20
–1
(52.2%) (amorphous powder). R 0.45 (A); [ꢄ] +45ꢅ (c 0.04; MeOH). C H N O . IR spectrum (ꢆ , cm ): 3600–3200
f
D
53 82
1
2
18
max
11
(ÎÍ, NH), 1750 (COOMe), 1720 (COOH), 1670 (C =O), 1560 (CONH). Í NMR spectrum (CD OD, , ppm): 0.81, 1.02,
1.05, 1.14, 1.27, 1.34, 1.40 (21Í, all s, 7ÑÍ ), 1.55–1.95 (ÑÍ, ÑÍ , m), 3.30, 3.60, 3.66 (9Í, s, ÎÑÍ ), 5.54 (1Í, s, Í-12).
Ñ NMR spectrum (CD OD, , ppm): 38.8 (Ñ-1), 26.8 (Ñ-2), 89.2 (Ñ-3), 39.2 (Ñ-4), 55.1 (Ñ-5), 17.0 (Ñ-6), 33.3 (Ñ-7), 45.3
3
3
2
3
13
3
(Ñ-8), 61.7 (Ñ-9), 36.6 (Ñ-10), 201.2 (Ñ-11), 127.5 (Ñ-12), 171.2 (Ñ-13), 43.2 (Ñ-14), 25.9 (Ñ-15), 26.1 (Ñ-16), 32.7 (Ñ-17),
49.8 (Ñ-18), 40.9 (Ñ-19), 43.9 (Ñ-20), 31.6 (Ñ-21), 37.6 (Ñ-22), 26.9 (Ñ-23), 15.2 (Ñ-24), 15.6 (Ñ-25), 17.9 (Ñ-26), 22.4
(Ñ-27), 27.1 (Ñ-28), 27.7 (Ñ-29), 177.3 (Ñ-30), 103.6 (Ñ-1ꢀ), 82.5 (Ñ-2ꢀ), 75.6 (Ñ-3ꢀ), 71.5 (Ñ-4ꢀ), 76.5 (Ñ-5ꢀ), 169.0 (Ñ-6ꢀ),
104.5 (Ñ-1ꢀꢀ), 74.8 (Ñ-2ꢀꢀ), 75.9 (Ñ-3ꢀꢀ), 71.8 (Ñ-4ꢀꢀ), 76.6 (Ñ-5ꢀꢀ), 169.1 (Ñ-6ꢀꢀ), 51.3 (Ñ-31); addl. reson.: 174.0, 174.1, 51.0,
50.8 (2ÑÎÎÑÍ ), 32.4, 32.1, 25.3, 25.2, 24.8, 24.4 (6ÑÍ ).
3
2
3-O-{2-O-[N-(ꢃ-D-Glucopyranosyluronoyl)-6-aminohexanoicAcid Methyl Ester]-N-(ꢃ-D-glucopyranosyluronoyl)-
6-aminohexanoicAcid Methyl Ester]-(3ꢃ,20ꢃ)-11-oxo-18ꢃ-olean-12-en-30-oicAcid (4). Asolution of GA(0.82 g, 1 mmol)
in dioxane (25 mL) was treated with HOSu (0.60 g, 5.2 mmol) and DCC (0.53 g, 2.5 mmol), stirred at this temperature for
2 h and at 20–22°C for 2 h, and left overnight in a refrigerator. The precipitate of dicyclohexylurea was filtered off. The
filtrate was treated with 6-aminohexanoic acid methyl ester hydrochloride (0.63 g, 2.5 mmol) and Et N (0.7 mL, 5 mmol) and
3
stored for 24 h at 20–22°C with periodic stirring. The mixture was worked up as described above to afford the crude conjugate
(1.0 g), which was chromatographed over a column of SG as described above. Yield 0.49 g (45.8%) (amorphous powder).
20
–1
R 0.46 (B), [ꢄ] +48ꢅ (c 0.06; MeOH). C H O N . IR spectrum (ꢆ , cm ): 3600–3200 (ÎÍ, NH), 1730 (COOMe),
f
D
56 88 18
1
2
max
11
1710 (COOH), 1660 (C =O), 1530 (CONH). Í NMR spectrum (CDCl + DMSO-d , , ppm): 0.66, 0.85, 0.96, 1.02, 1.15,
3
6
1.20, 1.62 (21Í, all s, 7ÑÍ ), 2.05–3.05 (m, ÑÍ, ÑÍ ), 3.50, 3.59 (6Í, br.s, 2ÑÎÎÑÍ ), 3.95–5.19 (m, Í-1ꢀ–Í-6ꢀ, Í-1ꢀꢀ–Í-6ꢀꢀ),
3
2
3
13
5.51 (1Í, s, Í-12). Ñ NMR spectrum (CDCl + DMSO-d , , ppm): 39.7 (C-1), 26.2 (Ñ-2), 89.5 (C-3), 40.0 (C-4), 55.1 (C-5),
3
6
18.5 (Ñ-6), 32.6 (Ñ-7), 45.2 (Ñ-8), 61.0 (Ñ-9), 36.5 (Ñ-10), 199.9 (C-11), 128.1 (C-12), 168.5 (C-13), 43.0 (C-14), 25.0 (Ñ-15),
26.0 (C-16), 32.1 (Ñ-17), 48.0 (Ñ-18), 41.0 (Ñ-19), 43.4 (Ñ-20), 31.6 (Ñ-21), 37.6 (Ñ-22), 27.3 (Ñ-23), 15.7 (Ñ-24), 16.2 (Ñ-25), 19.9
(Ñ-26), 23.2 (Ñ-27), 28.3 (Ñ-28), 28.4 (Ñ-29), 178.6 (C-30), 103.4 (C-1ꢀ), 82.5 (C-2ꢀ), 75.7 (Ñ-3ꢀ), 71.3 (Ñ-4ꢀ), 77.0 (Ñ-5ꢀ), 169.5
(C-6ꢀ), 105.0 (C-1ꢀꢀ), 72.5 (Ñ-2ꢀꢀ), 74.5 (Ñ-3ꢀꢀ), 71.3 (Ñ-4ꢀꢀ), 77.4 (Ñ-5ꢀꢀ), 169.6 (C-6ꢀꢀ);addl. reson.:169.9, 169.8, 52.1, 51.5 (2COOCH ),
3
39.4, 39.2, 31.2, 30.9, 29.2, 29.1, 17.2, 17.1, 13.7,13.4 (10ÑÍ ).
