M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 45 (2010) 4827e4837
4835
73.3, 73.2 (CH2-benzyl), 138.6, 138.1, 138.0, 137.7 (Cipso),
128.3e127.4 (Ar), 55.0 (OCH3).
(C-15), 74.4 (C-16), 46.2 (C-17), 12.8 (C-18), 11.8 (C-19), 43.9 (C-20),
17.0 (C-21), 212.5 (C-22), 38.2 (C-23), 27.2 (C-24), 33.1 (C-25), 74.8
(C-26), 17.0 (C-27), 103.6 (C-10), 82.2 (C-20), 84.7 (C-30), 77.9 (C-40),
74.8 (C-50), 68.9 (C-60), 170.6 (CH3CO2-3), 169.6 (CH3CO2-16), 21.4
(CH3CO2-3), 21.0 (CH3CO2-16),128.3e127.5 (Ar),138.4 (Cipso-PhCH2-
O-20), 138.6 (Cipso-PhCH2-O-30), 138.1 (Cipso-PhCH2-O-40), 138.2
(Cipso-PhCH2-O-60), 74.8 (PhCH2-O-20), 75.6 (PhCH2-O-30), 75.0
5.2.3. 2,3,4,6-tetra-O-Benzyl- -glucopyranose (11)
D
Compound 10 (1.0 g, 1.8 mmol) was dissolved in acetic acid
(20 mL) and 1N H2SO4 (2.5 mL) was added. The mixture was
refluxed for a period of 2 h and the organic phase was extracted
four times with CH2Cl2 and the combined extracts were washed
with a saturated solution of NaHCO3 (5 x 100 mL) and water
(3 x 100 mL), dried over Na2SO4, and concentrated under vacuum.
The product was purified by column chromatography with hexane/
(PhCH2-O-40), 73.4 (PhCH2-O-60). HRMS Calcd for C65H83O12
:
1055.5885, [M þ H]þ. Found: 1055.5878.
5.2.5. (25R)-3
-glucopyranoside (15)
To a solution of 14 (300 mg, 0.29 mmol) in MeOH:AcOEt (7:3)
b,16b-Diacetoxy-12,22-dioxo-5a-cholestan-26-yl b-
ethyl acetate (85:15) as eluent to give 11 as a solid (mixture of
anomers; 588 mg, 55%). 1H NMR (CDCl3):
7.49e7.09 (Ar),
4.98e4.45 (CH2-benzyl), 5.23 (1H, d, J1,2 ¼ 3.7 Hz, H-1 -anomer),
4.72 (1H, d, J1,2 ¼ 7.8 Hz, H-1
-anomer), 4.07e3.37 (H-2-6). 13C
NMR (CDCl3): 91.3 (C-1 -anomer), 97.5 (C-1 -anomer),
a
and
D
b
d
b
was added 10% Pd/C (200 mg) and the mixture was stirred under
hydrogen for 2 h at room temperature. The catalyst was removed by
filtration and the filtrate was evaporated to dryness. The residue
was purified by flash column chromatography (CHCl3/MeOH
a
d
a
b
84.6e68.4 (C-2-6), 75.7e73.2 (CH2-benzyl), 138.7e137.7 (Cipso),
128.5e127.6 (Ar).
(95:5)) to give 15 as a pale yellow powder (198 mg, 95%). [a]
D
þ57.5ꢀ (c 0.4, MeOH). IR: 3391 (OH), 2938 (CH, aliphatic), 1723 and
1726 (C]O, acetate), 1709 and 1711 (C]O, ketone). 1H NMR (CDCl3/
5.2.4. (25R)-3
2,3,4,6-tetra-O-benzyl-
solution of 2,3,4,6-tetra-O-benzyl-
513 mg, 0.95 mmol), CCl3CN (0.48 mL, 4.74 mmol), and DBU (15
b
,16
b
-Diacetoxy-12,22-dioxo-5
-glucopyranoside (14)
-glucopyranose (11,
L,
a-cholestan-26-yl
b-
D
CD3OD):
d
4.98 (1H, ddd, J16-17 ¼ J16-15a ¼ J16-15b ¼ 8.2 Hz, H-16),
4.66 (1H, m, H-3), 4.23 (1H, d, J1 ,2 ¼ 7.8 Hz, H-10), 3.86 (1H, dd,
0
0
A
D
J6 a,5 ¼ 1.9 Hz, Jgem ¼ 11.9 Hz, H-60a), 3.75 (1H, dd, J26a,25 ¼ 5.8 Hz,
0
0
m
0
0
0.1 mmol) in CH2Cl2 (5 mL) was stirred at room temperature for 3 h.
The mixture was concentrated under vacuum and the resulting
residue was purified by flash column chromatography with
hexane/ethyl acetate/triethylamine (90:9:1) as eluent to give the
corresponding trichloroacetimidate 12 as a syrup (575 mg, 89%).
