Practical synthesis of bredemolic acid, a natural inhibitor of glycogen phosphorylase

Article abstract of DOI:10.1021/np8003886

Bredemolic acid (3) is a naturally occurring 2β,3α-isomer of maslinic acid (1) that is an allosteric site inhibitor of glycogen Phosphorylase (GP). A practical synthesis of 3 was accomplished (18% yield) in five steps starting from the readily available 2β,3β-diol 6a. In a similar fashion, 2β,3α-dihydroxyurs-12-en-28-oic acid (4) was synthesized as a natural 2β,3α-isomer of corosolic acid (2). Compounds 3 and 4 exhibited significant inhibitory activity against rabbit muscle GPa with IC ₅₀ values of 6.25 and 1.1 μM, respectively.

Full text of DOI:10.1021/np8003886

J. Nat. Prod. 2008, 71, 1877–1880
1877
Practical Synthesis of Bredemolic Acid, a Natural Inhibitor of Glycogen Phosphorylase
Keguang Cheng,† Pu Zhang,† Jun Liu,‡ Juan Xie,§ and Hongbin Sun*,†
Center for Drug DiscoVery, College of Pharmacy, China Pharmaceutical UniVersity, 24 Tongjia Xiang, Nanjing 210009, People’s Republic of
China, Jiangsu Center for Drug Screening, China Pharmaceutical UniVersity, 1 Shennonglu, Nanjing 210038, People’s Republic of China, and
Laboratoire de Photophysique et Photochimie Supramole´culaires et Macromole´culaires, CNRS UMR 8531, Ecole Normale Supe´rieure de
Cachan, 61 AVenue du Pt Wilson, F-94235 Cachan, France
ReceiVed June 27, 2008
Bredemolic acid (3) is a naturally occurring 2ꢀ,3R-isomer of maslinic acid (1) that is an allosteric site inhibitor of
glycogen phosphorylase (GP). A practical synthesis of 3 was accomplished (18% yield) in five steps starting from the
readily available 2ꢀ,3ꢀ-diol 6a. In a similar fashion, 2ꢀ,3R-dihydroxyurs-12-en-28-oic acid (4) was synthesized as a
natural 2ꢀ,3R-isomer of corosolic acid (2). Compounds 3 and 4 exhibited significant inhibitory activity against rabbit
muscle GPa with IC50 values of 6.25 and 1.1 µM, respectively.
Maslinic acid (1) and corosolic acid (2) have recently attracted
much attention due to their reported antitumor, anti-HIV, anti-
inflammation, antioxidation, antihyperglycemia, and cardiovascular
activities.1 Previously we reported that 1, 2, and related pentacyclic
triterpenes represented a new class of allosteric site inhibitors of
glycogen phosphorylases (GP), and their glucose-lowering activity
could, at least in part, be due to modulation of glycogen
metabolism.2-6 Recently, Fukushima et al. proved that 2 exhibited
a glucose-lowering effect on postchallenge plasma glucose levels
in humans.7 Both 1 and 2 have 2R,3ꢀ-dihydroxy functions, and
the only structural difference lies in the positioning of E-ring
dimethyl groups. In our previous studies,2,3 it was shown that the
configuration of 2,3-dihydroxy groups had an impact on GP
inhibitory activity. In this regard, it seemed desirable to examine
how isomeric compounds having 2ꢀ,3R-dihydroxy functions, rather
than 2R,3ꢀ-dihydroxy groups as in 1 and 2, would affect biological
activity.
Bredemolic acid (3) (2ꢀ,3R-dihydroxyolean-12-en-28-oic acid)
was obtained by acidic hydrolysis of a crude sapogenin from
Bredemeyera floribunda (Polygalaceae).8 To our knowledge, there
has been no previous biological evaluation of this triterpene. 2ꢀ,3R-
Dihydroxyurs-12-en-28-oic acid (4), an ursane type of counterpart
of 3, is claimed to have been isolated from Lagerstroemia floribunda
(Lythraceae);9 however, no spectroscopic data are available for 4.
Herein, we report a practical synthesis and biological evaluation
of bredemolic acid (3) and 2ꢀ,3R-dihydroxyurs-12-en-28-oic acid
(4), which are naturally occurring 2ꢀ,3R-isomers of 1 and 2,
respectively.
opening of the resulting epoxide as the key steps.10,11 In our hands,
however, preparations of the required triterpene dienes were very
complex reactions, resulting in a variety of inseparable byproducts,
and the overall yields were very poor. We therefore developed a
new access to 3 and 4, starting from the readily available 2ꢀ,3ꢀ-
diols 6a and 6b,2,3 respectively (Scheme 1).
