Pentacyclic triterpenes. Part 3: Synthesis and biological evaluation of oleanolic acid derivatives as novel inhibitors of glycogen phosphorylase

Article abstract of DOI:10.1016/j.bmcl.2006.03.009

Oleanolic acid and its synthetic derivatives have been identified as novel inhibitors of glycogen phosphorylase. Within this series of compounds, 4 (IC₅₀ = 3.3 μM) is the most potent GPa inhibitor. Preliminary structure-activity relationships of the oleanolic acid derivatives are discussed.

Full text of DOI:10.1016/j.bmcl.2006.03.009

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2915–2919
Pentacyclic triterpenes. Part 3: Synthesis and biological
evaluation of oleanolic acid derivatives as novel inhibitors
of glycogen phosphorylase
Jun Chen,a Jun Liu,b Luyong Zhang,b Guanzhong Wu,c Weiyi Hua,a
Xiaoming Wua and Hongbin Suna,*
aCenter for Drug Discovery, College of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
bJiangsu Center for Drug Screening, China Pharmaceutical University, 1 Shennonglu, Nanjing 210038, China
cDepartment of Pharmacology, College of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
Received 18 January 2006; revised 16 February 2006; accepted 1 March 2006
Available online 20 March 2006
Abstract—Oleanolic acid and its synthetic derivatives have been identified as novel inhibitors of glycogen phosphorylase. Within
this series of compounds, 4 (IC50 = 3.3 lM) is the most potent GPa inhibitor. Preliminary structure–activity relationships of the
oleanolic acid derivatives are discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
Oleanolic acid (OA), which has been in active clinical
use as an anti-hepatitis drug in China for over 20 years,
possesses some attractive biological activities including
protection of the liver against toxic injury,1 anti-inflam-
mation,2 anti-HIV,3 and antitumor.4 Liu et al.5 reported
that OA exhibited hypoglycemic effect in alloxan-in-
duced hyperglycemic rats, meanwhile, significant accu-
mulation of hepatic glycogen in rats was observed in
OA treatment groups compared with the control
groups, indicating a possible inhibition of hepatic glyco-
genolysis. The action mechanism of OA’s effect on
hepatic glycogen metabolism, however, remains
unknown.
Very recently, we first reported that maslinic acid, a
natural pentacyclic triterpene abundant in olive fruit,
represented a new class of inhibitors of glycogen phos-
phorylase (GP).6 GP is the enzyme responsible for gly-
cogen breakdown to produce glucose and related
metabolites for energy supply.7 Due to its key role in
modulation of glycogen metabolism, pharmacological
inhibition of GP has been regarded as a promising ther-
apeutic approach for treating diseases caused by abnor-
malities in glycogen metabolism.8 Several structural
classes of GP inhibitors have been developed,8a,8b,9
and a couple of GP inhibitors have been in clinical or
pre-clinical trials, for example, PSN-357 is an oral GP
inhibitor which is currently in phase II clinical trial for
treatment of type 2 diabetes.10
H
H
Based on our previous studies, we further examined
other members of pentacyclic triterpene family with
respect to their inhibitory effects on GP. The enzyme
assay results showed that like maslinic acid
(IC50 = 28 lM), OA was also a naturally occurring GP
inhibitor (IC50 = 14 lM). Encouraged by this in vitro
result, we tested hypoglycemic activity of OA in adren-
aline-induced diabetic mice which had hyperglycemia
due to increased hepatic glycogenolysis. OA was admin-
istered orally at 100 mg/kg/day for 7 days, and on the
last day, after adrenaline was iv administered, blood
glucose levels of OA treatment groups and the control
COOH
COOH
HO
HO
HO
maslinic acid
oleanolic acid
Keywords: Oleanolic acid; Glycogen phosphorylase; Inhibitor; Deriv-
atives; Diabetes.
*
Corresponding author. Tel.: +86 25 85327950; fax: +86 25
83271335; e-mail: hbsun2000@yahoo.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.009