This compound was used immediately in the glycosylation reac-
tion. A mixture of the donor 12 (409 mg, 0.60 mmol), aglycon 7
(265 mg, 0.50 mmol) and 4 Å MS (200 mg) in dry CH2Cl2 (10 mL)
was stirred at room temperature for 15 min and then cooled to
Jgem ¼ 9.6 Hz, H-26a), 3.66 (1H, dd, J6 b,5 ¼ 5.3 Hz, Jgem ¼ 11.9 Hz, H-
60b), 3.36 (1H, dd, J26b,25 ¼ 6.0 Hz, Jgem ¼ 9.6 Hz, H-26b), 3.35 (1H,
dd, J3 ,2 ¼ J3 ,4 ¼ 9.1 Hz, H-30), 3.28 (1H, dd, J4 ,3 ¼ J4 ,5 ¼ 9.1 Hz, H-
0
0
0
0
0
0
0
0
40), 3.26 (1H, m, H-50), 3.18 (1H, dd, J2 ,3 ¼ 9.1 Hz, J2 ,1 ¼7.8 Hz, H-2 ),
0
0
0
0
0
2.91 (1H, dq, J20,21 ¼7.3 Hz, J20,17 ¼ 10.5 Hz, H-20), 2.77 (1H, m, 23a),
2.74 (1H, dd, J17,20 ¼ J17,16
¼
8.2 Hz, H-17), 2.68 (1H, dd,
J11ax,9 ¼ Jgem ¼ 12.9 Hz, H-11ax), 2.46 (1H, m, 15a), 2.46 (1H, m, H-
23b), 2.16 (1H, dd, J11eq,9 ¼ 4.8 Hz, Jgem ¼ 12.9 Hz, H-11eq), 2.00 (3H,
s, CH3CO2-3), 1.98 (3H, s, CH3CO2-16), 1.95 (1H, ddd, J8,7ax ¼ 7.2 Hz,
J8,7eq ¼ 3.6 Hz, J8,9 ¼ 10.8 Hz, H-8), 1.53 (1H, ddd, J2ax,1ax ¼ 17.0 Hz,
J2ax,1eq ¼ 4.0 Hz, Jgem ¼ 13.1 Hz, H-2ax), 1.23 (3H, s, CH3-18), 1.18 (1H,
ddd, J9,11ax ¼ 12.9 Hz, J9,11eq ¼ 4.8 Hz, J9,8 ¼ 10.8 Hz, H-9), 1.08 (3H, d,
J21,20 ¼ 7.3 Hz, CH3-21), 0.98 (3H, s, CH3-19), 0.94 (3H, d,
ꢃ20 ꢀC. A solution of TMSOTf in CH2Cl2 (10
mL, 0.05 mmol) was
added slowly to the reaction mixture. After stirring for 1 h, the
reaction was quenched by addition of triethylamine (0.1 mL) and
filtered. The filtrate was concentrated under vacuum to give
a residue of
a-(13) and
b
-(14) anomers in a 5:95 ratio (HPLC
J27,25 ¼ 6.6 Hz, CH3-27). 13C NMR (CDCl3/CD3OD):
d 37.5 (C-1), 28.3
acetonitrile/water 9:1) which was purified by flash column chro-
(C-2), 74.8 (C-3), 29.4 (C-4), 45.7 (C-5), 32.3 (C-6), 34.9 (C-7), 36.0
(C-8), 58.5 (C-9), 37.4 (C-10), 39.3 (C-11), 215.6 (C-12), 57.7 (C-13),
56.6 (C-14), 35.3 (C-15), 75.6 (C-16), 47.8 (C-17), 13.2 (C-18), 12.2 (C-
19), 45.0 (C-20), 17.7 (C-21), 216.2 (C-22), 39.5 (C-23), 28.5 (C-24),
34.3 (C-25), 75.9 (C-26), 17.3 (C-27), 104.6 (C-10), 75.2 (C-20), 78.2
(C-30), 71.7 (C-40), 77.9 (C-50), 62.9 (C-60), 172.5 (CH3CO2-3), 171.7
(CH3CO2-16), 21.3 (CH3CO2-3), 21.2 (CH3CO2-16). HRMS Calcd for
C37H59O12: 695.4006, [M þ H]þ. Found: 695.3998.