As shown in Scheme 1, treatment of 2ꢀ,3ꢀ-diol 6a, which was
readily prepared from oleanolic acid (5a),2 with tert-butyldimeth-
ylsilyl chloride (TBDMSCl) and imidazole in DMF gave the 2-O-
silylated product 7a as the major product (73%), together with the
3-O-silylated product 8a as a minor product (19%). The observed
stereoselectivity indicated that formation of 7a was kinetically more
favored than that of 8a, possibly due to less steric hindrance at
C2ꢀ-OH than at C3ꢀ-OH.12 The relative configurations of 7a and
8a have been determined by NOE experiments (see the Supporting
Information). Oxidation of 7a with pyridinium chlorochromate
(PCC) at slightly elevated temperature afforded ketone 9a (66%).
Considering that Meerwein-Pondorf reduction of the 3-oxo group
of pentacyclic triterpenes has been proved to be an efficient method
to prepare a 3R-hydroxy function,2,13 this methodology was
employed to provide the desired 3R-hydroxy function of the key
intermediate 10a. Reduction of 9a in the presence of aluminum
isopropoxide in isopropyl alcohol gave 10a as the major product
(46%), together with the 3ꢀ-hydroxy isomer 7a as the minor product
(40%). Deprotection of 10a with tetrabutylammonium fluoride
(TBAF) in THF at room temperature gave 2ꢀ,3R-diol 11a in high
yield (92%). Hydrogenolysis of 11a over palladium-carbon in THF
furnished bredemolic acid (3) in good yield (86%). In a similar
fashion, 2ꢀ,3R-dihydroxyurs-12-en-28-oic acid (4) was synthesized,
starting from 2ꢀ,3ꢀ-diol 6b.
The synthesized natural triterpenes 3 and 4 were evaluated for
their inhibitory activity against rabbit muscle GPa (RMGPa). The
activity of RMGPa was measured by detecting the release of
phosphate from glucose-1-phosphate in the direction of glycogen
synthesis.14 The bioassay results (Table 1) showed that both 3 (IC50
6.25 µM) and 4 (IC50 1.1 µM) exhibited significant inhibition against
RMGPa. Interestingly, while 3 was more potent than its 2R,3ꢀ-
isomer 1 (IC50 28 µM),6 4 was more potent than its 2R,3ꢀ-
counterpart 2 (IC50 20 µM)6 as well. That is to say, the 2ꢀ,3R-
dihydroxy function in pentacyclic triterpenes appears to be more
favorable than compounds having the 2R,3ꢀ-dihydroxy function
in terms of GP inhibition.
Results and Discussion
Tsehesche et al. carried out a partial synthesis of 3 by employing
epoxidation of a triterpene diene followed by acid-catalyzed ring
* To whom correspondence should be addressed. Tel: +86-25-85327950.
Fax: +86-25-83271335. E-mail: hbsun2000@yahoo.com.
† Center for Drug Discovery, College of Pharmacy, China Pharmaceutical
University.
Experimental Section
‡ Jiangsu Center for Drug Screening, China Pharmaceutical University.
§ Photophysique et Photochimie Supramole´culaires et Macromole´culaires,
CNRS UMR.
General Experimental Procedures. All commercially available
solvents and reagents were used without further purification. Melting
points of compounds were measured on a RY-1 melting point apparatus.
10.1021/np8003886 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/11/2008

1878 Journal of Natural Products, 2008, Vol. 71, No. 11
Cheng et al.
Scheme 1. Synthesis of Bredemolic Acid (3) and 2ꢀ,3R-Dihydroxyurs-12-en-28-oic acid (4)a
a (a) TBDMSCl, imidazole, DMF, 43 °C; (b) PCC, DCM, 40 °C; (c) Al(i-PrO)3, i-PrOH, AlCl3 (cat.), reflux; (d) TBAF, THF, rt; (e) H2, 10% Pd-C, THF, rt.
major product (6.62 g, 73%), together with benzyl 3ꢀ-(tert-butyldim-
ethylsilyloxy)-2ꢀ-hydroxyolean-12-en-28-oate (8a) as the minor product
(1.7 g, 19%).