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J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2915–2919
Table 1. Effect of oleanolic acid (OA) on fasted plasma glucose of
hyperglycemic mice induced by adrenaline (n = 10)
are outlined in Scheme 1. Treatment of OA with anhy-
dride/pyridine or RCOOH/DCC/DMAP or RCOCl/
Et3N afforded 3-O-acyl OA derivatives 1–5 in 40–91%
yields. C-28 esters 6–8 and 10 were prepared in high
yields (85–93%) by esterification of OA in the presence
of K2CO3 in DMF with bromoethane, allyl bromide,
ethyl bromoacetate, and ethyl 4-bromobutyrate, respec-
tively. Hydrolysis of 8 and 10 with 1 M NaOH in THF/
MeOH gave the corresponding carboxylic acids 9 and 11
in quantitative yields, respectively. Reaction of OA with
1,2-dibromoethane in the presence of K2CO3 in DMF
furnished bromide compound 12 (86%), which was fur-
ther converted to hydroxyl ethyl ester 13 (78%) by heat-
ing of 12 with aqueous ethanol in the presence of Et3N.
Treatment of 12 with aqueous methylamine solution in
the presence of K2CO3 in acetone at room temperature
afforded amine 14 in 75% yield. Reaction of 12 with
diethylamine in the presence of K2CO3 in acetone at re-
flux temperature gave amine 15 in 72% yield. Jones oxi-
dation of OA afforded oleanonic acid 16 (87%).
Reaction of 16 with hydroxylamine hydrochloride in
pyridine at 80 °C afforded oxime 17 (82%).
Compound Dose
(mg/kg)
Fasted plasma glucose (OD)
1 h 2 h
0 h
Vehicle
OA
0.138 0.028 0.212 0.032 0.149 0.037
0.114 0.020 0.153 0.040 0.063 0.025
100
groups were measured. Not surprisingly, the in vivo test
result (Table 1) showed that OA effectively inhibited the
increase of fasted plasma glucose of diabetic mice in-
duced by adrenaline.
The finding that OA is a natural GP inhibitor is of par-
ticular importance due to the following points: (1) it has
been disclosed herein that OA may lower blood glucose,
at least in part, through inhibiting GP and therefore
reducing hepatic glucose production. (2) OA, as a safe
nonprescription drug for the treatment of hepatitis,
may find its new clinical uses in treating diabetes charac-
terized by fasting hyperglycemia and other diseases
caused by disorders in glycogen metabolism. (3) OA,
which is widely distributed in plant kingdom, is very
cheap and easily available in large bulk, and therefore,
lead modification based on OA would be in a good
position for further drug development.
Next, we attempted to probe the effect of structural
modification at A-ring of OA on GP inhibitory activity.
In this regard, some oxime, oxadiazole, and isoxazole
derivatives of OA were synthesized as depicted in
Schemes 2 and 3. Esterification of OA with benzyl chlo-
ride followed by an oxidation reaction with PCC affor-
ded ketone 19 in high yields.6 Treatment of 19 with
hydroxylamine hydrochloride in pyridine gave oxime
With OA as a lead compound, we sought to identify a
series of OA derivatives as novel GP inhibitors. Initial
synthetic efforts were focused on structural modifica-
tions at C-3 and C-28 positions. The synthetic routes
H
H
H
COOR2
vii
COOH
COOH
O
HO
HON
vi
6
7
8
9
R2= C2H5
16
17
R2= CH2CH=CH2
R2= CH2COOC2H5
R2= CH2COOH
ii
iii
10 R2= CH2CH2CH2COOC2H5
11 R2= CH2CH2CH2COOH
H
H
iii
COOH
i
COOH
R1O
HO
OA
H
1
2
3
R1= COCH3
R1= COCH2CH2COOH
R1= COCH2CH2CH3
COOCH2CH2R3
iv
v
O
R1=
HO
4
H
13 R3 = OH
14 R3 = NHCH3
O
Cl
COOCH2CH2Br
5
R1=
15 R3 = N(C2H5)2
OCH3
HO
12
Scheme 1. Reagents and conditions: (i) anhydride/Pyr, 80 °C or RCOOH/DCC/DMAP, THF, rt, or RCOCl/Et3N, rt (40–91%); (ii) R2Br, K2CO3,
DMF, rt (85–93%); (iii) 1 M NaOH, THF/MeOH, rt (quant.); (iv) BrCH2CH2Br, K2CO3, DMF, 40 °C (86%); (v) for 13: Et3N, aqueous ethanol,
reflux (78%); for 14: aqueous methylamine solution, K2CO3, acetone, rt (75%); for 15: diethylamine, K2CO3, acetone, reflux, (72%); (vi) CrO3/H2SO4/
acetone/H2O, 0 °C (87%); (vii) HONH3Cl, pyridine, 80 °C (82%).

