Naturally occurring pentacyclic triterpenes as inhibitors of glycogen phosphorylase: Synthesis, structure-activity relationships, and X-ray crystallographic studies

Article abstract of DOI:10.1021/jm8000949

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity (7, 18-20) or no activity (21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

Full text of DOI:10.1021/jm8000949

3540
J. Med. Chem. 2008, 51, 3540–3554
Naturally Occurring Pentacyclic Triterpenes as Inhibitors of Glycogen Phosphorylase:
Synthesis, Structure-Activity Relationships, and X-ray Crystallographic Studies†
Xiaoan Wen,‡ Hongbin Sun,*,‡ Jun Liu,§ Keguang Cheng,‡ Pu Zhang,‡ Liying Zhang,‡ Jia Hao,‡ Luyong Zhang,§ Peizhou Ni,‡
Spyros E. Zographos,| Demetres D. Leonidas,| Kyra-Melinda Alexacou,| Thanasis Gimisis, Joseph M. Hayes,| and
Nikos G. Oikonomakos*,|
Center for Drug DiscoVery, College of Pharmacy, China Pharmaceutical UniVersity, 24 Tongjia Xiang, Nanjing 210009, China,
Jiangsu Center for Drug Screening, China Pharmaceutical UniVersity, 1 Shennonglu, Nanjing 210038, China, Institute of Organic and
Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vassileos Constantinou AVenue, Athens 11635, Greece, and
Organic Chemistry Laboratory, Department of Chemistry, UniVersity of Athens, Panepistimiopolis, Athens 15771, Greece
ReceiVed January 30, 2008
Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were
biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies,
the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity (7, 18-20)
or no activity (21, 22). These saponins, however, might find their value as potential natural prodrugs which
are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition,
we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The
X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator
AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that
exert hypoglycemic effects, at least in part, through GP inhibition.
Introduction
classes of GP inhibitors have been described, and at least six
potential regulatory binding sites have been identified in GP.7
Type 2 diabetes is associated with disorders in glucose
metabolism by the liver and periphery, and an ideal antidiabetic
agent should be capable of lowering blood glucose in both fed
and fasted states. Control of the hepatic and peripheral glycogen
metabolism is one of the key events through which insulin
maintains blood glucose homeostasis.1 Aiston et al. demon-
strated that inactivation of glycogen phosphorylase (GP)a but
not inhibition of glycogen kinase synthase-3 (GSK-3) could
mimic insulin stimulation of hepatic glycogen synthesis and that
a signaling pathway involving dephosphorylation of GPa leading
to both activation and translocation of glycogen synthase (GS)
was a critical component of the mechanism by which insulin
stimulated hepatic glycogen synthesis.2 In this regard, inactiva-
tion of GP would not only reduce glycogenolysis but would
also stimulate glycogen synthesis. GP inhibition has been
regarded as a therapeutic strategy for blood glucose control in
diabetes,3 and various studies have shown the efficacy of GP
inhibitors at lowering blood glucose in animal models of
diabetes4,5 and in clinical trials.6 To date, several structural
Pentacyclic triterpenes are widely distributed throughout the
plant kingdom, and a variety of biological properties have been
ascribed to this class of compounds.8 Pentacyclic triterpenes
can be classified into three major types based on their structural
skeleton (Figure 1): (a) oleanane type of triterpenes (e.g., 1-9,
18); (b) ursane type of triterpenes (e.g., 10-17, 19-22); (c)
lupane type of triterpenes (e.g., 23-25). The most well-known
member of this family of compounds is probably oleanolic acid
(1),9 which has been clinically used as a hepatoprotective/
antihepatitis drug in China for more than 20 years. Glycyrrhizic
acid (7) has also been marketed as an antihepatitis drug in China
and Japan. Moreover, several other pentacyclic triterpenes have
entered clinical trials as anti-HIV or antitumor agents.10–12 In a
recent human test, the Japanese researchers proved for the first
time that corosolic acid (12) exhibited a glucose-lowering effect
on postchallenge plasma glucose levels in humans.13 In previous
communications,14–17 we first reported that pentacyclic triter-
penes (e.g., 1, 3, and 12) represented a new class of inhibitors
of glycogen phosphorylase. Herein, we disclose the detailed
procedures for the semisynthesis of some natural pentacyclic
triterpenes, including maslinic acid (3), 3-epimaslinic acid (4),
augustic acid (5), corosolic acid (12), pygenic acid A (13), and
2-epicorosolic acid (14) (Figure 1). Moreover, SAR analysis
of a series of naturally occurring pentacyclic triterpenes as GP
inhibitors is discussed. The results of our X-ray crystallographic
studies are also presented to disclose the molecular basis of the
pentacyclic triterpenes binding to GP.
†
The coordinates of the new structures have been deposited with the
RCSB Protein Data Bank (http://www.rcsb.org/pdb) with the codes 2QN1
(GPb-asiatic acid complex) and 2QN2 (GPb-maslinic acid complex).
* To whom correspondence should be addressed. For H.S.: phone, +86-
25-85327950; fax, +86-25-83271335; e-mail, hbsun2000@yahoo.com. For
N.G.O.: phone, +30-210-7273-884 (761); fax, +30-210-7273831; e-mail,
ngo@eie.gr.
‡
Center for Drug Discovery, China Pharmaceutical University.
Jiangsu Center for Drug Screening, China Pharmaceutical University.
National Hellenic Research Foundation.
§
|
University of Athens.
a Abbreviations: GP, glycogen phosphorylase; 1,4-R-D-glucan, ortho-
phosphate R-glucosyltransferase (EC 2.4.1.1); GPb, rabbit muscle glycogen
phosphorylase b; GPa, rabbit muscle glycogen phosphorylase a; hlGPa,
human liver glycogen phosphorylase a; PLP, pyridoxal 5′-phosphate;
glucose, R-D-glucose; Glc-1-P, R-D-glucose 1-phosphate; Glc-6-P, D-glucose
6-phosphate; W1807, (-)-(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-
1-ethyl-2-methylpyridine-3,5,6-tricarboxylate; FR258900, (2R,3S)-2,3-
bis((E)-3-(4-hydroxyphenyl)acryloyloxy)pentanedioic acid; rms deviation,
root-mean-squared deviation.
Results and Discussion
Chemistry. 11-Deoxyglycyrrhetinic acid (9) was prepared
by deoxygenation of glycyrrhetinic acid (8) following the
literature procedure.18 Betulin (23),19 betulinic acid (24),20 and
23-hydroxybetulinic acid (25)21 were obtained following the
literature procedures. Oleanonic acid (6) and ursonic acid (17)
10.1021/jm8000949 CCC: $40.75
2008 American Chemical Society
Published on Web 06/03/2008

Triterpenes as Inhibitors of Glycogen Phosphorylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3541
Figure 1. Chemical structures of some naturally occurring pentacyclic triterpenes 1-25.
were easily prepared by oxidation of oleanolic acid (1) and
ursolic acid (10) with PCC, respectively.
Stereoselective syntheses of augustic acid (5) and 2-epicoro-
solic acid (14) are summarized in Scheme 3. Treatment of ketone
28 with a large excess of t-BuOK under air at room temperature
gave diketone 38, which existed in the form of R, ꢀ-unsaturated
ketone. Alternatively, treatment of R-hydroxyl ketone 34 with
KOH in MeOH-DMF at room temperature also afforded 38
in good yield. Reduction of 38 with NaBH4 gave 2ꢀ,3ꢀ-diol
40 in 61% yield. Interestingly, when 34 was treated with
NaHCO3 instead of KOH, it was found that a rearrangement
product was formed to give 3ꢀ-hydroxyketone 42. Reduction
of 42 with NaBH4 gave 2ꢀ,3ꢀ-diol 40, which was identical to
the product obtained by NaBH4 reduction of 38. Hydrogenolysis
of 40 over palladium/carbon in THF furnished augustic acid
(5). In the same fashion, 2-epicorosolic acid (14) was synthe-
sized via NaBH4 reduction of 39 (prepared by treatment of
ketone 29 with a large excess of t-BuOK as described for
preparation of 38), followed by hydrogenolysis of the resulting
2ꢀ,3ꢀ-diol 41.
In an initial effort14 for synthesis of 3 and 12, a modified
procedure based on Caglioti’s methodology22 was employed
using hydroboration-oxidation of enol acetates 30 and 31 as
the key step (Scheme 1). The disadvantages of the above
hydroboration-oxidation methodology were that the overall
yields were poor and that the cost (mainly due to the cost of
borane reagents) was relatively high in terms of large-scale
production, and thus, we explored another approach to 3 and
12 based on a ketone-hydroxylation strategy (Scheme 2).16,17,23
As depicted in Scheme 2, stereoselective hydroxylation of
28 with mCPBA catalyzed by H2SO4 in MeOH-CH2Cl2 at 0
°C gave 2R-hydroxyl ketone 34 (80%). Reduction of 34 with
NaBH4 in THF at 0 °C gave 2R,3ꢀ-diol 32 (77%) as the major
product, together with 2R,3R-diol 36 (18%) as a minor product.
On the other hand, Meerwein-Pondorf reduction24 of 34 gave
2R,3R-diol 36 as the major product (50%) together with 32 as
the minor product. Hydrogenolysis of 32 or 36 over palladium/
carbon in THF furnished maslinic acid (3) or 3-epimaslinic acid
(4) in quantitative yields, respectively. In the same fashion,
corosolic acid (12) and pygenic acid A (13) were synthesized
using R-hydroxyl ketone 35 as the key intermediate.
The synthetic route to 3-epioleanolic acid (2) and 3-epiursolic
acid (11) is outlined in Scheme 4. Meerwein-Pondorf reduction
of benzyloleanonic acid (28) afforded benzyl-3-epioleanolic acid
(43) as the major product (57%). Hydrogenolysis of 43 over
palladium/carbon in THF furnished 3-epioleanolic acid (2)

3542 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Wen et al.
Scheme 1. Synthesis of Maslinic Acid (3) and Corosolic Acid (12) Based on a Hydroboration-Oxidation Strategya
a Reagents and conditions: (a) K2CO3, BnCl, DMF, 55 °C; (b) PCC, CH2Cl2; (c) AcOCHdCH2, concentrated H2SO4 (cat.), 100 °C; (d) (a) BH3-THF;
(b) H2O2, NaOH; (e) H2, Pd/C, THF, room temp.
Scheme 2. Synthesis of Maslinic Acid (3), 3-Epimaslinic Acid (4), Corosolic Acid (12), and Pygenic Acid A (13) Based on a
Ketone-Hydroxylation Strategya
a Reagents and conditions: (a) mCPBA, concentrated H2SO4 (cat.), MeOH-CH2Cl2, 0 °C; (b) NaBH4, THF, 0 °C; (c) Al(O-i-Pr)3, i-PrOH, reflux; (d) H2,
Pd/C, THF, room temp.
(90%). In the same manner, 3-epiursolic acid (11) was prepared
via Meerwein-Pondorf reduction of benzylursonic acid (29),
followed by hydrogenolysis of the resulting benzyl ester 44.
For the preparation of triterpene acid ꢀ-D-glucopyranosyl ester
compounds, direct esterification of 1 with 2,3,4,6-tetra-O-acetyl-
R-D-glucosyl bromide (45)25 afforded oleanolic acid 2,3,4,6-

