Phytosterol Analogues as Anti-apoptotic Agent
457
(m, 1H), 2.35 (m, 2H), 2.07 (s, 3H), 2.05 (s, 3H), 2.02- CH2Cl2 (1.5 mL) was added dihydropyran (0.18 mL,
1.81 (m, 6H), 1.68 (m, 1H), 1.53 (m, 9H), 1.25-0.75 (m, 1.97 mmol) and -toluene sulfonic acid (12 mg, 0.07
22H), 0.67 (s, 3H); 13C-NMR (CDCl3, 75 MHz)
170.7, mmol). After stirring for 4 h, the reaction mixture was
p
δ
170.2, 140.6, 138.2, 129.2, 128.8, 128.0, 121.7, 92.7, quenched with H2O, and then diluted with EtOAc.
76.5, 66.7, 65.3, 63.1, 56.7, 55.8, 51.1, 50.1, 42.1, 40.4, The organic phase was washed with H2O and brine,
40.3, 39.5, 37.0, 36.6, 31.8, 28.8, 28.1, 25.3, 24.2, 21.1, dried over MgSO4 and concentrated in vacuo. Purifi-
21.0, 20.9, 20.9, 20.7, 19.2, 18.9, 12.2, 11.9; LR-MS cation of the residue via flash column chromatography
(FAB+) m/z 647 (M+Na).
on silica gel (EtOAc-hexanes = 1:20) afforded 82 mg
(68%) of as a white solid: FT-IR (neat) νmax 2921,
)- 1460, 1112 cm−1; H-NMR (CDCl3, 300 MHz)
5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4, (br, 1H), 4.69 (br, 1H), 3.88 (m, 1H), 3.54-3.43 (m, 2H),
7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1 -cy- 2.34-2.28 (m, 2H), 2.00-1.66 (m, 7H), 1.59-1.00 (m,
clopenta[ ]phenanthren-3-yloxy)-3,6-dihydro-2 24H), 0.98 (s, 3H), 0.86 (ddd, 9H, = 15.3, 6.4, 1.2 Hz),
7
1
((2
R
,3
S
)-3-Acetoxy-6-((3
S
,10
R,13R,17R)-17-((2R,5R
δ 5.32
H
α
H
-
J
pyran-2-yl)methyl acetate (8)
0.65 (s, 3H); LR-MS (FAB+) m/z 471 (M+H).
1
82%; white solid: H-NMR (CDCl3, 300 MHz)
(m, 2H), 5.32 (d, 1H,
δ
J
5.81
= 9.3
J
= 4.8 Hz), 5.25 (d, 1H,
MTT and morphological assay of viable endot-
helial cells
Human umbilical vein endothelial cells (HUVECs)
Hz), 5.14 (s, 1H), 4.16 (m, 3H), 3.51 (m, 1H), 2.34 (m,
2H), 2.06 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.00-1.81
(m, 5H), 1.74-1.00 (m, 22H), 0.97 (s, 3H), 0.88 (d, 3H, (3 × 105 cells/well) were plated onto 24-well plates in
J
= 6.2 Hz), 0.80 (q, 6H,
NMR (CDCl3, 100 MHz)
J
= 6.9 Hz), 0.64 (s, 3H); 13C- M199 media containing 20% fetal bovine serum. Next
170.4, 169.9, 140.5, 128.6, day, the cells were switched to serum free media and
δ
128.2, 121.6, 92.5, 77.9, 66.6, 65.1, 62.9, 56.2, 55.8, treated with or without various synthetic compounds
49.9, 45.5, 42.1, 40.2, 39.5, 36.9, 36.4, 35.9, 33.7, 31.7, at concentration of 10 g/mL. After 48 h, cells were
µ
31.6, 28.9, 28.0, 25.8, 24.1, 22.8, 20.8, 20.7, 20.6, 19.6, washed and photographed using optical microscope
19.1, 18.8, 18.6, 18.5, 12.0, 11.8; LR-MS (FAB+) m/z (Olympus 1X50-58F2) at 10X magnification. Cells were
649 (M+Na).
,3
then incubated with serum free media containing 100
g/mL MTT for 4 h. Upon incubation, the soluble yellow
MTT is converted to insoluble dark blue formazan
crystals by dehyrdrogenase enzymes of the viable
µ
((2
5-ethyl-6-methylheptan-2-yl)-10,13-dimethylhexa-
decahydro-1 -cyclopenta[
3,6-dihydro-2 -pyran-2-yl)methyl acetate (9)
78%; white solid: H-NMR (CDCl3, 300 MHz)
(m, 2H), 5.26 (d, 1H,
R
S
)-3-Acetoxy-6-((3
S
,10
S
,13
R
,17
R
)-17-((2
R
,5
R
)-
H
α]phenanthren-3-yloxy)- cells. The dark blue crystals were then solubilized by
H
adding DMSO-Ethanol (1:1) and the absorbance was
5.80 measured at 540 nm using ELISA plate reader (FLUO
1
δ
J
= 9.2 Hz), 5.14 (s, 1H), 4.16 (m, star Omega).
3H), 3.59 (m, 1H), 2.06 (s, 3H), 2.04 (s, 3H), 1.93 (m,
1H), 1.82-0.77 (m, 46H), 0.61 (s, 3H); LR-MS (FAB+)
m/z 651 (M+Na).
RESULTS AND DISCUSSION
Herein, we report design, synthesis and biological
evaluation of a series of phytosterol analogues as novel
((2
methyl-17-((
dro-1 -cyclopenta[
hydro-2 -pyran-2-yl)methyl acetate (10)
82%; white solid: H-NMR (CDCl3, 300 MHz)
(m, 2H), 5.26 (d, 1H,
3H), 3.60 (m, 1H), 2.07 (s, 3H), 2.05 (s, 3H), 1.93 (d, 1H, to the steroidal backbone of ginsenoside and choles-
= 12.1 Hz), 1.78-0.93 (m, 30H), 0.85 (dd, 9H, = 6.5, terol analogues. Our work commenced with compari-
10.08 Hz), 0.77 (s, 3H), 0.61 (s, 3H); LR-MS (FAB+) m/ son of activities of the phytosterols and their analogues
R,3S)-3-Acetoxy-6-((3S,5S,10S,13R,17R)-10,13-di-
R)-6-methylheptan-2-yl)hexadecahy- antiapoptotic agents. Considering the previous results
H
α
]phenanthren-3-yloxy)-3,6-di-
that ginsenoside and cholesterol analogues consisting
of steroidal backbone exhibited potent antiapoptotic
5.82 effects, we focused on the synthesis of the phytosterol
H
1
δ
J
= 9.2 Hz), 5.14, (s, 1H), 4.18 (m, analogues, which have the structurally similar skeleton
J
J
z
623 (M+Na).
connected to the dihydropyran moieties to elucidate
the influence of the carbohydrate moieties at C3
hydroxyl group as well as their steroidal backbones of
2-((3 ,10 ,13R,17R)-17-[(1R)-1,5-Dimethylhexyl]-
S
R
10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17- the analogues on antiapoptotic activities. Thus, we
tetradecahydro-1 -cyclopenta[ ]phenanthren-3- synthesized the 4,6-di- -acetyl-2,3-dideoxyhex-2-eno-
yloxy)tetrahydro-2 -pyran (11) pyran derivatives, which provided potent antiapoptotic
To a solution of cholesterol (100 mg, 0.26 mmol) in activity on HUVEC in preliminary studies. The struc-
H
H
α
O