Table 1. IC50 Values (µM) for the Inhibition of Rabbit Muscle
GPa
a
compd
GPa IC50
Compound 7a: white solid, mp 75-76 °C; [R]2D2 +57.9 (c 0.28,
1
CHCl3); IR (KBr) νmax 2950, 1726, 696 cm-1; H NMR (CDCl3, 300
3
4
6.25
1.1
MHz) δ 0.63, 0.90, 0.92, 0.95, 1.00, 1.11, 1.16 (each 3H, s), 0.09 and
0.10 (each 3H, s, Si(CH3)2), 0.91 (9H, s, CH3 of tert-butyl), 2.90 (1H,
dd, J ) 3.9, 13.6 Hz, H-18), 3.05 (1H, brs, H-3R), 4.06 (1H, m, H-2R),
5.06 (2H, s, CH2Ar), 5.28 (1H, t, J ) 3.5 Hz, H-12), 7.34 (5H, m,
H-Ar); 13C NMR (CDCl3, 75 MHz) δ -5.4, -4.1, 16.2, 16.9, 17.0,
18.0, 18.1, 23.0, 23.4, 23.6, 25.9, 26.0, 27.5, 29.7, 30.7, 32.4, 32.7,
33.1, 33.9, 36.8, 38.3, 39.4, 41.4, 41.9, 44.7, 45.9, 46.7, 47.9, 55.3,
65.9, 72.0, 78.1, 122.4, 127.8, 127.9, 128.4, 136.4, 143.8, 177.4; ESIMS
m/z 699.3 [M + Na]+, 715.3 [M + K]+; HRMS m/z 699.4796 (calcd
for C43H68NaO4Si, 699.4785); anal. calcd for C43H68O4Si·0.3H2O, C
75.67, H 10.13; found, C 75.42, H 10.18.
caffeine
83.1
a Values are means of three experiments.
Column chromatography was carried out on silica gel (200-300 mesh,
Qindao Ocean Chemical Company, China). IR spectra were recorded
1
on a Shimadzu FTIR-8400S spectrometer. H and 13C NMR spectra
were measured on Bruker AV-300 or AV-500 spectrometers. Chemical
shifts are reported as δ values from an internal tetramethylsilane
standard. Mass spectra were obtained on Agilent 1100 LC/DAD/MSD
or Q-Tof Micro MS/MS spectrometers. Elemental analyses were
measured on a Vario EL III instrument (Elementar, Germany).
Benzyl 2ꢀ-(tert-Butyldimethylsilyloxy)-3ꢀ-hydroxyolean-12-en-
28-oate (7a). Imidazole (7.48 g, 0.11 mol) was added to a solution of
6a2 (7.5 g, 13 mmol) in DMF (180 mL) at room temperature. Then
tert-butyldimethylsilyl chloride (TBDMSCl, 7.84 g, 52 mmol) was
added. The reaction mixture was heated at 43 °C for 4 h. After cooling
to room temperature, the mixture was diluted with water (200 mL)
and extracted with EtOAc (3 × 150 mL), and the combined organic
layers were washed with water and brine, dried over Na2SO4, filtered,
and concentrated to give a colorless oil. The oil was purified by flash
chromatography (EtOAc/petroleum ether, 1:160) to afford 7a as the
Compound 8a: white solid, mp 197-199 °C; [R]2D2 +51.8 (c 0.08,
1
CHCl3); IR (KBr) νmax 2950, 1724, 696 cm-1; H NMR (CDCl3, 300
MHz) δ 0.63, 0.90, 0.97, 1.11, 1.21 (each 3H, s), 0.92 (6H, s), 0.08
and 0.10 (each 3H, s, Si(CH3)2), 0.94 (9H, s, CH3 of tert-butyl), 2.90
(1H, dd, J ) 3.7, 13.8 Hz, H-18), 3.25 (1H, d, J ) 3.9 Hz, H-3R),
3.91 (1H, m, H-2R), 5.06 and 5.08 (each 1H, d, J ) 12.5 Hz, CH2Ar),
5.30 (1H, t, J ) 3.5 Hz, H-12), 7.34 (5H, m, H-Ar); ESIMS m/z 699.5
[M + Na]+, 715.5 [M + K]+; anal. calcd for C43H68O4Si·0.3CH3OH,
C 75.73, H 10.16; found, C 75.59, H 10.16.
Benzyl 2ꢀ-(tert-Butyldimethylsilyloxy)-3ꢀ-hydroxyurs-12-en-28-
oate (7b). According to the procedure for preparation of 7a, treatment
of 6b2,3 (2.4 g, 4.3 mmol) with TBDMSCl afforded 7b (2.0 g, 70%)

Practical Synthesis of Bredemolic Acid
Journal of Natural Products, 2008, Vol. 71, No. 11 1879
as the major product, together with benzyl 3ꢀ-(tert-butyldimethylsily-
loxy)-2ꢀ-hydroxyurs-12-en-28-oate (8b) as the minor product (0.75 g,
26%).
A mixture of freshly prepared aluminum isopropoxide (17 mL, 26
mmol) and AlCl3 (0.1 g, 0.7 mmol) was heated to 45 °C for half an
hour and cooled to 30 °C, and then a solution of 9a (2.8 g, 4.2 mmol)
in anhydrous i-PrOH (36 mL) was added. After the reaction mixture
was heated at reflux for 12 h, 1 M HCl (80 mL) was added at 0 °C,
and then the mixture was extracted with EtOAc (3 × 80 mL). The
combined organic layers were washed with water, saturated NaHCO3,
and brine, dried over Na2SO4, filtered, and concentrated to give a yellow
solid, which was purified by flash chromatography (EtOAc/petroleum
ether, 1:100) to afford 10a as the major product (1.29 g, 46%), together
with 7a (1.12 g, 40%) as the minor product.
