, 34.3, 33.4, 33.3, 33.2, 31.0, 28.4, 28.3, 26.2, 25.8, 23.9, 23.9, 23.8, 18.7, 17.5, 17.4, 16.8, 15.5; HRESIMS: m/z calcd. for C₃₆H₅₈O₇Na[M+Na⁺]: 625.4080; found: m/z 625.4059.

Benzyl oleanolate 3-O-2,3,4,6-tetra-O-benzoyl-β-D-galactopyranoside (34). Compound 12 (0.56 g, 0.8 mmol) and 9 [15] (0.6 g, 0.7 mmol) were coupled under the same conditions as that used for the preparation of 23 from 11 and 9, giving 34 [13] (0.9 g, 90%) as a foamy solid.

Oleanolic acid 3-O-β-D-galactopyranoside (3). Compound 3 [13] was prepared from 34 by the same procedure as for 1. Yield: 84%; white powder.

Benzyl oleanolate 3-O-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (36). Compound 13 [12] (1.5 g, 3.0 mmol) and 9 [10] (1.4 g, 2.5 mmol) were coupled under the same conditions as used for the preparation of 23 from 11 and 9, giving 36 (1.9 g, 86%) as a foamy solid. R_f = 0.16 (6:1 petroleum ether-EtOAc); +70.6 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 7.47-7.27 (m, 5 H, Bn-H), 5.35-5.24 (m, 3 H), 5.16-5.06 (m, 3 H), 4.97 (d, 1 H, J = 1.7 Hz, H-1'), 4.25 (dd, 1 H, J = 5.7, 12.5 Hz, H-3), 4.15-4.10 (m, 2 H), 3.21 (dd, 1 H, J = 4.0, 11.3 Hz, H-3), 2.90 (dd, 1 H, J = 4.0, 13.6 Hz, H-18), 2.16, 2.09, 2.05, 2.00 (s, 4 × 3 H, CH₃CO), 1.11, 1.00, 0.92, 0.89, 0.89, 0.82, 0.60 (s, 7 × 3 H, CH₃); ¹³C- NMR (CDCl₃): δ 177.4, 170.6, 170.2, 169.9, 169.8 (5 C=O), 143.6, 136.3, 128.3, 128.3, 127.9, 127.9, 122.4, 94.6 (C-1'), 84.7, 77.2, 70.7, 69.2, 69.0, 66.4, 66.3, 65.9, 62.6, 55.6, 47.6, 46.7, 45.8, 41.6, 41.3, 39.3, 38.3, 38.0, 36.8, 33.8, 33.0, 32.7, 32.3, 30.6, 28.7, 27.6, 25.8, 23.6, 23.4, 23.0, 22.1, 20.8, 20.6, 20.6, 18.2, 16.8, 16.4, 15.2; HRESIMS: m/z calcd. for C₅₁H₇₂O₈Na[M+Na⁺]: 835.5125; found: m/z 835.5118.

Oleanolic acid 3-O-α-D-mannopyranoside (4). Compound 4 was prepared from 36 by the same procedure as for 1. Yield: 87%; white powder, m.p. 250-252 °C, R_f = 0.07 (20:1:0.1 CHCl₃-MeOH-H₂O); +79.8 (c 0.5, MeOH); ¹H-NMR (pyridine-d₅): δ 5.54 (d, 1 H, J = 1.0 Hz, H-1'), 5.46 (br s, 1 H, H-12), 4.69 (m, 1 H), 4.59-4.50 (m, 3 H), 4.46-4.38 (m, 2 H), 3.47 (dd, 1 H, J = 4.2, 11.4 Hz, H-3), 3.28 (dd, 1 H, J = 4.0, 13.5 Hz, H-18), 1.24, 1.15, 1.00, 0.97, 0.94, 0.81, 0.79 (s, 7 × 3 H, CH₃); ¹³C-NMR (pyridine-d₅): δ 180.1 (C=O), 144.8, 124.1, 122.4, 97.7 (C-1'), 81.8, 75.8, 73.2, 72.9, 69.2, 63.4, 55.7, 47.9, 46.6, 46.4, 42.1, 41.9, 39.7, 38.5, 38.1, 37.1, 34.2, 33.2, 33.1, 33.1, 30.9, 29.0, 28.2, 26.1, 23.7, 23.6, 22.0, 18.5, 17.3, 16.9, 15.3; HRESIMS: m/z calcd. for C₃₆H₅₈O₈Na[M+Na⁺]: 641.4029; found: m/z 641.4037.

