Journal of Natural Products
Article
pump over 12 min. After 40 min had elapsed, saturated NH4Cl
solution (4 mL) was added, and the quenched reaction mixture was
warmed to room temperature and stirred overnight. A white
precipitate ensued, which was removed by filtration with a Celite-
(10S,13R,14R,17R)-4,4,10,13,14-Pentamethyl-17-((2R,5S,6S)-
5,6,7-trihydroxy-6-methylheptan-2-yl)-4,5,6,10,12,13,14,15,16,17-
decahydro-1H-cyclopenta[a]phenanthren-3(2H)-one (14): mp
139−141 °C; [α]D23 +34.3 (c 0.87, CHCl3); IR (KBr) νmax 3477
1
(br), 3024, 2969, 2872, 1699 cm−1; H NMR (CDCl3) δ 5.51 (d, J =
packed Buchner funnel. The contents of the funnel were washed with
̈
6.8 Hz, 1H), 5.38 (d, J = 6.1 Hz, 1H), 3.65 (d, J = 11.1 Hz, 1H), 3.55
(m, 1H), 3.49 (d, J = 11.2 Hz, 1H), 2.78 (dt, J = 5.7, 14.5 Hz, 1H),
2.34 (m, 1H), 2.30−1.96 (m, 7H), 1.76 (dt, J = 4.3, 13.2 Hz, 1H),
1.68−1.2 (9H), 1.19 (s, 3H), 1.12 (s, 3H), 1.10 (s, 3H), 1.09 (s, 3H),
0.91 (d, J = 6.5 Hz, 3H), 0.87 (s, 3H), 0.59 (s, 3H); 13C{1H} NMR
(CDCl3) δ 216.8, 144.5, 142.8, 119.9, 117.2, 78.5, 73.9, 67.6, 51.0,
50.7, 50.3, 47.5, 43.7, 37.4, 37.2, 36.6, 34.8, 33.1, 31.4, 28.5, 27.9, 25.4,
25.3, 23.6, 22.0, 21.0, 18.3, 15.7; HRMS calcd for C30H49O4 (M + H)+
473.3625, found 473.3616.
(10S,13R,14R,17R)-4,4,10,13,14-Pentamethyl-17-((2R,5R,6R)-
5,6,7-trihydroxy-6-methylheptan-2-yl)-4,5,6,10,12,13,14,15,16,17-
decahydro-1H-cyclopenta[a]phenanthren-3(2H)-one (15): mp
134−135 °C; [α]D23 +25.1 (c 0.42, CHCl3); IR (KBr) νmax 3335,
3018, 2964, 2924, 2883, 1707 cm−1; 1H NMR (CDCl3) δ 5.50 (d, J =
6.9 Hz, 1H), 5.38 (d, J = 6.5 Hz, 1H), 4.86 (br s, 1H), 3.64 (d, J = 11.2
Hz, 1H), 3.61 (m, 1H), 3.52 (d, J = 11.2 Hz, 1H), 2.77 (dt, J = 5.7,
14.6 Hz, 1H), 2.34 (ddd, J = 3.2, 4.4, 14.8 Hz, 1H), 2.21−1.97 (m,
6H), 1.75 (dt, J = 4.5 Hz, 13.7 Hz, 1H), 1.66−1.23 (m, 10H), 1.20 (s,
3H), 1.12 (s, 3H), 1.09 (s, 3H), 1.08 (s, 3H), 0.91 (d, J = 6.4 Hz, 3H),
0.88 (s, 3H), 0.59 (s, 3H); 13C{1H} NMR (CDCl3) δ 216.9, 144.5,
142.8, 119.9, 117.2, 76.2, 74.1, 69.4, 51.0, 50.7, 50.3, 47.5, 43.7, 43.4,
37.8, 37.2, 36.6, 34.8, 32.4, 31.4, 28.0, 27.9, 25.4, 25.3, 23.6, 22.0, 19.6,
18.3, 15.7; HRMS calcd for C30H49O4 (M + H)+ 473.3625, found
473.3615.