Compound 7b: white solid, mp 92-93 °C; [R]2D2 +62.3 (c 0.13,
1
CHCl3); IR (KBr) νmax 2950, 1723, 696 cm-1; H NMR (CDCl3, 300
MHz) δ 0.67, 0.94, 0.95, 1.00, 1.07, 1.18 (each 3H, s), 0.85 (3H, d, J
) 6.5 Hz), 0.09 and 0.10 (each 3H, s, Si(CH3)2), 0.91 (9H, s, CH3 of
tert-butyl), 2.28 (1H, d, J ) 11.4 Hz, H-18), 3.06 (1H, d, J ) 3.7 Hz,
H-3R), 4.07 (1H, m, H-2R), 5.02 and 5.06 (each 1H, d, J ) 12.5 Hz,
CH2Ar), 5.25 (1H, t, J ) 3.5 Hz, H-12), 7.34 (5H, m, H-Ar); 13C NMR
(CDCl3, 75 MHz) δ -5.2, -4.0, 16.5, 16.9, 17.0, 17.1, 18.1, 18.2,
21.1, 23.5, 23.7, 24.4, 26.0, 28.0, 30.0, 30.8, 33.2, 36.7, 36.9, 38.4,
39.0, 39.2, 39.9, 42.4, 45.2, 48.0, 48.3, 53.1, 55.5, 66.0, 72.3, 78.3,
125.8, 127.9, 128.2, 128.4, 136,6, 138.4, 177.2; ESIMS m/z 675.5 [M
- H]-; HRMS m/z 699.4787 (calcd for C43H68NaO4Si, 699.4785); anal.
calcd for C43H68O4Si·0.1CH3COOC2H5, C 76.00, H 10.11; found, C
76.16, H 10.06.
Compound 10a: white solid, mp 79-81 °C; [R]2D2 +82.6 (c 0.13,
1
CHCl3); IR (KBr) νmax 2950, 1724, 770 cm-1; H NMR (CDCl3, 300
MHz) δ 0.61, 0.90, 0.91, 0.92, 1.03, 1.10, 1.14 (each 3H, s), 0.06 and
0.07 (each 3H, s, Si(CH3)2), 0.89 (9H, s, CH3 of tert-butyl), 2.89 (1H,
m, H-18), 3.54 (1H, d, J ) 8.0 Hz, H-3ꢀ), 3.80 (1H, dd, J ) 6.8, 14.8
Hz, H-2R), 5.06 and 5.07 (each 1H, d, J ) 12.5 Hz, CH2Ar), 5.31
(1H, t, J ) 3.5 Hz, H-12), 7.33 (5H, m, H-Ar); 13C NMR (CDCl3, 75
MHz) δ -4.9, -4.2, 16.7, 18.0, 19.3, 20.0, 23.0, 23.2, 23.5, 23.7, 25.5,
25.9, 26.0, 27.6, 30.7, 32.4, 33.1, 34.0, 37.0, 37.5, 39.7, 41.6, 42.0,
46.0, 46.2, 46.9, 48.3, 50.4, 66.0, 71.4, 77.8, 122.7, 127.9, 128.0, 128.4,
136.5, 143.7, 177.4; ESIMS m/z 699.3 [M + Na]+, 715.3 [M + K]+;
anal. calcd for C43H68O4Si·0.25 CH3COOC2H5: C 75.53, H 10.09;
found, C 75.97, H 9.88.
Compound 8b: white solid, mp 134-135 °C; [R]2D2 +35.3 (c 0.09,
1
CHCl3); IR (KBr) νmax 2940, 1723, 696 cm-1; H NMR (CDCl3, 300
MHz) δ 0.65, 0.91, 0.92, 0.97, 1.06, 1.23 (each 3H, s), 0.85 (3H, d, J
) 6.4 Hz), 0.08 and 0.10 (each 3H, s, Si(CH3)2), 0.94 (9H, s, CH3 of
tert-butyl), 2.29 (1H, d, J ) 11.5 Hz, H-18), 3.26 (1H, d, J ) 3.9 Hz,
H-3R), 3.93 (1H, m, H-2R), 5.00 and 5.09 (each 1H, d, J ) 12.5 Hz,
CH2Ar), 5.25 (1H, t, J ) 3.4 Hz, H-12), 7.35 (5H, m, H-Ar); 13C NMR
(CDCl3, 75 MHz) δ -5.2, -4.1, 16.5, 17.0, 17.1, 17.8, 18.3, 21.1,
23.5, 23.6, 24.4, 26.0, 28.0, 30.0, 30.8, 33.2, 36.6, 36.7, 38.6, 39.0,
39.2, 39.8, 42.3, 43.3, 48.3, 53.1, 55.4, 66.0, 71.6, 80.3, 126.1, 128.0,
128.2, 128.4, 138.1, 177.2; ESIMS m/z 675.5 [M - H]-; anal. calcd
for C43H68O4Si·0.4 CH3COOC2H5, C 75.20, H 10.07; found, C 75.52,
H 9.89.