Benzyl oleanolate 3-O-2,3,4-tri-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl-6-deoxy-α-L-talopyranoside (38). Compound 16 (1.1 g, 1.3 mmol) and 14 [13] (0.9 g, 1.5 mmol) were coupled under the same conditions as that used for the preparation of 23 from 11 and 9, giving 38 (1.4 g, 90%) as a foamy solid. R_f = 0.13 (8:1 petroleum ether-EtOAc); -24.6 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.33-7.26 (m, 30 H, Ar-H), 5.67 (dd, 1 H, J = 8.2, 9.1 Hz, H-2''), 5.58 (dd, 1 H, J = 6.7, 9.1 Hz, H-3''), 5.48 (d, 1 H, J = 2.2 Hz, H-1'), 5.40 (dd, 1 H, J = 3.6, 3.6 Hz, H-3'), 5.28 (br s, 1 H, H-12), 5.07 (dd, 2 H, J = 12.5, 17.6 Hz, PhCH₂), 4.98-4.92 (m, 2 H), 4.70 (d, 1 H, J = 6.7 Hz, H-1''), 4.29-4.19 (m, 2 H), 3.50 (m, 1 H), 3.14-3.04 (m, 2 H), 2.89 (dd, 1 H, J = 4.1, 9.6 Hz, H-18), 1.19 (d, 3 H, J = 6.5 Hz, H-6'), 1.10, 0.97, 0.91, 0.89, 0.89, 0.81, 0.59 (s, 7 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 177.4, 166.3, 166.2, 165.6, 165.2, 164.9 (6 C=O), 143.7, 140.9, 138.8, 136.4, 133.3, 133.2, 133.1, 133.0, 130.4, 130.0, 130.0, 129.9, 129.8, 129.7, 129.2, 129.1, 128.6, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.0, 127.9, 127.8, 127.6, 127.5, 126.9, 122.4, 103.1, 100.7 (2 × C-1), 99.4, 89.5, 78.1, 77.3, 76.6, 76.4, 74.2, 73.7, 72.8, 72.3, 71.8, 71.6, 69.9, 68.6, 68.3, 65.9, 65.6, 65.3, 62.1, 60.2, 55.5, 55.4, 47.5, 46.7, 45.9, 41.7, 41.4, 39.3, 38.9, 38.4, 36.7, 33.1, 32.4, 30.7, 28.3, 27.6, 25.8, 23.6, 23.4, 23.1, 18.2, 16.9, 16.5, 16.1, 15.3; HRESIMS: m/z calcd. for C₈₃H₉₂O₁₆Na[M+Na⁺]: 1367.6283; found: m/z 1367.6290.

Oleanolic acid 3-O-β-D-xylopyranosyl-(1→4)-6-deoxy-α-L-talopyranoside (5). Compound 5 was prepared from 38 by the same procedure as for 1. Yield: 71%; white powder, m.p. 218-220 °C, R_f = 0.11 (10:1:0.1 CHCl₃-MeOH-H₂O); -30.7 (c 0.5, MeOH); ¹H-NMR (pyridine-d₅): δ 5.47 (br s, 1 H, H-12), 5.27 (s, 1 H, H-1'), 4.80 (d, 1 H, J = 7.4 Hz, H-1''), 4.34-4.30 (m, 2 H), 4.25-4.20 (m, 3 H), 4.13-3.93 (m, 3 H), 3.69 (dd, 1 H, J = 9.6, 10.9 Hz), 3.29 (dd, 1 H, J = 3.9, 13.6 Hz, H-3), 3.11 (dd, 1 H, J = 4.3, 11.6 Hz, H-18), 1.70 (d, 3 H, J = 6.6 Hz, H-6'), 1.29, 1.00, 1.00, 0.95, 0.92, 0.84, 0.79 (s, 7 × 3 H, CH₃); ¹³C-NMR (pyridine-d₅): δ 180.1 (C=O), 144.8, 122.4, 106.3, 104.9 (2 × C-1), 88.6, 83.3, 77.8, 74.7, 71.9, 70.5, 67.2, 67.1, 66.7, 55.5, 47.9, 46.6, 46.4, 42.1, 42.0, 39.7, 39.1, 38.4, 36.9, 34.2, 33.2, 33.2, 33.1, 30.9, 28.2, 26.1, 25.6, 23.7, 23.7, 23.7, 23.6, 18.5, 17.3, 17.0, 16.6, 15.4; HRESIMS: m/z calcd. for C₄₁H₆₆O₁₁Na[M+Na⁺]: 757.4503; found: m/z 757.4515.