matography with hexane/ethyl acetate (7:3) as eluent to afford the
pure
b
-anomer as a colorless foam (498 mg, 93%). [
a
]
D þ40.1ꢀ (c 0.5,
CHCl3). IR: 2939 (CH, aliphatic), 1726 and 1729 (C]O, acetate), 1709
and 1702 (C]O, ketone), 1506, 1086 and 3068 (CH aromatics). 1H
NMR (CDCl3): d 7.34e7.15 (20H, m, Ar), 5.00 (1H, m, H-16), 4.94 (1H,
d, Jgem ¼ 10.8 Hz, PhCH2-O-20a), 4.92 (1H, d, Jgem ¼ 11.4 Hz, PhCH2-
O-30a), 4.81 (1H, d, Jgem ¼ 10.8 Hz, PhCH2-O-40a), 4.78 (1H, d,
Jgem ¼ 10.8 Hz, PhCH2-O-30b), 4.71 (1H, d, Jgem ¼ 11.0 Hz, PhCH2-O-
20b), 4.67 (1H, m, H-3), 4.61 (1H, d, Jgem ¼ 12.6 Hz, PhCH2-O-60a),
5.3. Biological activity
4.55 (1H, d, Jgem
¼
12.6 Hz, PhCH2-O-60b), 4.53 (1H, d,
Jgem ¼ 10.8 Hz, PhCH2-O-40b), 4.37 (1H, d, J1 ,2 ¼ 7.8 Hz, H-10), 3.76
5.3.1. Cell culture
0
0
(1H, dd, J26a,25
¼
J26a,26b
¼
90.6 Hz, H-26a), 3.74 (1H, dd,
HeLa, CaSki and ViBo cell lines were purchased from the
American Type Culture Collection (ATCC Rockville, MD) and were
cultured in RPMI-1640 medium (GIBCO, USA) containing 5%
Newborn Calf Serum (NCS, GIBCO, USA) with phenol red supple-
mented by benzylpenicillin. Cultures were maintained in a humid-
ified atmosphere with 5% CO2 at 37 ꢀC. All cell-based assays were
performed using cells in the exponential growth phase.
0
0
0
0
0
0
J6 a,6 b ¼ 10.8 Hz, J6 a,5 ¼1.8 Hz, H-6 a), 3.67 (1H, dd, J6 b,6 a ¼ 10.8 Hz,
J6 b,5 ¼ 5.4 Hz, H-60b), 3.63 (1H, dd, J3 ,2 ¼ J3 ,4 ¼ 9.0 Hz, H-30), 3.57
0
0
0
0
0
0
(1H, dd, J4 ,3 ¼ 9.0 Hz, J4 ,5 ¼ 9.6 Hz, H-40), 3.45 (1H, m, H-50), 3.44
0
0
0
0
(1H, dd, J2 ,3 ¼ 9.0 Hz, J2 ,1 ¼ 7.8 Hz, H-20), 3.40 (1H, dd,
J26b,25 ¼ 5.4 Hz, J26b,26a ¼ 9.6 Hz, H-26b), 2.79 (1H, m, H-20), 2.79
(1H, m, H-17), 2.62 (1H, m, 23a), 2.53 (1H, m, H-15a), 2.53 (1H, dd,
J11ax,9 ¼ Jgem ¼ 12.6 Hz, H-11ax), 2.34 (1H, m, H-23b), 2.19 (1H, dd,
J11eq,9 ¼ 4.8 Hz, Jgem ¼ 12.6 Hz, H-11eq), 2.02 (3H, s, CH3CO2-3), 1.93
(3H, s, CH3CO2-16), 1.58 (1H, ddd, J1eq,2ax ¼ 4.2 Hz, J1eq,2ec ¼ 3.0 Hz,
0
0
0
0
5.3.2. Cell proliferation assay
Assays were performed by seeding 7500 cells/well in 96-well
Jgem
¼
13.2 Hz, H-1eq), 1.49 (1H, ddd, J2ax,1ax
¼
13.2 Hz,
tissue culture plates in a volume of 100
supplemented with 5% NCS per well. Cells were allowed to grow for
24 h in culture medium prior to exposure to 42 M or 65 M of 15.
mL of RPMI-1640 medium
J2ax,1eq ¼ 4.2 Hz, Jgem ¼ 16.2 Hz, H-2ax),1.15 (3H, s, CH3-18),1.08 (3H,
d, J21,20 ¼ 6.6 Hz, CH3-21), 0.96 (3H, d, J27,25 ¼ 6.6 Hz, CH3-27), 0.92
m
m
(3H, s, CH3-19). 13C NMR (CDCl3):
d
36.2 (C-1), 27.1 (C-2), 73.1 (C-3),
1% of vehicle (ethanol) was added to control cells. Antiproliferative
activity (IC50) was determined after 24 h by crystal violet staining
[20]. Growth inhibition was determined by measuring the
28.1 (C-4), 44.3 (C-5), 30.1 (C-6), 33.7 (C-7), 34.7 (C-8), 56.6 (C-9),
36.2 (C-10), 38.1 (C-11), 212.9 (C-12), 56.3 (C-13), 54.8 (C-14), 34.5