J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2915–2919
2917
H
H
H
COOR2
COOBn
COOBn iv
iii
ii
i
HON
O
OA
R1
HO
21 R2= Bn
22 R2= H
19 R1= O
20 R1= NOH
18
iii
v
vii
viii
H
H
COOR2
COOBn
HON
HON
HON
H
HO
COOBn
R3ON
23
26 R2= Bn
27 R2= H
v
O
vi
28 R3=
29 R3=
H2C
H
H
Cl
v
COOBn
COOH
N
N
N
O
N
H2C
Cl
O
25
24
Scheme 2. Reagents and conditions: (i) BnCl, K2CO3, DMF, 60 °C (95%); (ii) PCC, CH2Cl2, rt (89%); (iii) HONH3Cl, Pyr, 80 °C (80–87%); (iv)
NaNO2, H2SO4, CH3OH/THF/H2O, rt (58%); (v) H2, Pd/C, THF, rt; (vi) NaOH, 1,2-ethanediol, 200 °C, 30 min (82%); (vii) NaBH4, THF, 0 °C
(78%); (viii) benzyl chloride or 2,4-dichlorobenzyl chloride, K2CO3, DMF, 40 °C (50–55%).
H
H
H
COOBn
COOBn
COOH
ii
HO
i
iii
19
N
N
O
O
O
30
31
32
Scheme 3. Reagents and conditions: (i) HCOOEt, CH3ONa, CH2Cl2, rt (80%); (ii) HONH3Cl, aq EtOH, 80 °C, 2 h (95%); (iii) H2, Pd/C, EtOAc, rt,
2 h (12%).
20 (87%). Introduction of the oximino group to the C-2
position of 19 was carried out by reaction of 19 with
sodium nitrite under acidic conditions to give a-oximi-
noketone 21 (58%). Treatment of 21 with hydroxyl-
amine hydrochloride in pyridine at 80 °C afforded
dioxime 23 (80%). Cyclization of 23 was performed by
heating 23 with NaOH in 1,2-ethanediol at 200 °C to
give oxadiazole 24 (82%). Hydrogenolysis of 24 over
Pd/C in THF at room temperature gave oxadiazole car-
boxylic acid 2511 in quantitative yield. Reduction of 21
with NaBH4 in THF at 0 °C afforded a-oximinoalcohol
26 in 78% yield. Alkylation of 21 with benzyl chloride
and 2,4-dichlorobenzyl chloride in the presence of potas-
sium carbonate in DMF gave oxime ethers 28 and 29 in
moderate yields (50–55%), respectively. Hydrogenolysis
of 21 and 26 over Pd/C in THF at room temperature
gave the corresponding carboxylic acids 22 and 27 in
quantitative yield, respectively. Formylation of 19 with
ethyl formate in the presence of sodium methoxide in
CH2Cl2 gave compound 30 (80%). Treatment of 30 with
hydroxylamine hydrochloride in aqueous EtOH at
reflux temperature afforded isoxazole 31 (95%). Hydrog-
enolysis of 31 over Pd/C in EtOAc at room temperature
was a complex reaction to yield the desired isoxazole
carboxylic acid 32 in poor yield (12%).
The synthesized OA derivatives were evaluated in the
enzyme inhibition assay against rabbit muscle glycogen
phosphorylase a which shared considerable sequence
similarity with human liver GPa. As described previous-
ly,12 the activity of rabbit muscle GPa was measured
through detecting the release of phosphate from glu-
cose-1-phosphate in the direction of glycogen synthesis.
The assay results (Table 2) showed that many synthe-
sized OA derivatives exhibited moderate inhibitory
activity against rabbit muscle GPa.
Unlike maslinic acid derivatives which had a clear pref-
erence for hydrophobic groups at C-28 for GP inhibi-
tion,6b the OA derivatives showed some uncertainty in