Triterpenes as Inhibitors of Glycogen Phosphorylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3543
Scheme 3. Synthesis of Augustic Acid (5) and 2-Epicorosolic Acid (14)a
a Reagents and conditions: (a) t-BuOK, t-BuOH, air, room temp; (b) NaBH4, THF/MeOH, 0 °C; (c) H2, Pd/C, THF, room temp; (d) KOH, MeOH-DMF,
room temp; (e) NaHCO3, THF, room temp.
Scheme 4. Synthesis of 3-Epioleanolic Acid (2) and 3-Epiursolic
Acid (11)
inhibitory activity against GPa. It seems that the diversity of
structural skeleton of pentacyclic triterpenes does not have a
significant impact on GP inhibition, since all the three types of
tested triterpenes, including oleanane triterpenes (e.g., 1-9, 18),
ursane triterpenes (e.g., 10-15, 17, 19, 20), and lupane
triterpenes (e.g., 23-25), exhibited inhibitory activity against
GPa.
The amount and positioning of hydroxyl groups of triterpenes
seemed to have some impact on potency. Both 2R-hydroxy-
oleanolic acid (3, IC50 ) 28 µM) and 2ꢀ-hydroxyoleanolic aicd
(5, IC50 ) 34 µM) were less active than the parent compound
1 (IC50 ) 14 µM), suggesting that the introduction of hydroxyl
group at C-2 resulted in a loss of potency. The same tendency
was also observed with ursolic acid (10, IC50 ) 9 µM), since
both 2R-hydroxyursolic acid (12, IC50 ) 20 µM) and 2ꢀ-
hydroxyursolic aicd (14, IC50 ) 116 µM) were less potent than
10. On the other hand, in two cases examined (12 vs 15; 24 vs
25), incorporation of a hydroxyl group at C-23 resulted in slight
increases in potency. Surprisingly, as in the case of madecassic
acid (16), incorporation of hydroxyl group at C-6 resulted in a
complete loss of potency.
tetra-O-acetyl-ꢀ-D-glucopyranosyl ester (46), which was hy-
drolyzed with NaOH in MeOH/THF to give oleanolic acid ꢀ-D-
glucopyranosyl ester 18 (85% for two steps) (Scheme 5). In
the same fashion, ursolic acid ꢀ-D-glucopyranosyl ester 19 and
corosolic acid ꢀ-D-glucopyranosyl ester 20 were prepared.
Enzyme Assay and SAR Analysis. . Twenty-five naturally
occurring pentacyclic triterpenes were biologically evaluated for
their inhibitory activity against rabbit muscle GPa (RMGPa).
The activity of RMGPa was measured through detection of the
release of phosphate from glucose 1-phosphate in the direction
of glycogen synthesis.4 The bioassay results are summarized
in Table 1. Most of the tested triterpenes exhibited moderate
The effects of configuration of hydroxyl groups on GP
inhibition were further examined. In contrast to 3 or 12 having
2R,3ꢀ configuration, the potency decreased when both 2-hy-
droxyl and 3-hydroxyl groups were in the same side of A-ring
(e.g., 3 vs 4 and 5; 12 vs 13 and 14). Conversion of 3ꢀ hydroxyl
group of oleanolic acid (1) or ursolic acid (10) to 3R hydroxyl
group resulted in a slight loss of potency (1 vs 2; 10 vs 11).
Conversion of 3ꢀ hydroxyl group of 1 or 10 to 3-carbonyl group
did not result in a significant loss of potency (1 vs 6; 10 vs 17).

3544 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Wen et al.
Scheme 5. Synthesis of Triterpene Acid ꢀ-D-Glucopyranosyl Esters 18-20
for the liver isoform than the muscle isoform,27 we did not
observe a significant selectivity between liver GP inhibition and
muscle GP inhibition by the triterpenes.14 In our view, however,
GP subtype selectivity is not an essential issue here because of
the fact that pentacyclic triterpenes are mild GP inhibitors, and
thus, mechanism-related toxicity is avoidable.28 On the other
hand, it has been suggested that since the liver is the major site
of exposure to drugs administered orally, it should be possible
to achieve an appropriate drug dosage that targets predominantly
hepatic GP.29 In fact, not only hepatic glycogen metabolism
(mainly modulated by liver GP) contributes in glucose metabo-
lism but also peripheral glycogen metabolism (mainly modulated
by muscle GP and brain GP) plays an important role in
maintaining blood glucose homeostasis, especially under fed-
state conditions.
Table 1. IC50 Values (µM) for the Inhibition of Rabbit Muscle GPa
a
a
compd
GPa IC50
compd
GPa IC50
1
2
3
4
5
6
14
21
28
144
34
18
14
15
16
17
18
19
116
17
nab
57
293
97
7
8
9
10
11
12
13
822
66
82
9
19
20
21
22
23
24
25
caffeine
106
nab
nab
17
43
16
20
213
114
a Values are the mean of three experiments. b na ) no activity.
X-ray Crystallography. In order to elucidate the structural
basis of GP inhibition by pentacyclic triterpenes, we have
determined the crystal structures of GPb in complex with both
asiatic acid (15) and maslinic acid (3) (Figure 2). A summary
of crystallographic data collection and refinement statistics for
the GPb-asiatic acid and GPb-maslinic acid complex structures
is given in Supporting Information (SI, Tables S2 and S3). The
2Fo - Fc Fourier electron density maps indicated that both 15
and 3 were bound at the allosteric site (Figure 3). Portions of
the 2Fo - Fc electron density maps for asiatic acid and maslinic
acid molecules are shown in Figure 4.
Binding of Asiatic Acid (15). Asiatic acid and maslinic acid
both contain five fused six-membered rings (A-E) in a steroid-
like conformation, with ring E being cis fused to the 17-18
bond of ring D and containing an axial carboxylate substituent
in position 17 (Figure 2). Asiatic acid (15) binds at the interface
of the dimer forming the allosteric (AMP) site some 30 Å from
The presence of a sugar moiety in triterpene saponins resulted
in a markedly decreased activity (7, 18-20) or no activity (21,
22). Glycyrrhizic acid (7, IC50 ) 822 µM) was 12-fold less
potent than its aglycone 8 (IC50 ) 66 µM). 3ꢀ-Hydroxyolean-
12-en-28-oic acid ꢀ-D-glucopyranosyl ester (18, IC50 ) 293 µM)
was 20-fold less potent than its aglycone 1 (IC50 ) 14 µM).
The potency difference between 15 (IC50 ) 17 µM) and 21 (a
saponin derived from 15) was quite intriguing because of the
fact that while 15 was relatively quite active, 21 was inactive
at 2000 µM. Despite of the low potency or no activity of the
triterpene saponins, these saponins might find their value as
potential natural prodrugs which are much more water-soluble
than the corresponding aglycones. For example, asiaticoside (21)
was reported to undergo degradation in human bodies to release
the biologically active asiatic acid (15).26
Although some allosteric inhibitors such as phthalic acids and
dihydropyridine diacid were reported to have a higher potency

Triterpenes as Inhibitors of Glycogen Phosphorylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3545
Figure 2. Chemical structures of compounds 15 and 3 showing the numbering system used.
hydroxyl O23 make hydrogen bonding interactions with Gln72
NE2, Asp42′ OD1, and Asp42′ OD2. Carboxylate oxygens O28
and O29 exploit the allosteric effector phosphate-recognition
subsite by forming hydrogen bonding interactions with Arg310
NE and NH1. The phosphate-recognition subsite recognizes the
phosphate of a variety of phosphorylated compounds, such as
AMP, ATP, Glc-6-P, and the carboxylate of the nonphysiologi-
cal inhibitors W1807 and FR258900.32–34 The inhibitor becomes
buried on forming the complex with GPb. On the protein
surface, a total of 367 Å2 (309 Å2 in one subunit and 58 Å2 in
the symmetry related subunit) solvent accessible surface area
becomes inaccessible on binding of the ligand. The total buried
surface area (protein plus ligand) for the GPb-ligand complex
is 854 Å2.
Comparison with the Native T-State Structure. Superposi-
tion of the activation locus, residues 24-78, 94-111, and
118-125 from both subunits35 of the structure of the native
T-state GPb with the activation locus of structure of the GPb-15
complex gave an rms deviation of 0.34 Å for CR atoms,
indicating that the two structures have very similar overall
conformations within the limits of the 2.4 Å resolution data.
The major conformational changes on binding of 15 to rmGPb
occur in the vicinity of the allosteric site. Shifts for CR atoms
are observed for residues 41′-48′ (between 0.4 and 0.7 Å) and
residues 71-78 (between 0.4 and 0.7 Å) that affect the
subunit-subunit interface in the region between the cap′ and
the R2 helix. The major shifts for the side chain atoms are
observed for residue 72 by about 0.9-1.9 Å and for residue
196 by about 0.6-1.7 Å to optimize their hydrogen bonding
and van der Waals interactions with ligand. Similar shifts were
observed previously on binding of Glc-6-P,36 W1807,32,33,37
phenoxy-phthalates,38 acylureas,39,40 and FR25890034 to the
allosteric site. A comparison of the two structures in the vicinity
of the allosteric site is shown in Figure 6a.
Comparison with R-State GPa. Comparison of the GPb-15
complex with the R-state GPa30 suggests that the inhibitor is
likely to have lower affinity for the R-state conformation (Figure
6b). Superposition of the activation loci of the structure of the
R-state GPa (subunit A) with the activation locus of structure
of the GPb-15 complex gave an rms deviation of 1.18 Å for
CR atoms. If 15 were to be superimposed into the R-state GPa,
that would result in clashes with Asp42′ atoms OD1, OD2, CG,
and CB, Asn44′ atom CG, and Asn72 atoms CA, CB, and NE2.
Hence, it would be anticipated that the affinity of 15 for the R
state would be less than that of the T state, but there is no direct
experimental evidence.
Figure 3. Schematic diagram of the T-state GPb dimeric molecule,
for residues 10-837, viewed down the molecular dyad, showing the
positions for the catalytic and the allosteric binding sites. The catalytic
site (marked by glucose, shown in white), which includes the essential
cofactor PLP (not shown), is buried at the center of the subunit
accessible to the bulk solvent through a 15 Å long channel. The
allosteric site, which binds the activator AMP and the allosteric inhibitor
asiatic acid (shown in red), is situated at the subunit-subunit interface
some 30 Å from the catalytic site.
the catalytic site.30,31 On binding at the allosteric site, 15 makes
a total of six hydrogen bonds to the protein, involving all
potential hydrogen bonding groups except the hydroxyl group
O2, and 48 van der Waals interactions (8 polar/polar, 26 polar/
nonpolar, and 14 nonpolar/nonpolar interactions) (SI, Tables
S4 and S6). There are 13 contacts to residues Asp42′, Asn44′,
and Val45′ from the symmetry related subunit. The hydrogen
bonding interactions formed between the ligand and the protein
are illustrated in Figure 5a. Specifically, hydroxyl O3 and
Comparison with AMP. A structural comparison of the
R-state hlGPa-AMP complex41 with T-state GPb-15 com-