Benzyl oleanolate 3-O-2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1→4)-2,3-di-O-benzoyl-α-L-rhamnopyranoside (40). Compound 12 [11] (0.9 g, 1.2 mmol) and 31 (0.9 g, 1.0 mmol) were coupled under the same conditions as that used for the preparation of 23 from 11 and 9, giving 40 (1.3 g, 89%) as a foamy solid. R_f = 0.07 (8:1 petroleum ether-EtOAc); +64.5 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.07-7.05 (m, 35 H, Ar-H), 5.97 (d, 1 H, J = 3.1 Hz, H-4''), 5.75 (dd, 1 H, J = 7.9, 10.4 Hz, H-2''), 5.55-5.47 (m, 2 H, H-2', H-3'), 5.41 (dd, 1 H, J = 3.3, 10.4 Hz, H-3''), 5.30 (br s, 1 H, H-12), 5.13-5.07 (m, 3 H, H-1'', PhCH₂), 4.94 (d, 1 H, J = 1.4 Hz, H-1'), 4.71-4.37 (m, 3 H), 4.21-4.05 (m, 2 H), 3.14 (dd, 1 H, J = 7.5, 8.6 Hz, H-3), 2.91 (dd, 1 H, J = 3.9, 13.7 Hz, H-18), 1.50 (d, 1 H, J = 6.0 Hz, H-6'), 1.12, 0.96, 0.92, 0.92, 0.89, 0.85, 0.61 (s, 7 × 3 H, CH₃); ¹³C-NMR (CDCl₃): δ 177.4, 166.1, 165.5, 165.4, 165.4, 165.3, 164.7 (7 C=O), 143.7, 136.4, 133.5, 133.3, 133.2, 133.1, 132.8, 129.9, 129.7, 129.7, 129.7, 129.6, 129.6, 129.6, 129.6, 129.6, 129.5, 129.5, 129.4, 129.3, 129.0, 128.8, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 128.0, 128.0, 127.9, 122.5, 101.5, 99.8 (2 × C-1), 89.9, 77.6, 77.2, 72.5, 71.9, 71.1, 70.9, 69.7, 68.0, 67.1, 65.9, 62.0, 55.4, 47.6, 46.7, 45.9, 41.7, 41.4, 39.3, 38.9, 38.5, 36.7, 33.8, 33.0, 32.7, 32.4, 30.6, 28.3, 27.6, 25.8, 25.4, 23.6, 23.4, 23.1, 18.2, 18.1, 16.8, 16.5, 15.3; HRESIMS: m/z calcd. for C₉₁H₉₈O₁₈Na[M+Na⁺]: 1501.6651; found: m/z 1501.6629.

Oleanolic acid 3-O-β-D-galactopyranosyl-(1→4)-α-L-rhamnopyranoside (6). Compound 6 was prepared from 40 by the same procedure as for 1. Yield: 68%; white powder, m.p. 268-270 °C, R_f = 0.70 (10:2:0.1 CHCl₃-MeOH-H₂O); +24.6 (c 0.5, MeOH); ¹H-NMR (pyridine-d₅): δ 5.46 (br s, 1 H, H-12), 5.27 (s, 1 H, H-1'), 5.18 (d, 1 H, J = 7.8 Hz, H-1''), 4.60-4.24 (m, 8 H), 4.14 (dd, 1 H, J = 3.4, 9.5 Hz), 3.95 (dd, 1 H, J = 6.1, 6.4 Hz, H-2''), 3.29 (dd, 1 H, J = 4.2, 13.7 Hz, H-3), 3.11 (dd, 1 H, J = 4.5, 11.6 Hz, H-18),1.74 (d, 3 H, J = 6.2 Hz, H-6'), 1.27, 1.00, 0.99, 0.95, 0.89, 0.83, 0.76 (s, 7 × 3 H, CH₃); ¹³C-NMR (pyridine-d₅): δ 180.1 (C=O), 144.8, 122.7, 122.5, 107.3, 103.9 (2 × C-1), 88.5, 85.2, 76.9, 75.6, 74.1, 72.9, 71.9, 70.0, 68.1, 62.0, 55.5, 47.9, 46.6, 46.4, 42.1, 42.0, 39.7, 39.1, 38.4, 36.9, 34.2, 33.2, 33.2, 33.1, 30.9, 28.3, 28.2, 26.1, 25.7, 23.7, 23.7, 18.5, 18.3, 17.3, 16.7, 15.4; HRESIMS: m/z calcd. for C₄₂H₆₈O₁₂Na[M+Na⁺]: 787.4608; found: m/z 787.4579.

