Synthesis and biological evaluation of asiatic acid derivatives as inhibitors of glycogen phosphorylases

Article abstract of DOI:10.1002/cbdv.200800092

Twenty-four asiatic acid derivatives have been synthesized and biologically evaluated as inhibitors of glycogen phosphorylase (GP). Within this series of compounds, asiatic acid benzyl ester (23; IC₅₀=3.8 μm) exhibited more potent activity than

Full text of DOI:10.1002/cbdv.200800092

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Synthesis and Biological Evaluation of Asiatic Acid Derivatives as Inhibitors
of Glycogen Phosphorylases
by Liying Zhanga), Jun Chena), Yanchun Gonga), Jun Liub), Luyong Zhanga), Weiyi Huaa), and
Hongbin Sun*a)
a) Center for Drug Discovery, School of Pharmacy, China Pharmaceutical University, 24 Tongjia Xiang,
Nanjing 210009, P. R. China (phone: þ86-25-85327950; fax: þ86-25-83271335;
e-mail: hbsun2000@yahoo.com)
b) Jiangsu Center for Drug Screening, China Pharmaceutical University, 1 Shennong Road,
Nanjing 210038, P. R. China
Twenty-four asiatic acid derivatives have been synthesized and biologically evaluated as inhibitors of
glycogen phosphorylase (GP). Within this series of compounds, asiatic acid benzyl ester (23; IC50
¼
3.8 mm) exhibited more potent activity than its parent compound 1 (IC50 ¼17 mm). SAR Analysis showed
that asiatic acid (1) possessing a 2a-OH function exhibited more potent GP inhibitory activity than
eriantic acid B (27) which possesses a 2b-OH function. Further lead optimization based on 1 is needed to
find more effective asiatic acid derivatives as antidiabetic agents with protective effects against ischemic
diabetic complications.
Introduction. – Asiatic acid (1) is a member of the ursane family of pentacyclic
triterpenoids isolated from Centella asiatica, which is extensively used as a medicinal
herb in many countries [1]. Compound 1 was reported to possess a wide spectrum of
biological activities including antioxidation, anti-inflammation, antitumor, antidepres-
sion, anti-Alzheimerꢀs disease, cardiovascular protection, and hepatoprotective effect
[2–8]. Plant extracts containing 1 and related saponins have been in clinical uses for
decades mainly for treating injured skin (wound healing) and chronic ulcer
deformation of skin [9].
In previous communications [10–13], we first reported that pentacyclic triterpenes
such as maslinic acid (MA) and corosolic acid (CA) (Fig.) represented a new class of
inhibitors of glycogen phosphorylases (GP), and their glucose-lowering activity might
Figure. Structures of asiatic acid (1), maslinic acid, and corosolic acid
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢂrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
865
be, at least in part, due to modulation of hepatic and peripheral glycogen metabolism.
GP Inhibition has been regarded as a therapeutic approach to treating diabetes
[14][15], and various studies have shown the efficacy of GP inhibitors at lowering
blood glucose in animal models of diabetes [16] and in clinical trials [17].
Based on our previous studies, we tested the GP inhibition activity of 1 which is a
close analog of MA and CA, and, not surprisingly, the result showed that 1 was a
moderate GP inhibitor (IC50 ¼17 mm) [18]. To elucidate the mechanism of GP
inhibition by pentacyclic triterpenes, we have determined the X-ray crystal structure of
1 binding to GPb, which demonstrates that 1 binds at the allosteric activator site, where
the physiological activator AMP binds [18]. In this study, lead optimization based on 1
was carried out in order to find more potent triterpene GP inhibitors with good
pharmacokinetic properties. In this regard, amino acid derivatives of 1 were
synthesized in order to improve water solubility of 1. On the other hand, a series of
C(28) ester derivatives of 1 were synthesized in order to further explore structure–
activity relationships (SAR) at C(28) of 1. Moreover, eriantic acid B (27) [19], the
naturally occurring C(2)-epimer of 1, was synthesized to check the effect of 2-OH
configuration on GP inhibition.
Results and Discussion. – 1. Synthesis. As shown in Schemes 1 and 2, asiatic amide 5
and a series of amino acid derivatives of 1 were synthesized. Full acetylation of 1
afforded 2a,3b,23-O-triacetylasiatic acid (2) in good yield. Treatment of 2 with SOCl2
afforded the corresponding acyl chloride 3, which was used for the next reactions
Scheme 1
i) Ac2O/Pyr; 78%. ii) SOCl2. iii) NH3 ·H2O, THF; 83% over 2 steps. iv) 4n NaOH, MeOH/THF; 86%.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 2
i) RCH(NH2)COOMe·HCl, 4-(dimethylamino)pyridine (DMAP)/CH2Cl2; 52–87%. ii) 4n NaOH,
MeOH/THF; 68–89%.
without further purification. Reaction of 3 with concentrated NH3 solution at 08
furnished amide 4, which was hydrolyzed with aqueous NaOH to give asiatic amide 5
(Scheme 1). The amino acid derivatives 6–15 were readily prepared as described
above, using corresponding amino acid methyl esters as amine reactants (Scheme 2).
A series of C(28) ester derivatives of 1 were synthesized as depicted in Scheme 3.
Alkylation of 1 with 1,2-dibromoethane in the presence of K2CO3 in DMF gave 2-
bromoethyl ester 16 in 82% yield. Reaction of 16 with piperidine or morpholine gave 2-
(amino)ethyl esters 17 or 18, respectively. Similar to the preparation of 16, esters 19, 20,
and 21 were prepared in high yields. Ester 20 was further converted to the free acid 22
(59%) by saponification with 4n NaOH aqueous solution without affecting the C(28)
ester group.
It was noticed that the configurations of the OH groups at C(2) and C(3) had an
impact on GP inhibitory potency of pentacyclic triterpenes [13]. In the light of this
observation, it should be interesting to see how a 2b-OH function would affect the
potency in contrast to 1 with a 2a-OH function. In this regard, eriantic acid B (27),
possessing a 2b-OH function, was synthesized (Scheme 4). Reaction of 1 with BnCl
(Bn¼PhCH2) in the presence of K2CO3 in DMF at 608 gave benzyl ester 23 in high
yield. Treatment of 23 with 2,2-dimethoxypropane in the presence of TsOH afforded
24. Oxidation of 24 with pyridinium chlorochromate (PCC) in CH2Cl2 at room
temperature afforded ketone 25, which was used for the next reaction without further
purification. Reduction of 25 with NaBH4 in THF at 08, followed by acidic
deprotection, gave eriantic acid B benzyl ester 26 in 75% yield. Hydrogenolysis of 26
over Pd/C in THF furnished 27 in 92% yield. The spectroscopic data of 27 were
identical with those reported in [19].

