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5.1.18. Benzyl 2,3-dihydroxy-23-t-butyldimethylsilyloxy-lup-
20(29)-en-28-oate (16)
5.1.24. Benzyl 2-hydroxymethyl-3-hydroxy-23-t-
butyldimethylsilyloxy-lup-20(29)-en-28-oate (22)
To a solution of 12 (800 mg, 1.20 mmol) in THF (10 mL) and
CH3OH (15 mL) cooled at 0 ꢀC was added sodium borohydride
(100 mg, 2.7 mmol). The reaction mixture was then stirred at room
temperature for 3 h, quenched with water (1 mL) and diluted with
EtOAc (20 mL). The organic layer was washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. The crude product
was purified by column chromatography (petroleum ether-acetate
10:1) to afford the product 16 as a white powder (580 mg, 69.8%).
To a solution of 21 (600 mg, 0.85 mmol) was dissolved in THF
(30 mL), 10% HCl (2 mL) was added. After reaction completion, most
of the solvent was evaporated in vacuo and diluted with ethyl ac-
etate (30 mL). The mixture was washed with water and brine, dried
over anhydrous sodium sulfate, and concentrated in vacuo. The
residue was purified over silica gel (petroleum ether-acetone 4:1)
to give compound 22 as a white solid (343 mg, 68.6%).
5.1.25. Benzyl 2-hydroxymethyl-3,23-dihydroxy-lup-20(29)-en-28-
oate (23)
5.1.19. Benzyl 2,3,23-trihydroxy-lup-20(29)-en-28-oate (17)
To a solution of 22 (600 mg, 1.02 mmol) in THF (15 mL) and
CH3OH (10 mL) cooled at 0 ꢀC was added sodium borohydride
(150 mg, 3.96 mmol). The reaction mixture was stirred at room
temperature for 3 h. The mixture was then quenched with water
(1 mL) and diluted with EtOAc (30 mL), washed with water and
brine, dried over Na2SO4, and concentrated in vacuo. The crude
product was purified by column chromatography (petroleum ether-
acetone 2:1) to afford compound 23 as a white powder (370 mg,
61.2%).
To a solution of 16 (550 mg, 0.79 mmol) was dissolved in THF
(30 mL), 10% HCl (2 mL) was added and the resulting mixture was
stirred at room temperature. After reaction completion, most of the
solvent was evaporated in vacuo and diluted with ethyl acetate
(30 mL). The organic layer was washed with water and brine, dried
over anhydrous sodium sulfate, and concentrated in vacuo. The
crude product was purified over silica gel (petroleum ether-acetone
4:1) to give compound 17 as a white solid (310 mg, 67.8%).
5.1.26. Benzyl 2-acetoxymethyl-3,23-diacetoxy-lup-20(29)-en-28-
oate (24)
5.1.20. Benzyl 2,3,23-triacetoxy-lup-20(29)-en-28-oate (18)
To a solution of 17 (250 mg, 0.43 mmol) in dry pyridine (10 mL)
was added acetic anhydride (0.8 mL, 8.46 mmol). The reaction
mixture was stirred at room temperature for 12 h. The mixture was
diluted with EtOAc (25 mL), washed with 10% HCl and brine, dried
over Na2SO4, and concentrated in vacuo. The crude product was
purified by column chromatography (petroleum ether-acetate 7:1)
to afford the product 18 as a white amorphous powder (220 mg,
72.6%).
To a solution of 23 (280 mg, 0.47 mmol) in dry pyridine (10 mL)
was added acetic anhydride (0.8 mL). The reaction mixture was
stirred at 60 ꢀC for 8 h. The mixture was then diluted with EtOAc
(25 mL), washed 10% HCl, water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by column
chromatography (petroleum ether-acetate 5:1) to afford the prod-
uct 24 as a white amorphous powder (220 mg, 75.7%).
5.1.27. 2-Acetoxymethyl-3,23-diacetoxy-lup-20(29)-en-28-oic acid
(25)
5.1.21. 2,3,23-Triacetoxy-lup-20(29)-en-28-oic acid (19)
To a solution of 18 (150 mg, 0.21 mmol) in THF (10 mL) was
added 10% Pd on carbon (30 mg) at room temperature. The mixture
was subjected to 1 atm of H2 and was monitored by TLC. The
mixture was filtered after most of compound 18 was consumed,
and the filtrate was concentrated, chromatographed over silica gel
(petroleum ether-acetone 5:1) to give 19 as a white solid (80 mg,
62.0%).
To a solution of 24 (150 mg, 0.24 mmol) in THF (10 mL) was
added 10% Pd on carbon (30 mg) at room temperature. The mixture
was subjected to 1 atm of H2 and was monitored by TLC. The
mixture was filtered when most of compound 24 was consumed,
and the filtrate was concentrated, chromatographed over silica gel
(petroleum ether-ethyl acetate 3:1) to give 25 as a white solid
(70 mg, 51.6%).
5.1.28. 2-Hydroxymethyl-3,23-dihydroxy-lup-20(29)-en-28-oic
acid (26)
5.1.22. 2,3,23-Diacetoxy-lup-20(29)-en-28-oic acid (20)
To a solution of 19 (50 mg, 0.081 mmol) in THF (5 mL) and
CH3OH (3 mL) was added 4 N NaOH (1 mL) aqueous solution, the
resulting solution was stirred at room temperature for 2 h. The
mixture was diluted with ethyl acetate (15 mL), washed with water
and brine, dried over anhydrous sodium sulfate. Filtered, the filtrate
was concentrated and chromatographed over silica gel (dichloro-
methane-methanol 40:1) to afford 20 as a white solid (16 mg,
40.8%).
To a solution of 25 (50 mg, 0.077 mmol) in THF (5 mL) and
CH3OH (3 mL) was added 4 N NaOH (1 mL) aqueous solution, the
resulting solution was stirred at room temperature for 2 h. The
mixture was then diluted with ethyl acetate (15 mL), washed with
water and brine, dried over anhydrous sodium sulfate. Filtered, the
filtrate was concentrated and chromatographed over silica gel
(dichloromethane-methanol 30:1) to afford 26 as a white solid
(21 mg, 54.3%).
5.2. In vitro anti-proliferative activity
5.1.23. Benzyl 2-hydroxymethylene-3-oxo-23-t-
butyldimethylsilyloxy-lup-20(29)-en -28-oate (21)
5.2.1. Cell lines and culture conditions
To a solution of 4 (1.50 g, 2.22 mmol) in dry dichloromethane
(30 mL) was added sodium methoxide (300 mg, 5.55 mmol) and
ethyl formate (1.5 mL), the mixture was stirred for 16 h at room
temperature, then diluted with ethyl acetate (40 mL). The organic
layer was washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by column
chromatography (petroleum ether-acetate 6:1) to afford the prod-
uct 21 as a white powder. The crude product can be also used for
the next step without further purification.
HL-60 (human leukemia), BEL-7402 (human hepatoma) and SF-
763 (human cerebroma) originally obtained from American type of
cell culture collection (ATCC), USA and stock was maintained in
laboratory; B16 (mice melanoma) and HeLa (human cervical
adenocarcinoma) were obtained from Chinese Academy of Sciences
Committee Type Culture Collection. All cell lines were cultured in
RPMI 1640 (Gibco) containing 10% fetal bovine serum (Gibco) and
1% penicillin streptomycin (Gibco) at 37 ꢀC in the presence of 5%
CO2.