3546 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Wen et al.
plex is shown in Figure 7. The adenine and ribose partially
overlap with A and B rings of the steroid-like structure, and
the phosphate group partially overlaps with the D ring of
the cis-fused decalin moiety, while the carboxylate and the
phosphate groups are positioned very close to each other in
the phosphate-recognition subsite (P/C28 and O1P/O29
separations are respectively 2.2 and 1.8 Å).
Comparison with Other Types of Allosteric Site Inhibi-
tors. Compounds W1807, Novo4j,38 and AVE#2139 (SI, Figure
S1) are known AMP-site allosteric inhibitors for GP. Compari-
sonsofGPb-15withGPb-W1807(SI,FigureS2),GPb-Novo4j
(SI, Figure S3), and hlGPa-AVE#21 (SI, Figure S4) were
performed. The detailed comparisons are presented in the
Supporting Information. The results show that the allosteric site
displays broad specificity. For example, AVE#21 does not
exploit the allosteric phosphate-recognition site (composed of
Arg309, Arg310, and the more distant Arg242), which recog-
nizes phosphorylated compounds (e.g., AMP, ATP and Glc-6-
P) and the nonphysiological inhibitors such as W1807 and
Novo4j. Upon binding of the inhibitors W1807, Novo4j and
AVE#21, there are conformational changes in the vicinity of
the allosteric site that affect the plasticity of the site and appear
important in stabilizing an inactive T-state conformation. These
results show the ability of GP to distinguish among various
specific potent inhibitors, and this appears to originate in protein
conformational flexibility in the region of the allosteric site.42
Understanding such protein flexibility will be important in the
design of more specific inhibitors. The use of rabbit GP is a
good surrogate for human GP given the generally high
conservation of amino acid sequence for glycolytic enzymes
between different species. With the exception of Cys318 (which
is serine in the human liver isoenzyme), all AMP (or Glc-6-P)
binding residues are identical in the rabbit muscle and human
liver GPs.43
Binding of Maslinic Acid (3). The position and conforma-
tion of 3, bound at the allosteric site, are also similar to those
observed for 15 (Figures 5 and 8). The electron density
observed for maslinic acid exhibits many common features
with the corresponding density of asiatic acid. Interestingly,
a similar lack of density for C21, C22, and the methyl C24
is also observed. As expected, the main difference between
the two structures is the lack of density for O23 that is absent
in maslinic acid, although no density is observed for the
existing C23 either. The pattern of polar and nonpolar
interactions between the inhibitor and the enzyme is main-
tained when comparing the complex structures of derivatives
15 and 3 except that there is no a hydrogen bonding
interaction between O23 and Asp42′ OD1 in the 3 complex
(SI, Tables S5 and S7).
Figure 4. Diagrams of the 2Fo - Fc electron density map, contoured
at 1σ, for the bound compounds asiatic acid (a) and maslinic acid (b)
at the allosteric site of GPb. Electron density maps were calculated
using the standard protocol as implements in REFMAC65 before
incorporating ligand molecules.
Conclusions
In this study, 25 naturally occurring pentacyclic tritepenes, 15
of which were efficiently synthesized starting from commercial
products, were biologically evaluated as inhibitors of glycogen
phosphorylase. The bioassay results show that most of the tested
triterpenes exhibit moderate inhibitory activity against GPa. X-ray
analyses of the GPb-asiatic acid and GPb-maslinic acid com-
plexes show that compounds 15 and 3, on binding to GPb, promote
conformational changes that stabilize the inactive T-state quaternary
conformation of the enzyme, form direct hydrogen bonds with
Gln72, Arg310, Asp42′, and make nonpolar interactions with the
side chains of Tyr75, Phe196 and Val45′ (from the symmetry
related subunit). These interactions appear important in stabilizing
the inactive T state and may explain why 15 and 3 are micromolar
inhibitors of the enzyme.
Taken together, pentacyclic triterpenes may exert hypogly-
cemic effects,9,13–15,44–46 at least in part, through GP inhibition.
Other mechanisms47–49 may also account for the hypoglycemic
activity of pentacyclic triterpenes. Further studies are needed
to address the detailed molecular mechanisms and to biologically
evaluate natural and synthetic pentacyclic triterpenes as promis-
ing antidiabetic agents with preventive effects against diabetic
complications such as ischemic heart and brain diseases.
Experimental Section
Chemistry. Materials and General Methods. All commercially
available solvents and reagents were used without further purifica-

Triterpenes as Inhibitors of Glycogen Phosphorylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3547
Figure 5. (a) Interactions between asiatic acid and GPb at the allosteric site. (b) Interactions between maslinic acid and GPb at the allosteric site.
Residues from subunit 1 are shown in green, and residues from subunit 2 are shown in orange.
tion. The purity of commercial triterpene products (1, 7, 8, 10, 15,
16, 21, 22) used in this study was at least 95%. Melting points of
compounds were measured on a RY-1 melting point apparatus.
Column chromatography was carried out on silica gel (200-300
mesh, Qindao Ocean Chemical Company, China). IR spectra were
recorded on Shimadzu FTIR-8400S spectrometer. 1H and 13C NMR
spectra were measured on a Bruker AV-300 or AV-500 spectrom-
eter. Chemical shifts are reported as δ values from an internal
tetramethylsilane standard. Mass spectral data were obtained on
Agilent 1100 LC/DAD/MSD or Q-Tof Micro MS/MS spectrometer.
Elemental analysis was carried out on Vario EL III instrument
(Elementar, Germany).
to give 17 as a white solid (66%). Mp: 265-267 °C. IR (KBr,
cm-1): 2975, 2940, 2871, 1691, 1459, 1385, 1316, 1276, 1258,
1235, 1111, 747. 1H NMR (CDCl3, 500 MHz): δ 0.85, 1.04, 1.06,
1.095, 1.104 (each 3 H, s), 0.88 (3 H, d, J ) 6.4 Hz), 0.95 (3 H,
d, J ) 6.2 Hz), 2.23 (1 H, d, J ) 11.2 Hz), 2.41 and 2.54 (each 1
H, m), 5.28 (1 H, t, J ) 3.5 Hz). ESI-MS m/z: 477.3 [M + Na]+.
Benzyl-3ꢀ-hydroxyolean-12-en-28-oic Acid (26). A mixture of
oleanolic acid (100 g, 0.22 mol) and K2CO3 (61 g, 0.44 mol) in
DMF (800 mL) was heated to 55 °C, and then benzyl chloride (33
mL, 0.29 mol) was added dropwise over a period of 20 min. After
being stirred at 55 °C for 4 h, the reaction mixture was cooled to
room temperature, filtered, and washed with DMF (50 mL × 3).
The filtrate was poured into ice-water to give a white precipitate.
The precipitate was filtered, washed with water, and dried to give
7 (115 g, 96%), which was almost a pure product, and was used
for the next reaction without further purification. Pure product was
obtained by recrystallization of the crude product from EtOH. Mp:
3-Oxoolean-12-en-28-oic Acid (Oleanonic Acid, 6). To a
mixture of oleanolic acid (0.46 g, 1.01 mmol) and pyridine (10
mL) was added PCC (0.52 g, 2.42 mmol) at room temperature.
After the mixture was stirred at room temperature for 48 h, EtOAc
was added to the reaction mixture. The resulting mixture was
washed with 1 N HCl and brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was purified by column
chromatography [petroleum ether-EtOAc (6:1)] to give 6 as a white
solid (0.35 g, 76%). Mp: 199-201 °C. IR (KBr, cm-1): 3449, 2945,
1
190-192 °C. IR (KBr, cm-1): 3415, 2941, 1695, 1253, 1170. H
NMR (CDCl3, 500 MHz): δ 0.62, 0.78, 0.88, 0.90, 0.92, 0.98, 1.13
(each 3 H, s), 2.91 (1 H, dd, J ) 4.4, 13.9 Hz), 3.20 (1 H, dd, J )
4.5, 11.2 Hz), 5.04 and 5.09 (each 1 H, d, J ) 12.5 Hz), 5.28 (1 H,
t, J ) 3.6 Hz), 7.28-7.36 (5 H, m). ESI-MS m/z: 569 [M + Na]+.
Benzyl-3ꢀ-hydroxyurs-12-en-28-oic Acid (27). Following the
procedure for preparation of 26, benzylation of ursolic acid gave
27 as a white solid. Mp: 180-182 °C. IR (KBr, cm-1): 2924, 2870,
1721, 1455, 1377, 1267, 1224, 1138, 1110, 1029, 996, 749, 696.
1H NMR (CDCl3, 300 MHz): δ 0.67, 0.81, 0.95, 1.01, 1.10 (each
1
2866, 1701, 1460, 1385, 1363, 1267, 1161, 1009, 642. H NMR
(CDCl3, 300 MHz): δ 0.81, 0.91, 0.93, 1.03, 1.05, 1.09, 1.15 (each
3 H, s), 2.39 and 2.53 (each 1 H, m), 2.82 (1 H, dd, J ) 4.1, 13.8
Hz), 5.30 (1 H, brs). ESI-MS m/z: 453.3 [M - H].
3-Oxours-12-en-28-oic Acid (Ursonic Aicd, 17). Following the
procedure for preparation of 6, ursolic acid was oxidized by PCC

3548 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Wen et al.
Figure 6. (a) Comparison between GPb-asiatic acid complex (shown in green-subunit 1 and orange-subunit 2) and T-state GPb (shown in mauve-
subunit 1 and cyan-subunit 2). (b) GPa complex in the R state (shown in mauve-subunit 1 and orange-subunit 2) viewed in an orientation similar
to that of the GPb-asiatic acid complex. The position of asiatic acid is superimposed.
Figure 7. Comparison between GPb-asiatic acid complex (shown in green-subunit 1 and orange-subunit 2) and R-state hlGPa-AMP (shown in
mauve-subunit 1 and cyan-subunit 2).
3 H, s), 0.89 (3 H, d, J ) 6.4 Hz), 0.97 (3 H, d, J ) 5.4 Hz), 2.31
(1 H, d, J ) 11.3 Hz), 3.23 (1 H, dd, J ) 4.6, 10.4 Hz), 5.03 and
5.11 (each 1 H, d, J ) 12.5 Hz), 5.26 (1 H, t, J ) 3.5 Hz), 7.35 (5
H, m). ESI-MS m/z: 547.1 [M + H]+.
Benzyl-3-oxoolean-12-en-28-oic Acid (28). To a solution of 26
(111 g, 0.2 mol) in CH2Cl2 (588 mL) was added PCC (60.4 g,
0.28 mol) at 0 °C. After being stirred at 0 °C for 4 h, the reaction
mixture was warmed to room temperature and stirred overnight.