Benzyl 2ꢀ-(tert-butyldimethylsilyloxy)-3r-hydroxyurs-12-en-28-
oate (10b). According to the procedure for preparation of 10a, reduction
of 9b (1.5 g, 2 mmol) in the presence of aluminum isopropoxide
afforded 10b as the major product (0.7 g, 47%), together with 7b as
the minor product (0.62 g, 41%).
Compound 10b: white solid, mp 73-75 °C; [R]2D2 +77.2 (c 0.07,
1
CHCl3); IR (KBr) νmax 2927, 1723, 758 cm-1; H NMR (CDCl3, 300
Benzyl 2ꢀ-(tert-Butyldimethylsilyloxy)-3-oxoolean-12-en-28-oate
(9a). To a solution of 7a (5.87 g, 8.7 mmol) in CH2Cl2 (26 mL) was
added pyridinium chlorochromate (PCC, 3.59 g, 17.4 mmol) at 0 °C.
Then the mixture was stirred at 40 °C for 12 h. The mixture was filtered
through silica gel, and the insoluble material was washed several times
with CH2Cl2. The filtrate was concentrated to give a crude product,
which was purified by flash chromatography (EtOAc/petroleum ether,
1:40) to afford 9a (3.85 g, 66%).
MHz) δ 0.64, 0.92, 0.95, 1.03, 1.08, 1.12 (each 3H, s), 0.86 (3H, d, J
) 6.6 Hz), 0.08 (6H, s, Si(CH3)2), 0.90 (9H, s, CH3 of tert-butyl), 2.25
(1H, d, J ) 11.4 Hz, H-18), 3.52 (1H, d, J ) 7.8 Hz, H-3ꢀ), 3.81 (1H,
dd, J ) 6.9, 14.1 Hz, H-2R), 5.00 and 5.08 (each 1H, d, J ) 12.5 Hz,
CH2Ar), 5.26 (1H, t, J ) 3.2 Hz, H-12), 7.34 (5H, m, H-Ar); 13C NMR
(CDCl3, 75 MHz) δ -4.9, -4.3, 16.9, 17.0, 18.0, 19.1, 19.6, 21.1,
22.9, 23.3, 23.7, 24.3, 25.7, 25.9, 27.9, 30.7, 32.7, 36.7, 37.0, 37.4,
38.9, 39.2, 39.8, 42.3, 46.1, 48.2, 48.3, 50.3, 53.0, 66.0, 71.5, 77.9,
125.9, 128.0, 128.2, 128.4, 136.4, 138.2, 177.2; ESIMS m/z 675.5 [M
- H]-; anal. calcd for C43H68O4Si, C 76.28, H 10.12; found, C 76.26,
H 10.26.
Compound 9a: white solid, mp 80-82 °C; [R]2D2 +81.8 (c 0.13,
1
CHCl3); IR (KBr) νmax 2952, 1725, 697 cm-1; H NMR (CDCl3, 300
MHz) δ 0.61, 0.80, 0.91, 0.93, 1.07, 1.10, 1.19 (each 3H, s), 0.004
and 0.13 (each 3H, s, Si(CH3)2), 0.89 (9H, s, CH3 of tert-butyl), 2.95
(1H, dd, J ) 3.9, 13.7 Hz, H-18), 4.66 (1H, dd, J ) 7.9, 11.2 Hz,
H-2R), 5.06 and 5.08 (each 1H, d, J ) 12.5 Hz, CH2Ar), 5.33 (1H, t,
J ) 3.4 Hz, H-12), 7.33 (5H, m, H-Ar); 13C NMR (CDCl3, 75 MHz)
δ -5.5, -4.7, 16.3, 18.0, 18.5, 19.6, 20.0, 23.1, 23.5, 23.6, 25.7, 25.8,
27.6, 29.9, 30.7, 31.8, 32.3, 33.1, 33.9, 36.9, 39.3, 41.7, 42.0, 45.9,
46.3, 46.9, 47.0, 51.4, 52.1, 66.0, 70.8, 122.5, 127.9, 128.0, 128.4, 136,4,
143.5, 177.3, 217.3; ESIMS m/z 697.3 [M + Na]+, 713.3 [M + K]+;
anal. calcd for C43H66O4Si·1.3CH3COOC2H5, C 73.32, H 9.75; found,
C 73.58, H 9.70.