Reproducible proton shifts were found in DMSO-d6 for the
hydroxyl proton on C-24 of purified triols (1, δ 4.33; 13, δ 4.25; 14, δ
4.11; and 15, δ 4.05; all doublets, J = 6.4 ± 0.4 Hz). These signals were
integrated with a relaxation delay of 2 s to determine the de for the
asymmetric dihydroxylation reactions.
Cell Culture. The human breast cancer cell lines MCF-7 and
MDA-MB-231, obtained from ATCC (Manassas, VA, USA), were
cultivated in Dulbecco’s modified Eagle’s medium (DMEM)
containing penicillin (50 U/mL), streptomycin (50 U/mL), and
10% fetal bovine serum. Media and supplements were from Invitrogen
(Grand Island, NY, USA).
Cell Proliferation Assay. Ganodermanontriol, 13, 14, and 15
(isomers) were dissolved in DMSO (Sigma; St. Louis, MO, USA) at a
concentration of 10 mM and stored at 4 °C. Dulbecco’s phosphate
buffered saline was purchased from Cambrex Bio Science Walkersville,
Inc. (Walkersville, MD, USA).
Cell proliferation was determined by the tetrazolium salt method
(MTT method), according to the manufacturer’s instructions
(Promega, Madison, WI, USA). Briefly, MCF-7 and MDA-MB-231
cells (2.5 × 103/well) were cultured in a 96-well plate and treated with
ganodermanontriol, 13, 14, and 15 (0−100 μM) for 24, 48, and 72 h.
At the end of the incubation period, the cells were harvested and
absorption was determined with an ELISA plate reader at 570 nm.
Data points represent mean ± SD in triplicate determinations repeated
at least twice. IC50 values were determined by using SigmaPlot (Systat
Software Inc., San Jose, CA, USA).
CH2Cl2 (15 mL), and the combined organic layers were then
separated from the aqueous solution and washed with saturated brine
(30 mL). The CH2Cl2 solution was dried over MgSO4, vacuum
filtered, and concentrated with a rotary evaporator to give 11E as pure
white crystals (0.197 g, 96% yield). Alcohol 11Z was made by an
analogous procedure in 90% yield.
Deprotection of Ketals. Alcohol 11E (0.165 g, 0.342 mmol, 1.0
equiv) was dissolved in acetone (10 mL). p-Toluenesulfonic acid
(0.0065 g, 0.034 mmol, 0.01 equiv) was added, and the mixture was
stirred for 24 h at reflux. A rotary evaporator was used to remove the
acetone, and the residual solid was then diluted with CH2Cl2 (10 mL)
and washed with saturated sodium bicarbonate solution (2 × 10 mL)
followed by saturated brine (10 mL). The organic layer was dried over
MgSO4 and vacuum filtered through a pad of Celite, and the resulting
filtrate was then concentrated using a rotary evaporator to give 12E as
off-white crystals (0.124 g, 83% yield). Ketone 12Z was made by an
analogous procedure in 93% yield.
AD-Mix. Alcohol 12Z (0.0138 g, 0.0325 mmol, 1.0 equiv) was
dissolved in t-BuOH (0.33 mL) and water (0.33 mL). To this solution
was added AD-mix-α (0.091 g, 2.8 g/mmol) followed by
methanesulfonamide (0.0062 g, 0.065 mmol, 2.0 equiv). The solution
was allowed to stir for 6 h at 25 °C. Saturated aqueous sodium sulfite
(5 mL) was added, and the quenched reaction mixture was allowed to
stir for 1 h. The mixture was then diluted with ethyl acetate (5 mL),
the phases were separated, and the aqueous layer was then washed
with ethyl acetate (4 × 5 mL). The combined organic layers were
washed with saturated brine (20 mL), dried over MgSO4, and vacuum
filtered through a pad of Celite, and the resulting filtrate was
evaporated to dryness using a rotary evaporator. The solid was then
purified using silica gel chromatography (preabsorbed on silica gel,
step gradient of 3:1 hexane/EtOAc followed by 100% MeOH) to
afford 1 as a pure white crystalline solid (0.0114 g, 76% yield). The
other isomeric triols were made by an analogous procedure to afford
13 (0.0100 g, 56% yield from 12Z with AD-mix-β), 14 (0.0137 g, 64%
yield 12Z with AD-mix-α), and 15 (0.0169 g, 76% yield from 12E with
AD-mix-β).