Triterpenes as Inhibitors of Glycogen Phosphorylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3549
33.8, 33.0, 32.4, 32.3, 30.6, 27.6, 25.8, 24.7, 23.6, 23.5, 23.0, 21.6,
19.1, 17.0, 15.9. EI-MS: 561 [M + H]+. HRMS calcd for C37H51O4
[M - H]: 559.3787. Found: 559.3815. Anal. Calcd for C37H52O4:
C, 79.24, H, 9.35. Found: C, 78.98, H, 9.31.
Benzyl-2r-hydroxy-3-oxours-12-en-28-oic Acid (35). Follow-
ing the procedure for preparation of 34, hydroxylation of 29 with
m-CPBA gave 35 as a white solid (70%). Mp: 95-97 °C. IR (KBr,
cm-1): 3479, 2945, 2926, 2872, 1718, 1456, 1387, 1263, 1225,
1
1144, 1103, 993, 744, 596. H NMR (CDCl3, 300 MHz): δ 0.68,
1.04, 1.11, 1.15, 1.24 (each 3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 0.94
(3 H, d, J ) 5.9 Hz), 2.30 (1 H, d, J ) 11.1 Hz), 2.43 (1 H, dd, J
) 6.6, 12.5 Hz), 4.53 (1 H, dd, J ) 6.6, 12.6 Hz), 5.01 and 5.09
(each 1 H, d, J ) 12.5 Hz), 5.24 (1 H, t, J ) 3.6 Hz), 7.34 (5 H,
m). 13C NMR (CDCl3, 75 MHz): δ 16.1, 17.0, 17.2, 19.2, 21.1,
21.6, 23.45, 23.53, 24.2, 24.8, 27.9, 30.6, 32.7, 36.6, 37.6, 38.8,
39.1, 39.7, 42.1, 47.3, 47.7, 48.1, 49.7, 52.8, 57.8, 66.0, 69.2, 125.2,
128.0, 128.2, 128.4, 136.4, 138.4, 177.2, 216.6. ESI-MS m/z: 561.4
[M + H]+, 583.4 [M + Na]+, 599.4 [M + K]+. HRMS Calcd for
C37H51O4 [M - H]: 559.3787. Found: 559.3811. Anal. Calcd for
C37H52O4: C, 79.24, H, 9.35. Found: C, 79.10, H, 9.30.
Benzyl-2r,3ꢀ-dihydroxyolean-12-en-28-oic Acid (32). To a
solution of 34 (8.9 g, 0.016 mol) in THF (100 mL) and ethanol
(20 mL) was added NaBH4 (0.7 g, 0.018 mol) at 0 °C. After the
mixture was stirred at 0 °C for 5 h, 1 N HCl (200 mL) was added
dropwise, and the mixture was extracted with EtOAc (150 mL ×
1, 80 mL × 3). The organic layer was washed with saturated
NaHCO3 (80 mL × 3) and brine (80 mL × 3), dried over Na2SO4,
filtered, and concentrated to afford a white powder, which was
purified by flash chromatography [petroleum ether-EtOAc (6:1)]
to give 32 as a white solid (6.94 g, 77%), together with 36 as a
minor product (1.62 g, 18%). Data for 32: mp 155-157 °C. IR
(KBr, cm-1): 3371, 2945, 1725, 1461, 1385, 1261, 1159, 1122,
Figure 8. Comparison of the position of the asiatic acid (red) to that
of the maslinic acid (orange) at the allosteric site after superimposing
the GPb-maslinic acid complex structure onto the GPb-asiatic acid
complex structure.
The mixture was filtered through a pad of Celite and washed with
CH2Cl2 (50 mL × 8). The filtrate was concentrated in vacuo to
give a brown solid. Crystallization from ethanol gave 28 as a white
solid (96.8 g, 89%). Mp: 160-162 °C. IR (KBr, cm-1): 3417, 2935,
1724, 1460. 1H NMR (CDCl3, 500 MHz): δ 0.62, 0.90, 0.92, 1.01,
1.04, 1.08, 1.13 (each 3 H, s), 2.36-2.51 (2 H, m), 2.92 (1 H, dd,
J ) 4.3, 13.8 Hz), 5.07 and 5.08 (each 1 H, d, J ) 12.5 Hz), 5.30
(1 H, t, J ) 3.6 Hz), 7.25-7.36 (5 H, m). ESI-MS m/z: 567 [M +
Na]+. HRMS calcd for C37H51O3 [M - H]: 543.3838. Found:
543.3865.
1
1049, 1031, 994, 745, 697. H NMR (CDCl3, 300 MHz): δ 0.60,
0.82, 0.90, 0.92, 0.95, 1.02, 1.12 (each 3 H, s), 2.92 (1 H, dd, J )
4.1, 13.7 Hz), 3.01 (1 H, d, J ) 9.5 Hz), 3.68 (1 H, m), 5.06 and
5.08 (each 1 H, d, J ) 12.6 Hz), 5.29 (1 H, t, J ) 3.5 Hz), 7.34 (5
H, m). ESI-MS m/z: 585.2 [M + Na]+. HRMS Calcd for
C37H53O4[M - H]: 561.3944. Found: 561.3929.
Benzyl-2r,3ꢀ-dihydroxyurs-12-en-28-oic Acid (33). Reduction
of 35 with NaBH4 was carried out following the procedure for
reduction of 34 with NaBH4 to give 33 (68%) as a major product,
together with 37 (14%) as a minor product. Data for 33: mp
163-165 °C. IR (KBr, cm-1): 3401, 2925, 2870, 1723, 1455, 1377,
1267, 1225, 1140, 1109, 1048, 1031, 995, 961, 746, 696. 1H NMR
(CDCl3, 500 MHz): δ 0.63, 0.82, 0.96, 1.03, 1.07 (each 3 H, s),
0.85 (3 H, d, J ) 6.4 Hz), 0.94 (3 H, d, J ) 6.3 Hz), 2.28 (1 H, d,
J ) 11.3 Hz), 3.00 (1 H, d, J ) 9.4 Hz), 3.69 (1 H, m), 5.00 and
5.08 (each 1 H, d, J ) 12.5 Hz), 5.24 (1 H, t, J ) 3.5 Hz),
7.28-7.37 (5 H, m). 13C NMR (CDCl3, 125 MHz): δ 16.7, 16.8,
16.97, 17.00, 18.3, 21.1, 23.3, 23.6, 24.2, 27.9, 28.6, 30.6, 32.9,
36.6, 38.2, 38.8, 39.07, 39.13, 39.6, 42.1, 46.6, 47.5, 48.1, 52.8,
55.3, 66.0, 69.0, 83.9, 125.5, 127.9, 128.1, 128.4, 136.3, 138.2,
177.3. ESI-MS m/z: 585 [M + Na]+. HRMS Calcd for C37H53O4[M
- H]: 561.3944. Found: 561.3969.
2r,3ꢀ-Dihydroxyolean-12-en-28-oic Acid (Maslinic Acid)
(3). A mixture of 32 (2.8 g, 5 mmol) and 10% Pd/C (0.4 g) in
THF (27 mL) was stirred at room temperature under H2 at
atmospheric pressure for 18 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 3 as
a white solid (2.2 g, 95%). Mp: 269-271 °C (lit. mp 266-269
°C50). IR (KBr, cm-1): 3414, 2943, 2878, 1695, 1460, 1389, 1366,
1269, 1184, 1051, 1031, 822, 658. 1H NMR (pyridine-d5, 300
MHz): δ 0.93, 0.98, 0.99, 1.01, 1.06 (each 3 H, s), 1.26 (6 H, s),
3.30 (1 H, dd, J ) 3.7, 13.5 Hz), 3.39 (1 H, d, J ) 9.3 Hz), 4.08
(1 H, m), 5.46 (1 H, brs). 13C NMR (pyridine-d5, 75 MHz): δ 16.9,
17.5, 17.7, 18.9, 23.7, 23.8, 23.9, 26.2, 28.3, 29.3, 31.0, 33.2, 33.3,
34.3, 38.6, 39.8, 42.0, 42.2, 46.5, 46.7, 47.8, 48.2, 55.9, 68.6, 83.8,
122.5, 144.9, 180.2. ESI-MS m/z: 471 [M - H]. HRMS Calcd for
C30H47O4 [M - H]: 471.3474. Found: 471.3465.
Benzyl-3-oxours-12-en-28-oic Acid (29). Following the proce-
dure for preparation of 28, ursolic acid benzyl ester 27 was oxidized
with PCC to give 29 as a white solid. Mp: 156-157 °C. IR (KBr,
cm-1): 2924, 2869, 1722, 1704, 1454, 1383, 1268, 1225, 1140,
1
1110, 1030, 749, 697. H NMR (CDCl3, 300 MHz): δ 0.71, 1.05,
1.07 (each 3 H, s), 0.89 (3 H, d, J ) 6.4 Hz), 0.97 (3 H, d, J ) 5.6
Hz), 1.11 (6 H, s), 2.29 (1 H, d, J ) 11.3 Hz), 2.41 and 2.54 (each
1 H, m), 5.04 and 5.12 (each 1 H, d, J ) 12.5 Hz), 5.28 (1 H, t, J
) 3.5 Hz), 7.37 (5 H, m). ESI-MS m/z: 567 [M + Na]+. HRMS
Calcd for C37H51O3 [M - H]: 543.3838. Found: 543.3851.
Benzyl-2r-hydroxy-3-oxoolean-12-en-28-oic Acid (34). To a
mixture of 28 (10.88 g, 0.02 mol), CH2Cl2 (100 mL), methanol
(200 mL), and concentrated sulfuric acid (0.1 mL) was added
m-CPBA (85%, 6.2 g, 0.03 mol) at 0 °C. The reaction mixture
was stirred in darkness at room temperature for 21 h. To the reaction
mixture was added an aqueous solution containing NaHSO3 (1.2
g, 0.012 mol), and the resulting mixture was stirred for 30 min.
After most organic solvents were removed by evaporation in vacuo
at 35 °C, the residue was acidified with 1 N HCl (100 mL) and
extracted with EtOAc. The combined extract was washed with
saturated NaHCO3 (125 mL × 3) and brine (125 mL × 3), dried
over Na2SO4, filtered, and concentrated in vacuo to afford a yellow
oil, which was purified by column chromatography [petroleum
ether-EtOAc (30:1)] to give 34 as a white solid (8.9 g, 80%). Mp:
180-182 °C. 1H NMR (CDCl3, 300 MHz):δ 0.59, 0.83, 0.89, 1.09,
1.10, 1.15, 1.23 (each, 3 H, s), 2.38 (1 H, dd, J ) 6.6, 12.5 Hz),
2.90 (1 H, dd, J ) 4.1, 13.8 Hz), 4.54 (1 H, dd, J ) 6.6, 12.6 Hz),
5.07 and 5.08 (each 1 H, d, J ) 12.5 Hz), 5.29 (1 H, m), 7.34 (5
H, m). 13C NMR (CDCl3, 75 MHz): δ 216.6, 177.3, 143.8, 136.4,
128.4, 128.0, 127.9, 122.0, 69.1, 65.9, 57.7, 49.5, 47.3, 45.8, 41.3,