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J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2915–2919
Table 2. Inhibition of rabbit muscle GPa by compounds 1–32
In summary, we have identified oleanolic acid and its
synthetic derivatives as novel GPa inhibitors. As a pos-
sible result of this finding, oleanolic acid, a nonprescrip-
tion anti-hepatitis drug, may find its new clinical uses in
treating fasting hyperglycemia and other diseases caused
by abnormalities in glycogen metabolism.13 Lead opti-
mization based on oleanolic acid resulted in a series of
triterpene class of GP inhibitors, among which, 4 was
the most potent GPa inhibitor (IC50 = 3.3 lM). Further
research and drug development on pentacyclic triterpene
compounds as promising GPa inhibitors are ongoing in
our laboratory and the results will be reported in due
course.
a
Compound
RMGPa IC50 (lM)
1
NIb
34.3
NI
2
3
4
3.3
5
16.9
NI
6
7
NI
NI
8
9
62.6
76.7
NI
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
OA
Caffeine
NI
NI
53.2
NI
Acknowledgments
17.9
225
461
66.6
20.8
46.8
26.1
22.3
1002
11.2
179.1
28.2
61.3
8.0
This work was supported by program for New Century
Excellent Talents in University (NCET), Jiangsu Natu-
ral Science Foundation (Grant BK2005101), Jiangsu
Outstanding Researcher Grant, and Chinese University
Ph.D. Program Foundation (No. Y051018).
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6.3
19.6
12.7
14
114
a Values are means of three experiments.
b NI, no inhibition.
terms of inhibitory potency correlated with the hydro-
phobic properties of C-28 substituents (e.g., 8 vs 9 and
10 vs 11). Introduction of aminoethyl esters at C-28 in
order to improve aqueous solubility resulted in decreas-
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boxylic acids were more potent than their benzyl esters
(e.g., 21 vs 22, 24 vs 25, 26 vs 27, and 31 vs 32), indicat-
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C-28.
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analogues 4 (IC50 = 3.3 lM) and 5 (IC50 = 16.9 lM)
were potent GPa inhibitors, while acetate 1 and butyrate
3 were inactive. Compound 4 was 3-fold more potent
than OA, indicating that A-ring of OA might be a good
target for further lead optimization. 2-Oximino series of
compounds (21–23 and 26–28) were less potent than
OA, except for 29 (IC50 = 8 lM). Oxadiazole 25
(IC50 = 11.2 lM) and isoxazole 32 (IC50 = 12.7 lM)
exhibited similar enzyme inhibitory potency with OA,
indicating that A-ring fused heterocyclic analogues
might deserve some attention for further inhibitor
design. Conversion of OA to oleanonic acid (16,
IC50 = 17.9 lM) did not result in a significant change
in potency.
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11. Analytical data for compound 4: 1H NMR (CDCl3,
300 MHz): d 0.78 (3H, s), 0.91 (6H, s), 0.93 (6H, s), 0.97
(3H, s), 1.15 (3H, s), 2.81–2.85 (1H, m), 4.64 (1H, t,
J = 8.0 Hz), 5.29 (1H, br s), 6.41 (1H, d, J = 16.0 Hz), 7.35
(2H, d, J = 8.5 Hz), 7.46 (2H, d, J = 8.6 Hz), 7.61 (1H, d,
J = 16.0 Hz); 13CNMR (CDCl3, 300 MHz): 15.4, 16.8,
17.2, 18.2, 23.0, 23.4, 23.6, 25.9, 27.7, 28.1, 30.7, 32.6, 32.8,
33.1, 33.8, 37.0, 38.0, 38.1, 39.3, 41.0, 41.6, 45.9, 46.5, 47.6,
55.4, 81.2, 119.5, 122.6, 129.1, 129.2, 133.1, 136.1, 142.9,
143.6, 166.6, 182.8; ESIMS: 619 [MÀH]À. Analytical data
for compound 25: IR (KBr, cmÀ1) 2947, 2869, 1693, 1460,
1386, 1002, 881; 1HNMR (CDCl3, 300 MHz): d 0.83, 0.84,
0.92, 0.94, 1.18, 1.31, 1.41 (each 3H, s), 2.17 (1H, d,
J = 16.3 Hz, Ha-1), 3.10 (1H, d, J = 16.2 Hz, Hb-1), 2.87
(1H, dd, J = 3.9, 13.6 Hz, H-18), 5.36 (1H, t, J = 3.5 Hz,
H-12); 13CNMR (CDCl3, 300 MHz): 15.4, 16.6, 19.1, 22.9,
23.3, 23.5, 24.7, 25.7, 27.7, 30.7, 31.3, 31.8, 32.4, 33.0, 33.2,
33.8, 35.3, 38.5, 39.4, 41.1, 41.9, 45.8, 45.9, 46.6, 53.1,
122.1, 143.7, 150.5,159.7,183.3; ESIMS: 481 [M+H]+, 503
[M+Na]+.
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