(10S,13R,14R,17R)-4,4,10,13,14-Pentamethyl-17-((2R,5S,6R)-
5,6,7-trihydroxy-6-methylheptan-2-yl)-4,5,6,10,12,13,14,15,16,17-
decahydro-1H-cyclopenta[a]phenanthren-3(2H)-one (ganoderma-
nontriol, 1): mp 145−147 °C; [α]D23 +33.6; IR (KBr) νmax 3343 (br),
1
3020, 2958, 2933, 2882, 1706, 1150, 1112, 1048, 812 cm−1; H NMR
(CDCl3) δ 5.51 (br d, J = 6.5 Hz, 1H), 5.38 (br d, J = 6.2 Hz, 1H),
4.86 (br s, 2H), 3.83 (d, J = 11.2 Hz, 1H), 3.49 (d, J = 11.3 Hz, 1H),
3.45 (br d, 1H), 2.77 (dt, J = 5.7, 14.6 Hz, 1H), 2.34 (m, 1H), 2.30−
1.96 (m, 7H), 1.85−1.30 (m, 11H), 1.20 (s, 3H), 1.12 (s, 3H), 1.11 (s,
3H), 1.08 (s, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.88 (s, 3H), 0.59 (s, 3H);
13C{1H} NMR (CDCl3) δ 216.9, 144.5, 142.8, 119.9, 117.2, 79.3, 74.0,
67.6, 51.0, 50.7, 50.3, 47.5, 43.8, 43.4, 37.8, 37.2, 36.6, 36.5, 34.8, 33.5,
31.4, 28.9, 27.9, 25.4, 25.3, 23.7, 22.0, 21.0, 18.6, 15.7; HRMS calcd for
C30H49O4 (M + H)+, 473.3625, found 473.3641.
(10S,13R,14R,17R)-4,4,10,13,14-Pentamethyl-17-((2R,5R,6S)-
5,6,7-trihydroxy-6-methylheptan-2-yl)-4,5,6,10,12,13,14,15,16,17-
decahydro-1H-cyclopenta[a]phenanthren-3(2H)-one (13): mp
142−145 °C; [α]D23 +36.3 (c 0.60, CHCl3); IR (KBr) νmax 3418
ASSOCIATED CONTENT
■
S
* Supporting Information
1
(br), 3040, 2964, 2927, 2881, 1700 cm−1; H NMR (CDCl3) δ 5.51
Biological assay results for 1, 13, 14, and 15 and analytical data
for all of the newly synthesized compounds are available free of
(d, J = 6.7 Hz, 1H), 5.39 (d, J = 6.3 Hz, 1H), 4.76 (br s, 1H), 3.84 (d, J
= 11.0 Hz, 1H), 3.50 (m, 2H), 2.80 (dt, J = 5.8, 14.6 Hz, 1H), 2.50 (br
m, 2H), 2.34 (m, 1H), 2.31−1.98 (m, 6H), 1.76 (dt, J = 4.5, 14.0 Hz,
1H), 1.70−1.22 (m, 10H), 1.20 (s, 3H), 1.13 (s, 3H), 1.11 (s, 3H),
1.09 (s, 3H), 0.91 (d, J = 6.4 Hz, 3H), 0.88 (s, 3H), 0.60 (s, 3H);
13C{1H} NMR (CDCl3) δ 216.9, 144.5, 142.8, 119.9, 117.2, 77.2, 74.1,
69.3, 50.9, 50.7, 50.3, 47.5, 43.7, 37.8, 37.2, 36.6, 36.5, 34.8, 32.8, 31.4,
28.3, 27.9, 25.4, 25.3, 23.7, 22.7, 22.5, 22.0, 19.6, 18.6, 15.7; HRMS
calcd for C30H49O4 (M + H)+ 473.3625, found 473.3619.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (317) 274-6869. Fax: (317) 274-4701. E-mail: rminto@
2336
dx.doi.org/10.1021/np200205n|J. Nat. Prod. 2011, 74, 2332−2337