3550 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Wen et al.
2r,3ꢀ-Dihydroxyurs-12-en-28-oic Acid (Corosolic Acid) (12).
Corosolic acid was prepared in quantitative yield via hydrogenolysis
of 33 following the procedure for preparation of 3. Mp 253-255
°C (lit. mp 255-258 °C51). IR (KBr, cm-1): 3421, 2966, 2945,
2926, 2872, 1695, 1456, 1389, 1230, 1049, 1032, 997, 662. 1H
NMR (pyridine-d5, 300 MHz): δ 0.96, 1.02, 1.05, 1.19, 1.25 (each
3 H, s), 0.94 (3 H, d, J ) 6.0 Hz), 0.97 (3 H, d, J ) 4.7 Hz), 2.61
(1 H, d, J ) 11.3 Hz), 3.38 (1 H, d, J ) 9.4 Hz), 4.06 (1 H, m),
5.44 (1 H, t, J ) 3.1 Hz). 13C NMR (pyridine-d5, 75 MHz): δ
17.0, 17.50, 17.52, 17.7, 18.9, 21.4, 23.8, 23.9, 24.9, 28.7, 29.4,
31.1, 33,5, 37.5, 38.5, 39.4, 39.5, 39.8, 40.1, 42.5, 48.0, 48.06,
48.13, 53.6, 56.0, 68.6, 83.8, 125.6, 139.3, 179.8. ESI-MS m/z:
495.4 [M + Na]+. HRMS Calcd for C30H47O4[M - H]: 471.3474.
Found: 471.3453.
Benzyl-2r,3r-dihydroxyolean-12-en-28-oic Acid (36). A mix-
ture of 34 (1.2 g, 2.14 mmol), freshly prepared Al(O-i-Pr)3 (42.8
mmol), catalytic amount of AlCl3, and dry i-PrOH (20 mL) was
refluxed for 4 h. After the mixture was cooled to room temperature,
1 N HCl (50 mL) was added to the reaction mixture, and the mixture
was extracted with EtOAc (50 mL × 3). The combined extract
was washed with saturated NaHCO3 (50 mL × 3) and brine (50
mL × 3), dried over Na2SO4, filtered, and concentrated to afford a
yellow solid, which was purified by flash chromatography [petro-
leum ether-EtOAc (6:1)] to give 36 as a white solid (0.6 g, 50%),
together with 32 as a minor product. Data for 36: mp 110-112
°C. IR (KBr, cm-1): 3447, 2947, 2870, 1726, 1632, 1460, 1387,
1258, 1161, 1036, 941, 824, 743, 694, 596, 465. 1H NMR (CDCl3,
300 MHz): δ 0.60, 0.85, 0.90, 0.919, 0.925, 1.01, 1.16 (each 3 H,
s), 2.92 (1 H, dd, J ) 4.6, 14.1 Hz), 3.42 (1 H, d, J ) 2.6 Hz),
4.00 (1 H, m), 5.06 and 5.07 (each 1 H, d, J ) 12.6 Hz), 5.29 (1
H, t, J ) 3.5 Hz), 7.35 (5 H, m). 13C NMR (CDCl3, 75 MHz): δ
16.3, 16.9, 18.0, 21.8, 23.0 23.4, 23.6, 26.0, 27.6, 28.5, 30.7, 32.4,
32.5, 33.1, 33.9, 38.2, 38.4, 39.5, 41.4, 41.7, 41.8, 45.9, 46.7, 47.3,
48.1, 65.9, 66.5, 78.9, 122.3, 127.87, 127.94, 128.4, 136.5, 143.8,
177.4. ESI-MS m/z: 585.3 [M + Na]+. HRMS Calcd for C37H53O4
[M - H]: 561.3944. Found: 561.3962.
d, J ) 11.1 Hz), 3.74 (1 H, d, J ) 2.6 Hz), 4.28 (1 H, m), 5.44 (1
H, t, J ) 3.3 Hz). 13C NMR (pyridine-d5, 75 MHz): δ 16.8, 17.4,
17.5, 18.5, 21.4, 22.3, 23.7, 23.9, 24.9, 28.7, 29.5, 31.1, 33.5, 37.4,
38.6, 38.8, 39.4, 39.5, 40.2, 42.6, 43.0, 47.9, 48.1, 48.7, 53.6, 66.1,
79.4, 125.6, 139.3, 179.9. ESI-MS m/z: 495.3 [M + Na]+, 511.3
[M + K]+. HRMS Calcd for C30H47O4 [M - H]: 471.3474. Found:
471.3485.
Benzyl-2-hydroxy-3-oxoolean-1,12-dien-28-oic Acid (38).
Method A (Preparation from 34). A mixture of 34 (0.6 g, 1.07
mmol), DMF (8 mL), MeOH (8 mL), and KOH (1.5 g, 27 mmol)
was stirred at room temperature for 4 h. The reaction was quenched
by adding 2 N HCl (20 mL), and the sample was extracted with
EtOAc (50 mL × 3). The combined extract was washed with
saturated NaHCO3 (50 mL × 3) and brine (50 mL × 3), dried
over Na2SO4, filtered, and concentrated to afford a yellow oil, which
was purified by flash chromatography [petroleum ether-EtOAc (30/
1, v/v)] to give 38 as a white solid (0.55 g, 92%).
Method B (Preparation from 28). To a solution of 28 (2.0 g,
3.68 mmol) in t-BuOH (80 mL) and THF (10 mL) was added
t-BuOK (2.5 g, 22.28 mmol) at room temperature under air. After
the mixture was stirred overnight, most of the solvents were
removed by evaporation in vacuo, and the residue was diluted with
water, acidified with 1 N HCl, and extracted with EtOAc. The
combined extract was washed with saturated brine, dried over
Na2SO4, filtered, and concentrated in vacuo to afford a pale-yellow
oil, which was purified by column chromatography [petroleum
ether-EtOAc (30:1)] to give 38 as a white solid (1.72 g, 84%).
Mp: 88-90 °C. IR (KBr, cm-1): 3441, 2945, 2870, 1726, 1666,
1460, 1389, 1236. 1H NMR (CDCl3, 300 MHz): δ 0.64, 0.90, 0.93,
1.115, 1.122, 1.19, 1.21 (each 3 H, s), 2.95 (1 H, dd, J ) 4.4, 13.6
Hz), 5.07 and 5.09 (each 1 H, d, J ) 12.5 Hz), 5.34 (1 H, t, J )
3.5 Hz), 5.92 (1 H, s), 6.33 (1 H, s), 7.34 (5 H, m). 13C NMR
(CDCl3, 75 MHz): δ 17.3, 18.7, 19.6, 21.8, 23.0, 23.4, 23.6, 25.8,
27.2, 27.6, 32.3, 32.5, 33.1, 33.9, 38.4, 40.0, 41.5, 42.1, 43.1, 43.9,
45.7, 46.8, 53.9, 66.0, 121.9, 127.95, 128.04, 128.2, 128.4, 136.4,
143.7, 144.1, 177.4, 201.1. ESI-MS m/z: 559.4 [M + H]+, 581.3
[M + Na]+, 597.3 [M + K]+. HRMS Calcd for C37H49O4 [M -
H]: 557.3631. Found: 557.3657.
Benzyl-2-hydroxy-3-oxours-1,12-dien-28-oic Acid (39). In the
same fashion as described for preparation of 38, treatment of 35
with KOH gave 39 (62%) as a white solid. Alternatively, direct
oxidation of 29 in the presence of t-BuOK also afforded 39 (85%).
Mp: 90-92 °C. IR (KBr, cm-1): 3435, 2970, 1724, 1664, 1454,
1404, 1232, 1144. 1H NMR (CDCl3, 300 MHz): δ 0.69, 1.06, 1.12,
1.20, 1.21 (each 3 H, s), 0.86 (3 H, d, J ) 6.4 Hz), 0.95 (3 H, d,
J ) 6.1 Hz), 2.31 (1 H, d, J ) 11.1 Hz), 5.01 and 5.09 (each 1 H,
d, J ) 12.4 Hz), 5.29 (1 H, t, J ) 3.5 Hz), 5.93 (1 H, s), 6.35 (1
H, s), 7.34 (5 H, m). 13C NMR (CDCl3, 75 MHz): δ 17.0, 17.6,
18.7, 19.7, 21.1, 21.8, 23.3, 23.5, 24.3, 27.2, 28.0, 30.7, 32.9, 36.6,
38.3, 38.9, 39.1, 40.3, 42.5, 43.1, 43.9, 48.2, 53.1, 54.0, 66.0, 125.1,
128.0, 128.2, 128.4, 136.4, 138.7, 143.8, 177.1, 201.1. ESI-MS m/z:
559.4 [M + H]+, 581.4 [M + Na]+, 597.4 [M + K]+. HRMS
Calcd for C37H49O4[M - H]: 557.3631. Found: 557.3651.
Benzyl-2r,3r-dihydroxyurs-12-en-28-oic Acid (37). Meer-
wein-Pondorf reduction of 35 was carried out following the
procedure for Meerwein-Pondorf reduction of 34 to give 37 as a
major product (51%), together with 33 (6%) as a minor product.
Data for 37: mp 106-108 °C. IR (KBr, cm-1): 3450, 3431, 2947,
2926, 2870, 1724, 1454, 1383, 1271, 1225, 1140, 1036, 993, 746,
1
696, 665, 598. H NMR (CDCl3, 300 MHz): δ 0.63, 0.85, 0.94,
1.01, 1.08 (each 3 H, s), 0.84 (3 H, d, J ) 5.4 Hz), 0.93 (3 H, d,
J ) 4.7 Hz), 2.29 (1 H, d, J ) 11.3 Hz), 3.43 (1 H, d, J ) 2.7 Hz),
4.01 (1 H, m), 5.00 and 5.08 (each 1 H, d, J ) 12.5 Hz), 5.24 (1
H, t, J ) 3.5 Hz), 7.34 (5 H, m). 13C NMR (CDCl3, 75 MHz):
δ16.5, 17.0, 17.1, 18.1, 21.2, 21.9, 23.3 23.7, 24.3, 28.0, 28.5, 30.7,
32.9, 36.7, 38.3, 38.4, 38.9, 39.1, 39.8, 42.1, 42.2, 47.4, 48.2, 53.0,
66.0, 66.5, 79.0, 125.6, 127.9, 128.2, 128.4, 136,5, 138.3, 177.3.
ESI-MS m/z: 585.3 [M + Na]+, 601.2 [M + K]+. HRMS Calcd
for C37H53O4 [M - H]: 561.3944. Found: 561.3964.
2r,3r-Dihydroxyolean-12-en-28-oic Acid (4). Hydrogenolysis
of 36 was carried out in quantitative yield following the procedure
for hydrogenolysis of 32 to give 4 as a white solid. Mp: 295-297
°C. IR (KBr, cm-1): 3445, 2947, 2874, 1693, 1458, 1385, 1267,
1182, 1036, 995, 941, 881, 822, 667, 646, 567. 1H NMR (pyridine-
d5, 300 MHz): δ 0.89, 0.91, 0.96, 0.98, 1.01, 1.17, 1.26 (each 3 H,
s), 3.26 (1 H, dd, J ) 4.4, 13.9 Hz), 3.75 (1 H, d, J ) 2.6 Hz),
4.29 (1 H, m), 5.46 (1 H, t, J ) 3.3 Hz). 13C NMR (pyridine-d5,
75 MHz): δ 16.6, 17.5, 18.5, 22.3, 23.7, 23.8, 23.9, 26.1, 28.3,
29.5, 30.9, 33.2, 34.3, 38.7, 38.8, 40.0, 42.0, 42.3, 42.8, 46.5, 46.7,
48.0, 48.8, 66.1, 79.4, 122.5, 144.9, 180.2. ESI-MS m/z: 473.4 [M
+ H]+, 495.4 [M + Na]+, 511.3 [M + Na]+. HRMS Calcd for
C30H47O4 [M - H]: 471.3474. Found: 471.3495.
2r,3r-Dihydroxyurs-12-en-28-oic Acid (Pygenic Acid A)
(13). Hydrogenolysis of 37 was carried out following the procedure
for hydrogenolysis of 32 to give 1352 as a white solid. Mp: 230-232
°C. IR (KBr, cm-1) 3454, 2928, 1688, 1630, 1383. 1H NMR
(pyridine-d5, 300 MHz): δ 0.88, 0.94, 1.03, 1.11, 1.25 (each 3 H,
s), 0.92 (3 H, d, J ) 6.2 Hz), 0.96 (3 H, d, J ) 6.3 Hz), 2.61 (1 H,
Benzyl-2ꢀ,3ꢀ-dihydroxyolean-12-en-28-oic Acid (40). To a
solution of 38 (4 g, 7.2 mmol) in THF (43 mL) and ethanol (8.9
mL) was added NaBH4 (0.62 g, 15.7mmol) at 0 °C. The reaction
mixture was stirred at 0 °C for 5 h, and then 1 N HCl (100 mL)
was added, extracted with EtOAc (100 mL × 1, 50 mL × 3). The
combined extract was washed with saturated NaHCO3 (50 mL ×
3) and brine (50 mL × 3), dried over Na2SO4, filtered, and
concentrated to afford a white powder, which was purified by flash
chromatography [petroleum ether-EtOAc (8:1)] to give 40 as a
white solid (2.47 g, 61%). Mp: 98-100 °C. IR (KBr, cm-1): 3448,
2947, 2876, 1726, 1632, 1458, 1383, 1159. 1H NMR (CDCl3, 300
MHz): δ 0.63, 0.90, 0.92, 1.00, 1.01, 1.12, 1.21 (each 3 H, s), 2.93
(1 H, dd, J ) 4.1, 13.8 Hz), 3.21 (1 H, d, J ) 3.9 Hz), 4.09 (1 H,
m), 5.07 and 5.08 (each 1 H, d, J ) 12.5 Hz), 5.30 (1 H, t, J ) 3.5
Hz), 7.34 (5 H, m). 13C NMR (CDCl3, 75 MHz): δ 16.3, 16.9,
17.3, 18.1, 23.1, 23.5, 23.6, 25.9, 27.5, 29.7, 30.7, 32.4, 32.7, 33.1,
33.9, 36.7, 38.1, 39.4, 41.4, 41.8, 44.1, 45.9, 46.8, 48.1, 55.2, 65.9,
71.1, 78.5, 122.6, 127.9, 128.0, 128.4, 136.5, 143.7, 177.4. ESI-