Benzyl 2ꢀ,3r-Dihydroxyolean-12-en-28-oate (11a). To a solution
of 10a (0.29 g, 4.3 mmol) in THF (2 mL) was added dropwise 1 M
TBAF (3 mL, 3 mmol). After stirring at room temperature for 8 h, the
reaction mixture was diluted with water (20 mL) and extracted with
EtOAc (3 × 10 mL). The combined organic layers were washed with
water and brine, dried over Na2SO4, filtered, and concentrated to give
a colorless oil, which was purified by flash chromatography (EtOAc/
petroleum ether, 1:5) to afford 11a (0.22 g, 92%).
Compound 11a: white solid, mp 106-108 °C; [R]2D2 +99.2 (c 0.08,
1
CHCl3); IR (KBr) νmax 2947, 1723, 756 cm-1; H NMR (CDCl3, 300
Benzyl 2ꢀ-(tert-Butyldimethylsilyloxy)-3-oxours-12-en-28-oate (9b).
According to the procedure for preparation of 9a, oxidation of 7b (2.0
g, 3 mmol) with PCC afforded 9b (1.6 g, 80%).
MHz) δ 0.60, 0.92, 1.00, 1.06, 1.13 (each 3H, s), 0.90 (6H, s), 2.92
(1H, dd, J ) 4.1, 13.7 Hz, H-18), 3.65 (1H, d, J ) 10.4 Hz, H-3ꢀ),
3.75 (1H, m, H-2R), 5.06 and 5.07 (each 1H, d, J ) 12.5 Hz, CH2Ar),
5.31 (1H, t, J ) 3.4 Hz, H-12), 7.33 (5H, m, H-Ar); 13C NMR (CDCl3,
75 MHz) δ 16.6, 20.0, 20.8, 23.2, 23.3, 23.4, 23.6, 24.0, 25.9, 27.6,
29.7, 30.7, 32.3, 32.4, 33.1, 34.0, 37.4, 37.6, 39.7, 41.6, 42.0, 45.9,
46.9, 47.1, 48.3, 51.1, 66.0, 69.1, 78.3, 122.6, 127.9, 128.0, 128.4, 136.5,
143.7, 177.4; ESIMS m/z 585.5 [M + Na]+, 561.5 [M - H]-; anal.
calcd for C37H54O4 ·0.1H2O, C 78.71, H 9.68; found, C 78.43, H 9.53.
Benzyl 2ꢀ,3r-Dihydroxyurs-12-en-28-oate (11b). According to the
procedure for preparation of 11a, treatment of 10b (0.5 g, 0.74 mmol)
with TBAF afforded 11b (0.39 g, 93%).
Compound 9b: white solid, mp 93-95 °C; [R]2D2 +106.7 (c 0.12,
1
CHCl3); IR (KBr) νmax 2932, 1725, 992 cm-1; H NMR (CDCl3, 500
MHz) δ 0.63, 0.81, 0.87, 0.96, 1.07, 1.10, 1.13 (each 3H, s), 0.005
and 0.13 (each 3H, s, Si(CH3)2), 0.90 (9H, s, CH3 of tert-butyl), 2.31
(1H, d, J ) 18.6 Hz, H-18), 4.67 (1H, dd, J ) 13.1, 18.7 Hz, H-2R),
5.00 and 5.08 (each 1H, d, J ) 20.7 Hz, CH2Ar), 5.27 (1H, t, J ) 6.0
Hz, H-12), 7.34 (5H, m, H-Ar); 13C NMR (CDCl3, 125 MHz) δ -5.3,
-4.5, 16.6, 17.0, 18.1, 18.5, 19.7, 20.1, 21.1, 23.5, 23.6, 24.4, 25.9,
28.0, 30.0, 30.8, 32.2, 36.7, 37.0, 39.0, 39.3, 39.7, 42.4, 46.3, 47.1,
48.4, 51.7, 52.4, 53.3, 66.1, 70.9, 125.8, 128.0, 128.3, 128.4, 136.5,
138.2, 177.1, 217.1; ESIMS m/z 675.5 [M + H]+, 697.4 [M + Na]+;
anal. calcd for C43H66O4Si·0.1H2O, C 76.30, H 9.86; found, C 75.94,
H 9.89.
Benzyl 2ꢀ-(tert-Butyldimethylsilyloxy)-3r-hydroxyolean-12-en-
28-oate (10a). Preparation of aluminum isopropoxide: finely cut
aluminum (2.5 g, 0.09 mol) and anhydrous AlCl3 (0.3 g, 2 mmol) was
added to anhydrous isopropyl alcohol (60 mL), and the resulting mixture
was refluxed until aluminum was deliquescent. Reflux was continued
for 2 h, and then the mixture was cooled to room temperature.