Benzyl oleanolate 3-O-2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl-6-deoxy-α-L-talopyranoside (42). Compound 16 (0.3 g, 0.3 mmol) and 15 (0.41 g, 0.4 mmol) were coupled under the same conditions as used for the preparation of 23 from 11 and 9, giving 42 (0.5 g, 90%) as a foamy solid. R_f = 0.11 (4:1 petroleum ether-EtOAc); -26.6 (c 0.5, CHCl₃); ¹H-NMR (CDCl₃): δ 8.28-7.16 (m, 45 H, Ar-H), 5.78 (d, 1 H, J = 3.5 Hz), 5.59 (dd, 1 H, J = 7.9, 10.4 Hz), 5.47-5.41 (m, 2 H), 5.26-5.20 (m, 2 H), 5.06 (dd, 2 H, J = 12.5, 17.3 Hz, PhCH₂), 4.93-4.86 (m, 3 H), 4.44 (d, 1 H, J = 1.4 Hz), 4.39-3.92 (m, 6 H), 3.30 (dd, 1 H, J = 5.6, 12.1 Hz, H-3), 3.06-2.87 (m, 3 H), 0.99 (d, 1 H, J = 6.5 Hz, H-6'), 1.09, 0.93, 0.91, 0.89, 0.86, 0.77, 0.58 (s, 7 × 3 H, CH₃); ¹³C-NMR (CDCl₃) δ 177.4, 166.3, 166.2, 165.8, 165.4, 165.3, 165.0, 164.9, 163.9 (9 C=O), 143.7, 136.4, 133.3, 133.3, 133.3, 133.2, 133.2, 133.1, 133.0, 132.9, 130.5, 130.0, 129.9, 129.9, 129.8, 129.8, 129.7, 129.7, 129.7, 129.7, 129.7, 129.7, 129.6, 129.6, 129.6, 129.5, 129.5, 129.4, 129.4, 129.4, 129.4, 129.2, 129.0, 128.7, 128.6, 128.5, 128.5, 128.5, 128.5, 128.5, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 127.8, 122.4, 103.3, 101.0, 100.6 (3 × C-1), 89.3, 77.3, 73.2, 71.7, 70.9, 70.2, 69.7, 68.5, 68.0, 67.5, 65.9, 65.4, 62.4, 61.2, 55.3, 47.5, 46.7, 45.9, 41.7, 41.4, 39.3, 38.9, 38.3, 36.6, 33.8, 33.0, 32.6, 32.3, 30.6, 28.2, 27.6, 25.8, 25.1, 23.6, 23.3, 23.0, 18.2, 16.8, 16.4, 15.9, 15.3; HRESIMS: m/z calcd. for C₁₁₀H₁₁₄O₂₄Na[M+Na⁺]: 1841.7598; found: m/z 1841.7579.

Oleanolic acid 3-O-β-D-galactopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-6-deoxy-α-L-talo-pyranoside (7). Compound 7 was prepared from 42 by the same procedure as for 1. Yield: 75%; white powder, m.p. 202-204 °C, R_f = 0.82 (10:2:0.1 CHCl₃-MeOH-H₂O); -36.8 (c 0.5, MeOH); ¹H- NMR (pyridine-d₅): δ 5.47 (br s, 1 H, H-12), 5.27-5.25 (m, 2 H), 4.72 (d, 1 H, J = 7.8 Hz), 4.54-4.50 (m, 2 H), 4.40-3.91 (m, 12 H), 3.85 (dd, 1 H, J = 7.0, 7.0 Hz), 3.59 (dd, 1 H, J = 10.2, 11.2 Hz), 3.28 (dd, 1 H, J = 4.2, 10.0 Hz, H-3), 3.09 (dd, 1 H, J = 4.4, 11.4 Hz, H-18), 1.67 (d, 3 H, J = 6.5 Hz, H-6'), 1.28, 1.00, 0.99, 0.95, 0.90, 0.83, 0.78 (s, 7 × 3 H, CH₃); ¹³C-NMR (pyridine-d₅): δ 180.2, 144.9, 122.5, 106.3, 105.9, 104.9 (3 × C-1), 88.8, 86.8, 83.6, 77.3, 75.2, 73.5, 73.1, 72.0, 70.2, 69.0, 67.1, 66.7, 66.5, 62.1, 57.4, 55.6, 48.0, 46.7, 46.5, 42.2, 42.1, 39.8, 39.2, 38.5, 37.0, 34.3, 33.3, 33.2, 31.0, 28.4, 28.3, 26.2, 25.7, 23.8, 23.7, 19.2, 18.6, 17.4, 17.0, 16.8, 15.5; HRESIMS: m/z calcd. for C₄₇H₇₆O₁₆Na[M+Na⁺]: 919.5031; found: m/z 919.5018.

3.3. Fungicidal activity bioassay

We used the mycelium growth rate test [20]. The culture media, with known concentration of the test compounds, were obtained by mixing the soln of compounds 1-7 in methanol with potato dextrose agar (PDA), on which fungus cakes were placed. The blank test was made using methanol. The culture was carried out at 24 ± 0.5 °C. Three replicates were performed.

4. Conclusions

Seven glycoconjugates of oleanolic acid were designed and efficiently synthesized. The bioassays showed that they had some fungicidal activity against four fungi. All of the compounds exhibited more fungicidal activity against R. solani, and the compounds 1 and 2 had better activity against B. cinerea and P. CapasiciLeonian than the other compounds.