Triterpenes as Inhibitors of Glycogen Phosphorylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3551
MS m/z: 585.3 [M + Na]+, 601.3 [M + K]+. HRMS Calcd for
C37H53O4 [M - H]: 561.3944. Found: 561.3971.
0.89, 0.90, 0.92, 0.95, 1.14 (each 3 H, s), 2.89 (1 H, dd, J ) 4.6,
13.6 Hz), 3.40 (1 H, t, J ) 2.7 Hz), 5.06 and 5.08 (each 1 H, d, J
) 12.6 Hz), 5.29 (1 H, t, J ) 3.5 Hz), 7.34 (5 H, m). ESI-MS m/z:
569.3 [M + Na]+.
Benzyl-3r-hydroxyurs-12-en-28-oic Acid (44). Meerwein-
Pondorf reduction of 29 was carried out following the procedure
described for the preparation of 36 to afford 44 as a white solid
(52%). Mp: 103-105°. IR (KBr, cm-1): 3359, 2923, 2870, 1722,
1454, 1386, 1274, 1226, 1139, 1108. 1H NMR (CDCl3, 300 MHz):
δ 0.65, 0.84, 0.91, 0.95, 1.09 (each 3 H, s), 0.86 (3 H, d, J ) 6.8
Hz), 0.94 (3 H, d, J ) 6.3 Hz), 2.28 (1 H, d, J ) 11.4 Hz), 3.40
(1 H, t, J ) 2.8 Hz), 5.00 and 5.08 (each 1 H, d, J ) 12.5 Hz),
5.24 (1 H, t, J ) 3.6 Hz), 7.34 (5 H, m). ESI-MS m/z: 569.4 [M +
Na]+.
3r-Hydroxyolean-12-en-28-oic Acid (3-Epioleanolic Acid)
(2). Hydrogenolysis of 43 was carried out following the procedure
for hydrogenolysis of 32 to give 256,57 as a white solid (90%). Mp:
293 °C (dec). IR (KBr, cm-1): 3450, 2947, 2868, 1691, 1462, 1385,
1279, 1209, 1068. 1H NMR (pyridine-d5, 300 MHz): δ 0.88, 0.91,
0.92, 0.99, 1.05, 1.18, 1.22 (each 3 H, s), 3.27 (1 H, dd, J ) 4.0,
13.8 Hz), 3.60 (1 H, brs), 5.49 (1 H, t, J ) 3.3 Hz). ESI-MS m/z:
479.4 [M + Na]+.
Benzyl-2ꢀ,3ꢀ-dihydroxyurs-12-en-28-oic Acid (41). In the same
fashion as described for the preparation of 40, reduction of 39 with
NaBH4 gave 41 as a white solid (69%). Mp: 85-87 °C. IR (KBr,
cm-1): 3452, 2924, 2872, 1724, 1456, 1381, 1225, 1142. 1H NMR
(CDCl3, 300 MHz): δ 0.66, 1.00, 1.01, 1.06, 1.22 (each 3 H, s),
0.86 (3 H, d, J ) 6.4 Hz), 0.94 (3 H, d, J ) 5.9 Hz), 2.29 (1 H, d,
J ) 11.3 Hz), 3.43 (1 H, d, J ) 4.0 Hz), 4.01 (1 H, m), 5.00 and
5.09 (each 1 H, d, J ) 12.5 Hz), 5.24 (1 H, t, J ) 3.6 Hz), 7.34 (5
H, m). 13C NMR (CDCl3, 75 MHz): δ 16.4, 16.95, 17.00, 17.3,
18.1, 21.1, 23.4 23.6, 24.2, 27.8, 29.7, 30.7, 33.0, 36.6, 36.7, 38.1,
38.8, 39.1, 39.7, 42.2, 44.3, 48.0, 48.1, 52.9, 55.2, 66.0, 71.1, 78.5,
125.8, 127.9, 128.2, 128.4, 136,4, 138.1, 177.2. ESI-MS m/z: 585.2
[M + Na]+, 601.2 [M + K]+. HRMS Calcd for C37H53O4 [M -
H]: 561.3944. Found: 561.3959.
2ꢀ,3ꢀ-Dihydroxyolean-12-en-28-oic Acid (Augustic Acid) (5).
Hydrogenolysis of 40 was carried out following the procedure for
hydrogenolysis of 32 to give 553,54 as a white solid. Mp: 308-310
°C. IR (KBr, cm-1): 3485, 3433, 2949, 1703, 1464, 1385, 1263,
1194, 1063. 1H NMR (pyridine-d5, 300 MHz): δ 0.98, 1.04, 1.11,
1.29, 1.34, 1.38, 1.54 (each 3 H, s), 3.35 (1 H, dd, J ) 3.4, 13.4
Hz), 3.46 (1 H, d, J ) 3.6 Hz), 4.42 (1 H, m), 5.54 (1 H, brs). 13
C
3r-Hydroxyurs-12-en-28-oic Acid (3-Epiursolic Acid) (11).
Hydrogenolysis of 44 was carried out following the procedure for
hydrogenolysis of 32 to give 1158 as a white solid (94%). Mp: 279
°C (dec). IR (KBr, cm-1): 3448, 2945, 2926, 2870, 1697, 1454,
1383, 1311, 1232, 1067. 1H NMR (pyridine-d5, 300 MHz): δ 0.88,
0.92, 1.07, 1.11, 1.22 (each 3 H, s), 0.93 (3 H, d, J ) 3.7 Hz), 0.95
(3 H, d, J ) 6.5 Hz), 2.60 (1 H, d, J ) 11.5 Hz), 3.60 (1 H, brs),
5.49 (1 H, t, J ) 3.4 Hz). ESI-MS m/z: 479.4 [M + Na]+.
2,3,4,6-Tetra-O-acetyl-ꢀ-D-pyranoglucosyl 3ꢀ-Hydroxyolean-
12-en-28-oate (46). A mixture of oleanolic acid (1) (2 g), 2,3,4,6-
tetra-O-acetyl-R-D-glucosyl bromide25 (45, 3.51 g), K2CO3 (2.42
g), and DMF (40 mL) was stirred overnight at room temperature.
The solids were filtered off, and to the filtrate was added 200 mL
of water. The resulted precipitate was collected by filtration, washed
with water, and dried. The crude product was purified by column
chromatography to give 46 as a white solid (3.15 g, 92%). Mp:
163-165 °C. IR (KBr, cm-1): 2647, 1759, 1462, 1366, 1226, 1076,
1037. 1H NMR (300 MHz, CDCl3): δ 0.74, 0.78, 0.98, 1.13 (each,
3 H, s), 0.91 (9 H, s), 2.01 (3 H, s), 2.02 (6 H, s), 2.07 (3 H, s),
2.80 (1 H, dd, J ) 4.5, 13.9 Hz), 3.20 (1 H, dd, J ) 4.3, 11.0 Hz),
3.80 (1 H, m), 4.07 (1 H, dd, J ) 2.1, 12.4 Hz), 4.26 (1 H, dd, J
) 4.4, 12.5 Hz), 5.13 (1 H, t, J ) 9.2 Hz), 5.18 (1 H, t, J ) 7.8
Hz), 5.25 (1 H, t, J ) 9.1 Hz), 5.28 (1 H, brs), 5.57 (1 H, d, J )
7.8 Hz). 13C NMR (CDCl3, 75 MHz): δ 15.3,15.6, 16.9, 18.3, 20.6,
20.7, 22.8, 23.38, 23.42, 25.7, 27.2, 27.7, 28.1, 29.7, 30.6, 31.7,
32.9, 33.0, 33.7, 37.0, 38.5, 38.7, 39.3, 41.0, 41.7, 45.7, 46.8, 47.6,