Compound 11b: white solid, mp 84-86 °C; [R]2D2 +80.2 (c 0.12,
1
CHCl3); IR (KBr) νmax 3401, 1723, 757 cm-1; H NMR (CDCl3, 300
MHz) δ 0.63, 0.91, 0.94, 1.01 (each 3H, s), 0.86 (3H, d, J ) 6.4 Hz),
1.08 (6H, s), 2.25 (1H, d, J ) 11.2 Hz, H-18), 3.62 (1H, d, J ) 10.4
Hz, H-3ꢀ), 3.73 (1H, m, H-2R), 5.00 and 5.08 (each 1H, d, J ) 12.4
Hz, CH2Ar), 5.26 (1H, t, J ) 3.5 Hz, H-12), 7.33 (5H, m, H-Ar); 13C
NMR (CDCl3, 75 MHz) δ 16.8, 17.0, 19.9, 21.07, 21.14, 23.27, 23.33,
23.6, 24.0, 24.3, 27.9, 30.8, 32.6, 36.7, 37.3, 37.6, 38.9, 39.2, 39.8,
42.4, 47.4, 48.3, 51.1, 53.1, 66.0, 69.1, 78.3, 125.8, 128.0, 128.2, 128.4,

1880 Journal of Natural Products, 2008, Vol. 71, No. 11
Cheng et al.
136.4, 138.2, 177.3; ESIMS m/z 561.3 [M - H]-; anal. calcd for
C37H54O4, C 78.96, H 9.67; found, C 78.66, H 9.95.
absorbance was measured at 655 nm. The IC50 values were estimated
by fitting the inhibition data to a dose-dependent curve using a logistic
derivative equation.
2ꢀ,3r-Dihydroxyolean-12-en-28-oic acid (Bredemolic acid, 3). A
mixture of 11a (0.17 g, 0.3 mmol) and 10% Pd/C (0.13 g) in THF (3
mL) was stirred at room temperature under H2 at atmospheric pressure
for 24 h. The reaction mixture was filtered through Celite, and the
insoluble substance was washed with THF (10 mL × 3). The filtrate
was concentrated in vacuo to give a white solid, which was purified
by flash chromatography (EtOAc/petroleum ether, 1:3) to afford 3 (0.12
g, 86%).
Acknowledgment. This program was financially supported by the
National Natural Science Foundation (grants 30672523 and 90713037),
research grants from the Ministry of Education (grants 706030 and
20050316008), and program for New Century Excellent Talents in
University (NCET-05-0495).
Compound 3: white solid, mp 292-294 °C (lit.8 288-292 °C);
[R]2D2 +93.5 (c 0.17, pyridine) (lit.8 [R]2D2 +100.5, c 1.0, pyridine); IR
1
Supporting Information Available: Copies of H NMR and 13C
NMR spectra of 3, 4, and synthetic intermediates 7a, 7b, 8a, 8b, 9a,
9b, 10a, 10b, 11a, and 11b. Copies of NOE spectra of 7a and 8a. This
material is available free of charge via the Internet at http://pubs.acs.org.
1
(KBr) νmax 3397, 1741, 1007 cm-1; H NMR (pyridine-d5, 300 MHz)
δ 0.93, 0.99, 1.06, 1.24, 1.28, 1.30, 1.31 (each 3H, s), 3.32 (1H, m,
H-18), 3.99 (1H, d, J ) 7.3 Hz, H-3ꢀ), 4.36 (1H, dd, J ) 6.3, 12.9 Hz,
H-2R), 5.51 (1H, s, H-12); 13C NMR (pyridine-d5, 75 MHz) δ 17.3,
19.8, 23.5, 23.8, 24.0, 26.2, 26.9, 28.3, 30.0, 31.0, 32.1, 33.1, 33.3,
34.4, 37.8, 37.9, 40.2, 42.2, 42.5, 46.0, 46.6, 46.8, 48.8, 51.0, 70.7,
78.4, 122.8, 144.9, 180.1; ESIMS m/z 495.4 [M + Na]+, 471.5 [M -
H]-; HRMS m/z 471.3473 (calcd for C30H47O4, 471.3474); anal. calcd
for C30H48O4 ·0.2H2O, C 75.65, H 10.24; found, C 75.21, H 10.64.
2ꢀ,3r-Dihydroxyolean-12-en-28-oic acid (4). According to the
procedure for preparation of 3, hydrogenolysis of 11b (0.25 g, 4.4
mmol) afforded 4 (0.19 g, 91%).
References and Notes
(1) Dzubak, P.; Hajduch, M.; Vydra, D.; Hustova, A.; Kvasnica, M.;
Biedermann, D.; Markova, L.; Urban, M.; Sarek, J. Nat. Prod. Rep.
2006, 23, 394–411.