2
3-O-{2-O-[N-(ꢃ-D-Glucopyranosyluronoyl)-11-aminoundecanoic Acid Methyl Ester]-N-(ꢃ-D-
glucopyranosyluronoyl)-11-aminoundecanoic Acid Methyl Ester}-(3ꢃ,20ꢃ)-11-oxo-18ꢃ-olean-12-en-30-oic Acid (5).
A solution of GA (0.82 g, 1 mmol) in dioxane (20 mL) at 0–5°C was treated with HOSu (0.60 g, 5.2 mmol) and DCC (0.52 g,
2.5 mmol), stirred at this temperature for 2 h and at 20–22°C for 2 h, and left overnight in a refrigerator. The precipitate of
dicyclohexylurea was filtered off. The filtrate was treated with DMF (10 mL), 11-aminoundecanoic acid methyl ester
hydrochloride (0.55 g, 2.2 mmol), and NEM (0.6 mL, 0.6 mmol). The reaction mixture was worked up as described above to
afford the crude conjugate (0.9 g), which was chromatographed over a column of SG as described above. Yield 0.54 g (44.3%)
20
–1
(amorphous compound). R 0.45 (B). [ꢄ] +50ꢅ (c 0.02,MeOH). C H N O . IR spectrum (ꢆ , cm ): 3500–3200 (ÎÍ,
f
D
66 108
1
2
18
max
11
NH), 1740 (COOMe), 1710 (COOH), 1650 (C =O), 1560 (CONH). Í NMR spectrum (Py-d , , ppm): 0.78, 1.08, 1.12, 1.15,
5
1.18, 1.35, 1.46 (21Í, all s, 7ÑÍ ), 2.40–2.55 (m, ÑÍ, ÑÍ ), 3.64, 3.74 (6Í, br.s, ÎÑÍ ), 3.82–4.78 (m, Í-1ꢀ–Í-6ꢀ, Í-1ꢀꢀ–
3
2
3
13
Í-6ꢀꢀ), 5.98 (1Í, s, Í-12). Ñ NMR spectrum (Py-d , , ppm): 39.6 (C-1), 26.6 (C-2), 89.4 (C-3), 40.0 (C-4), 55.2 (Ñ-5), 17.4
5
(C-6), 33.9 (C-7), 46.3 (Ñ-8), 61.9 (Ñ-9), 37.1 (C-10), 199.3 (C-11), 128.4 (C-12), 166.9 (C-13), 43.9 (Ñ-14), 25.0 (C-15),
26.4 (C-16), 32.7 (C-17), 48.5 (Ñ-18), 41.4 (C-19), 45.3 (C-20), 31.9 (C-21), 38.2 (C-22), 27.6 (C-23), 16.6 (C-24, C-25), 18.6
(C-26), 23.3 (C-27), 28.4 (C-28), 28.5 (C-29), 178.9 (C-30), 104.9 (C-1ꢀ), 83.0 (C-2ꢀ), 75.5 (C-3ꢀ), 70.6 (C-4ꢀ), 77.2 (C-5ꢀ),
169.5 (C-6ꢀ), 106.0 (C-1ꢀꢀ), 73.7 (C-2ꢀꢀ), 75.2 (C-3ꢀꢀ), 71.8 (C-4ꢀꢀ), 77.5 (C-5ꢀꢀ), 169.7 (C-6ꢀꢀ); addl. reson.: 171.0, 170.5, 52.0,
51.0 (2ÑÎÎÑÍ ), 39.3, 39.2, 29.2–14.1 (ì, ÑÍ ).
3
2
Study of in vitro Cytotoxicity. The cytotoxicity of 4 was evaluated in tests on MDCK cell culture. A series of two-
fold dilutions of the compound were prepared at concentrations 500–3 ꢁg/mL in Eagle’s MEM. Cells were incubated at 37°C
for 48 h in an incubator with a 5% CO atmosphere. Then, MTT cell proliferation assays were carried out in 96-well plates.
2
Cells were rinsed twice with normal saline (0.9% NaCl) and treated (100 ꢁL/well) with MTT [3-(4,5-dimethylthiazole-2)-2,5-
diphenyltetrazolium bromide] at a concentration of 0.5 ꢁg/mL in normal saline. The plates were incubated at 37°C for 1 h,
after which the liquid was removed. The wells were treated with DMSO (0.1 mL). The optical density of the wells was
measured on a Victor 2 1440 spectrophotometer at 535 nm. The compound concentration destroying 50% of cells in culture
(CTD ) was calculated from the results.
50
319