55.2, 61.5, 68.0, 69.9, 72.4, 72.8, 79.0, 91.6, 122.9, 142.8, 168.9,
169.4, 170.1, 170.6, 175.6. ESI-MS m/z: 809.4 [M + Na]+.
2,3,4,6-Tetra-O-acetyl-ꢀ-D-pyranoglucosyl 3ꢀ-Hydroxyurs-
12-en-28-oate (47). Reaction of ursolic acid (10) with 45 was
carried out following the procedure described for the preparation
of 46 to afford 47 as a white solid (90%). Mp: 174-175 °C. IR
(KBr, cm-1): 2926, 1758, 1454, 1367, 1224, 1069, 1037. 1H NMR
(CDCl3, 300 MHz): δ 0.77, 0.78, 0.92, 0.95, 0.98, 1.07 (each 3 H,
s), 0.85 (3 H, d, J ) 6.4 Hz), 2.01, 2.02, 2.03, 2.06 (each 3 H, s),
2.21 (1 H, d, J ) 11.8 Hz), 3.20 (1 H, dd, J ) 4.7, 10.4 Hz), 3.78
(1 H, m), 4.05 (1 H, dd, J ) 2.3, 12.3 Hz), 4.25 (1 H, dd, J ) 4.4,
12.3 Hz), 5.11 (1 H, t, J ) 9.5 Hz), 5.16 (1 H, t, J ) 8.5 Hz), 5.24
(1 H, t, J ) 9.2 Hz), 5.28 (1 H, t, J ) 3.9 Hz), 5.54 (1 H, d, J )
7.9 Hz). 13C NMR (CDCl3, 75 MHz): δ 15.5, 15.6, 16.9, 17.1,
18.3, 20.58, 20.64, 20.7, 21.1, 23.3, 23.4, 24.1, 27.3, 28.2, 30.6,
33.3, 36.0, 37.0, 38.7, 38.8, 38.9, 39.1, 39.6, 42.1, 47.6, 48.2, 52.7,
55.3, 61.7, 68.2, 70.0, 72.5, 73.0, 79.0, 91.6, 126.2, 137.3, 169.0,
169.4, 170.1, 170.6, 175.4. ESI-MS m/z: 809.1 [M + Na]+.
2,3,4,6-Tetra-O-acetyl-ꢀ-D-pyranoglucosyl 2r,3ꢀ-Dihydrox-
yurs-12-en-28-oate (48). Reaction of corosolic acid (12) with 45
was carried out following the procedure described for the prepara-
tion of 46 to afford 48 as a white solid (94%). Mp: 165-167 °C.
IR (KBr, cm-1): 2925, 1758, 1454, 1367, 1224, 1068, 1037, 749.
NMR (pyridine-d5, 75 MHz): δ 16.6, 17.5, 18.1, 18.7, 23.8, 24.0,
26.3, 28.3, 30.3, 31.0, 33.3, 33.4, 34.3, 37.4, 38.8, 40.0, 42.1, 42.4,
45.0, 46.6, 46.7, 48.6, 56.0, 71.5, 78.4, 122.8, 144.9, 180.2. ESI-
MS m/z: 495.3 [M + Na]+. HRMS Calcd for C30H47O4 [M - H]:
471.3474. Found: 471.3470.
2ꢀ,3ꢀ-Dihydroxyurs-12-en-28-oic Acid (2-Epicorosolic Acid)
(14). Hydrogenolysis of 41 was carried out following the procedure
for hydrogenolysis of 32 to give 1455 as a white solid. Mp: 284 °C
(dec). IR (KBr, cm-1): 3439, 2926, 1691, 1634, 1454, 1381, 1051,
1
999. H NMR (pyridine-d5, 300 MHz): δ 0.97 (3 H, d, J ) 5.7
Hz), 1.03 (3 H, d, J ) 6.4 Hz), 1.11, 1.25, 1.27, 1.36, 1.51 (each
3 H, s), 2.66 (1 H, d, J ) 11.3 Hz), 3.46 (1 H, d, J ) 3.9 Hz), 4.41
(1 H, m), 5.50 (1 H, brs). 13C NMR (pyridine-d5, 75 MHz): δ 16.8,
17.5, 18.2, 18.7, 21.4, 23.9, 24.0, 25.0, 28.7, 30.3, 31.2, 33.7, 37.3,
37.5, 38.8, 39.5, 39.6, 40.2, 42.8, 45.2, 48.1, 48.5, 53.7, 56.0, 71.5,
78.4, 125.9, 139.3, 179.9. ESI-MS m/z: 495.2 [M + Na]+, 511.2
[M + K]+. HRMS Calcd for C30H47O4 [M - H]: 471.3474. Found:
471.3481.
Benzyl-3ꢀ-hydroxy-2-oxoolean-12-en-28-oic Acid (42). A mix-
ture of 34 (0.54 g, 0.96 mmol), NaHCO3 (2 g), CH2Cl2 (20 mL),
MeOH (20 mL), and water (10 mL) was heated at reflux for 48 h.
The reaction mixture was then evaporated under reduced pressure,
and to the residue was added water, extracted with EtOAc (50 mL
× 3). The combined extract was washed with 1 N HCl and brine,
dried over Na2SO4, filtered, and concentrated. Purification by flash
chromatography [petroleum ether-EtOAc (10:1)] and recrystalli-
zation from EtOH gave 42 as a white crystal (80 mg, 15%). Mp:
184-186 °C. IR (KBr, cm-1): 3483, 2959, 2912, 2876, 1720, 1705,
1454, 1389, 1304, 1254, 1213, 1157. 1H NMR (CDCl3, 300 MHz):
δ 0.58, 0.69, 0.84, 0.90, 0.92, 1.17, 1.18 (each 3 H, s), 2.07 (1 H,
d, J ) 12.1 Hz), 2.43 (1 H, d, J ) 12.2 Hz), 2.94 (1 H, m), 3.88
(1 H, s), 5.06 and 5.07 (each 1 H, d, J ) 12.5 Hz), 5.29 (1 H, brs),
7.34 (5 H, m). 13C NMR (CDCl3, 75 MHz): δ 16.1, 16.45, 16.53,
18.5, 23.0, 23.3, 23.6, 25.8, 27.6, 29.4, 30.7, 32.3, 32.4, 33.0, 33.8,
39.7, 41.4, 41.8, 43.6, 45.6, 45.9, 46.7, 47.6, 53.1, 54.5, 65.9, 82.8,
121.7, 127.9, 128.0, 128.4, 136.4, 143.9, 177.2, 210.9. ESI-MS m/z:
561.3 [M + H]+, 583.3 [M + Na]+, 599.3 [M + K]+. HRMS
Calcd for C37H51O4 [M - H]: 559.3787. Found: 557.3808. Anal.
Calcd for C37H52O4: C, 79.24, H, 9.35. Found: C, 79.14, H, 9.20.
Preparation of 40 via Reduction of 42. Reduction of 42 with
NaBH4 was carried out following the procedure described for
reduction of 38 to give 2ꢀ,3ꢀ-diol 40, which was identical with
the product obtained by NaBH4 reduction of 38.
Benzyl-3r-hydroxyolean-12-en-28-oic Acid (43). Meerwein-
Pondorf reduction of 28 was carried out following the procedure
described for the preparation of 36 to afford 43 as a white solid
(57%). Mp: 196-198 °C. IR (KBr, cm-1): 2946, 2863, 1723, 1456,
1386, 1365, 1262, 1159. 1H NMR (CDCl3, 300 MHz): δ 0.61, 0.83,