(2) Wen, X. A.; Sun, H. B.; Liu, J.; Cheng, K. G.; Zhang, P.; Zhang,
L. Y.; Hao, J.; Zhang, L. Y.; Ni, P. Z.; Zographos, S. E.; Leonidas,
D. D.; Alexacou, K. M.; Gimisis, T.; Hayes, J. M.; Oikonomakos,
N. G. J. Med. Chem. 2008, 51, 3540–3554.
(3) Wen, X. A.; Xia, J.; Cheng, K. G.; Liu, J.; Zhang, L. Y.; Ni, P. Z.;
Sun, H. B. Bioorg. Med. Chem. Lett. 2007, 17, 5777–5782.
(4) Chen, J.; Liu, J.; Zhang, L. Y.; Wu, G. Z.; Hua, W. Y.; Wu, X. M.;
Sun, H. B. Bioorg. Med. Chem. Lett. 2006, 16, 2915–2919.
(5) Wen, X. A.; Zhang, P.; Liu, J.; Zhang, L. Y.; Wu, X. M.; Ni, P. Z.;
Sun, H. B. Bioorg. Med. Chem. Lett. 2006, 16, 722–726.
(6) Wen, X. A.; Sun, H. B.; Liu, J.; Wu, G. Z.; Zhang, L. Y.; Wu, X. M.;
Ni, P. Z. Bioorg. Med. Chem. Lett. 2005, 15, 4944–4948.
(7) Fukushima, M.; Matsuyama, F.; Ueda, N.; Egawa, K.; Takemoto, J.;
Kajimoto, Y.; Yonaha, N.; Miura, T.; Kaneko, T.; Nishi, Y.; Mitsui,
R.; Fujita, Y.; Yamada, Y.; Seino, Y. Diabetes Res. Clin. Pract. 2006,
73, 174–177.
(8) Tschesche, R.; Sengupta, A. K. Chem. Ber. 1960, 93, 1903–1913.
(9) Dou, H.; Zhang, R. P.; Lou, X.; Jia, J.; Zhou, C. X.; Zhao, Y. Biochem.
Syst. Ecol. 2005, 33, 639–642.
(10) Tsehesche, R.; Henckel, E.; Snatzke, G. Ann. 1964, 676, 175–187.
(11) Tschesche, R.; Henckel, E.; Snatzke, G. Tetrahedron Lett. 1963, 10,
613–617.
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7405–7408.
(13) Bore, L.; Honda, T.; Gribble, G. W. J. Org. Chem. 2000, 65, 6278–
6282.
Compound 4: white solid, mp 257-259 °C; [R]2D2 +60.9 (c 0.06,
pyridine); IR (KBr) νmax 3430, 1696, 664 cm-1; 1H NMR (pyridine-d5,
300 MHz) δ 0.96, 1.08, 1.19, 1.29 (each 3H, s), 1.01 (3H, d, J ) 6.4
Hz), 1.30 (6H, s), 2.66 (1H, d, J ) 11.2 Hz, H-18), 4.00 (1H, d, J )
7.5 Hz, H-3ꢀ), 4.36 (1H, dd, J ) 6.6, 13.7 Hz, H-2R), 5.50 (1H, t, J
) 3.3 Hz, H-12); 13C NMR (pyridine-d5, 75 MHz) δ 17.4, 17.5, 19.7,
19.9, 21.4, 23.5, 23.8, 23.9, 25.0, 27.0, 28.6, 31.2, 33.4, 37.5, 37.6,
37.9, 39.47, 39.54, 40.3, 42.8, 46.1, 48.2, 48.7, 50.9, 53.7, 70.7, 78.4,
125.9, 139.3, 179.9; ESIMS m/z 495.3 [M + Na]+, 471.3 [M - H]-;
HRMS m/z 471.3484 (calcd for C30H47O4, 471.3474); anal. calcd for
C30H48O4 ·0.5CH3OH, C 74.96, H 10.31; found, C 74.66, H 10.80.
Enzyme Assay. Inhibitory activity of the compounds against rabbit
muscle glycogen phosphorylase a (GPa) was monitored using a
microplate reader (BIO-RAD) based on the published method.14 In brief,
GPa activity was measured in the direction of glycogen synthesis by
the release of phosphate from glucose-1-phosphate. Each test compound
was dissolved in DMSO and diluted at different concentrations for IC50
determination. The enzyme was added into 100 µL of buffer containing
50 mM Hepes (pH 7.2), 100 mM KCl, 2.5 mM MgCl2, 0.5 mM glucose-
1-phosphate, 1 mg/mL glycogen, and the test compound in 96-well
microplates (Costar). After the addition of 150 µL of 1 M HCl
containing 10 mg/mL ammonium molybdate and 0.38 mg/mL malachite
green, reactions were run at 22 °C for 25 min, and then the phosphate
(14) Martin, W. H.; Hoover, D. J.; Armento, S. J.; Stock, I. A.; McPherson,
R. K.; Danley, D. E.; Stevenson, R. W.; Barrett, E. J.; Treadway, J.
L Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1776–1781.
NP8003886