3552 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Wen et al.
1H NMR (CDCl3, 300 MHz): δ 0.76, 0.82, 0.95, 0.99, 1.03, 1.07
(each, 3 H, s), 0.84 (3 H, d, J ) 6.4 Hz), 2.01 (3 H, s), 2.02 (6 H,
s), 2.06 (3 H, s), 2.17 (1 H, d, J ) 11.4 Hz), 3.01 (1 H, d, J ) 9.5
Hz), 3.69 (1 H, m), 3.77 (1 H, m), 4.05 (1 H, dd, J ) 2.2, 12.4
Hz), 4.25 (1 H, dd, J ) 4.4, 12.4 Hz), 5.12 (1 H, t, J ) 9.5 Hz),
5.16 (1 H, t, J ) 8.5 Hz), 5.25 (1 H, t, J ) 9.1 Hz), 5.28 (1 H, t,
J ) 3.4 Hz), 5.54 (1 H, d, J ) 7.9 Hz). 13C NMR (CDCl3, 75
MHz): δ 16.8, 16.9, 17.1, 18.3, 20.57, 20.60, 20.67, 20.69, 21.1,
23.3, 24.1, 28.1, 28.6, 30.5, 33.2, 36.0, 38.2, 38.8, 39.06, 39.13,
39.7, 42.1, 46.7, 47.5, 48.2, 52.6, 55.3, 61.6, 68.1, 69.0, 70.0, 72.5,
72.9, 84.0, 91.6, 125.9, 137.3, 168.9, 169.4, 170.1, 170.6, 175.3.
ESI-MS m/z: 825.4 [M + Na]+.
ꢀ-D-Pyranoglucosyl 3ꢀ-Hydroxyolean-12-en-28-oate (18). To
a solution of 46 (0.14 g, 0.19 mmol) in THF (3 mL) and MeOH (2
mL) was added dropwise 4 N NaOH aqueous solution (0.5 mL),
and then the reaction mixture was stirred at room temperature for
45min. The mixture was diluted with water (30 mL) and extracted
with EtOAc-THF (2:1) three times (each time 30 mL). The
combined extract was washed with 1 N HCl (20 mL) and brine
(20 mL), dried over Na2SO4, filtered, and concentrated. The crude
product was purified by flash chromatography [CH2Cl2-MeOH (20:
1)] to give 1859 as a white solid (0.11 g, 92%). Mp: 224-226 °C.
IR (KBr, cm-1): 3435, 2944, 2873, 1735, 1460, 1385, 1072, 1029.
1H NMR (pyridine-d5, 300 MHz): δ 0.88, 0.90, 0.92, 1.02, 1.03,
1.22, 1.24 (each, 3 H, s), 3.22 (1 H, dd, J ) 4.5, 13.9 Hz), 3.43 (1
H, dd, J ) 5.6, 10.0 Hz), 4.02 (1 H, m), 4.19 (1 H, t, J ) 8.3 Hz),
4.27 (1 H, t, J ) 8.6 Hz), 4.34 (1 H, t, J ) 8.9 Hz), 4.40 (1 H, dd,
J ) 4.2, 11.8 Hz), 4.44 (1 H, dd, J ) 2.5, 11.9 Hz), 5.45 (1 H,
brs), 6.31 (1 H, d, J ) 7.9 Hz). 13C NMR (pyridine-d5, 75 MHz):
δ 15.7, 16.5, 17.6, 18.9, 23.5, 23.7, 23.9, 26.1, 28.1, 28.3, 28.8,
30.8, 32.6, 33.1, 33.3, 34.1, 37.4, 39.0, 39.4, 40.0, 41.8, 42.2, 46.3,
47.1, 55.9, 62.3, 71.2, 74.2, 78.2, 79.0, 79.3, 95.8, 144.2, 176.4.
ESI-MS m/z: 641.4 [M + Na]+.
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 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.
X-ray Crystallography. Rabbit muscle GPb was isolated,
purified, recrystallized, and assayed as described.62 Binding studies
performed by diffusion of pentacyclic triterpenes into freshly
prepared GPb crystals, grown in the tetragonal lattice, space group
P43212, as described previously,63 resulted in crystal dissolution.
Crystal dissolution was in part overcome by using old preformed
GPb crystals, since in most experiments soaking resulted in
calculated 2Fo- Fc and Fo- Fc electron density maps with very
poor or no electron density for the bound ligand. Good results were
obtained with asiatic (15) and maslinic (3) acids. Binding of 15 at
the allosteric site showed a concentration and soaking time
dependence. Thus, at 10 mM and a soaking time of 2.5 h, there
was no binding of either 15 or 3 at the allosteric site. Also, with
2.5 and 5 mM 15 and a soaking time of 2.5-85 h there was no
ligand binding. As the concentration of 15 was increased, binding
was observed at 10 mM or higher. Similarly with 10 mM 3 and a
soaking time of 2.5 h, there was no binding. Finally, soaking with
corosolic acid (12), glycyrrhetinic acid (8), ursolic acid (10), and
oleanolic acid (1) at the highest concentration achieved (5 mM)
and at a soaking time of 7 h resulted in no binding of these ligands.
The best crystallographic results were obtained with 10 mM 15
(soaked for 7 h) in a buffered solution (10 mM Bes, 10% DMSO,
pH adjusted to ∼8 with NaOH) prior to data collection. Similarly,
crystallographic data for the GPb-maslinic acid complex were
collected from a single crystal soaked with 10 mM maslinic acid
(for 7 h) in 10 mM Bes, 10% DMSO, pH adjusted to ∼8 with
NaOH. The high B-factors for both inhibitors probably indicate a
rather low occupancy of the allosteric site. Diffraction data were
collected either at Daresbury Laboratory SRS (beamline PX10.1)
or at EMBL-Hamburg outstation (beamline X13). Crystal orienta-
tion, integration of reflections, interframe scaling, partial reflection
summation, data reduction, and postrefinement were all performed
using DENZO and SCALEPACK.64
ꢀ-D-Pyranoglucosyl 3ꢀ-Hydroxyurs-12-en-28-oate (19). Hy-
drolysis of 47 was carried out following the procedure described
for the preparation of 18 to afford 1960 as a white solid (75%).
Mp: 186-188 °C. IR (KBr, cm-1): 3437, 2925, 1736, 1458, 1388,
1
1072, 1028, 996. H NMR (pyridine-d5, 300 MHz): δ 0.91, 1.03,
1.18, 1.19, 1.24 (each, 3 H), 0.95 (6 H, s), 0.98 (3 H, d, J ) 7.6
Hz), 2.52 (1 H, d, J ) 11.4 Hz), 3.45 (1 H, m), 4.04 (1 H, m), 4.11
(1 H, t, J ) 8.1 Hz), 4.28 (1 H, t, J ) 8.6 Hz), 4.26 (1 H, t, J )
9.0 Hz), 4.40 (1 H, m), 4.35 (1 H, m), 5.47 (1 H, brs), 6.26 (1 H,
d, J ) 7.8 Hz). 13C NMR (pyridine-d5, 75 MHz): δ 15.8, 16.6,
17.4, 17.7, 18.8, 21.3, 23.7, 23.8, 24.7, 28.2, 28.7, 28.8, 30.9, 33.6,
36.8, 37.3, 39.17, 39.22, 39.4, 40.2, 42.5, 48.2, 48.4, 53.4, 55.9,
62.4, 71.3, 74.1, 78.2, 78.9, 79.2, 95.8, 126.2, 138.5, 176.2. ESI-
MS m/z: 641.1 [M + Na]+.
Crystallographic refinement of the complexes was performed by
maximum-likelihood methods using REFMAC.65 The starting
model employed for the refinement of the complexes was the
structure of the native T state GPb complex determined at 1.9 Å
resolution (Oikonomakos et al., unpublished results). The 2Fo -
Fc and Fo - Fc electron density maps calculated were visualized
using the program for molecular graphics “O”.66 Ligand models
of asiatic acid (15) and maslinic acid (3), optimized by quantum
mechanics using DFT (B3LYP/6-31G*) with Jaguar 7.0, were fitted
to the electron density maps after adjustment of their torsion angles.
Alternate cycles of manual rebuilding with “O” and refinement with
REFMAC improved the quality of the models.
ꢀ-D-Pyranoglucosyl 2r,3ꢀ-Dihydroxyurs-12-en-28-oate (20).
Hydrolysis of 48 was carried out following the procedure described
for the preparation of 18 to afford 2061 as a white solid. Mp: 275
°C (dec). IR (KBr, cm-1): 3414, 2925, 2873, 1730, 1455, 1380,
1
1072, 1030, 997. H NMR (pyridine-d5, 300 MHz): δ 0.82, 0.96,
The stereochemistry of the protein residues was validated by
PROCHECK.67,68 Hydrogen bonds and van der Waals interactions
were calculated with the program CONTACT as implemented in
CCP4,68 applying a distance cutoff of 3.3 and 4.0 Å, respectively.
Protein structures were superimposed using LSQKAB.68 Solvent-
accessible areas were calculated with the program NACCESS.69
The figures were prepared with the program MolScript70 and
rendered with Raster3D71 and with the program MG.72,73 The
coordinates of the new structures have been deposited with the
RCSB Protein Data Bank (http://www.rcsb.org/pdb) with codes
2QN1 (GPb-asiatic acid complex) and 2GN2 (GPb-maslinic
complex).
1.00, 1.18 (each, 3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 1.10 (6 H, s),
2.43 (1 H, d, J ) 11.3 Hz), 3.29 (1 H, d, J ) 9.4 Hz), 3.95 (2 H,
m), 4.11 (1 H, t, J ) 8.2 Hz), 4.19 (1 H, t, J ) 8.5 Hz), 4.26 (1 H,
t, J ) 9.2 Hz), 4.31 (1 H, dd, J ) 4.3, 11.7 Hz), 4.35 (1 H, dd, J
) 2.8, 11.8 Hz), 5.36 (1 H, brs), 6.17 (1 H, d, J ) 7.9 Hz). 13C
NMR (pyridine-d5, 75 MHz): δ 17.1, 17.4, 17.7, 17.8, 18.9, 21.3,
23.8, 24.7, 28.7, 29.4, 30.9, 33.6, 36.8, 38.5, 39.2, 39.4, 39.8, 40.3,
42.6, 48.1, 48.2, 48.4, 53.3, 56.0, 62.4, 68.6, 71.3, 74.1, 78.9, 79.2,
83.9, 95.8, 126.1, 138.5, 176.2; ESI-MS m/z: 657.1 [M + Na]+.
Enzyme Kinetics. The inhibitory activity of the test compounds
against rabbit muscle glycogen phosphorylase a (GPa) was moni-
tored using microplate reader (BIO-RAD) based on a published
method.4 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 to 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-
Acknowledgment. This work was made possible by an
EFSD grant supported by Bristol-Myers Squibb Company,
National Natural Science Foundation of China (Grants 30672523
and 90713037), research grants from Chinese Ministry of
Education (Grants 706030 and 20050316008), program for New
Century Excellent Talents in University (Grant NCET-05-0495),

Triterpenes as Inhibitors of Glycogen Phosphorylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3553
(9) Liu, J. Pharmacology of oleanolic acid and ursolic acid. J. Ethnop-
harmacol. 199549, 57–68.
EU Marie Curie Early Stage Training (EST) (Contract No.
MEST-CT-020575), Marie Curie Host Fellowships for the
Transfer of Knowledge (ToK) (Contract No. MTKD-CT-2006-
042776), the EMBL-Hamburg outstation under FP6 “Structuring
the European Research Area Programme” (Contract No. RII3/
CT/2004/5060008), and SRS Daresbury Laboratory (Contract
IHPP HPRI-CT-1999-00012).
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Supporting Information Available: Crystallographic data col-
lection details for compounds 15 and 3, hydrogen bonding and van
der Waals interactions between the inhibitors and GPb residues at
the catalytic site, detailed structural comparisons of GPb-asiatic
acid with GPb-W1807, GPb-Novo4j and hlGPa-AVE#21, and
analytical data for the synthesized compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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as multifunctional agents for the prevention and treatment of cancer.
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