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
867
Scheme 3
i) BrCH2CH2Br, K2CO3, DMF, 408; 82%. ii) Piperidine or morpholine, K2CO3, acetone, reflux; 72–77%.
iii) R2Br, K2CO3, DMF, r.t.; 82–92%. iv) 4n NaOH, MeOH/THF; 59%.
2. Biological Activity. The synthesized asiatic acid derivatives were evaluated in the
enzyme-inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa).
The activity of RMGPa was measured through detecting the release of phosphate from
glucose-1-phosphate in the direction of glycogen synthesis [16]. Caffeine was routinely
used as a positive control in GP assay in our studies and others [10–13][16][18].
Although caffeine is not a very potent GP inhibitor, it is a fairly convincing positive
control. More importantly, caffeine is a known allosteric GP inhibitor, and thus it shares
the same binding site with the triterpene compounds, since our X-ray crystal-structure
study shows that 1 binds at the allosteric site of GP [18].
The bioassay results are summarized in the Table. Some of the tested triterpene
compounds exhibited good-to-moderate inhibitory activity against GPa. Within this
series of compounds, 23 (IC50 ¼3.8 mm) was the most potent (more potent than its
parent compound 1 (IC50 ¼17 mm)). Incorporation of amino acid moiety at C(28) of 1
resulted in loss of potency (e.g., 11–15). Even though benzyl ester 23 exhibited strong
GP inhibition activity, data analysis indicated no clear SAR for structural modifications
at C(28). Interestingly, 2b-OH function of 27 (2-epiasiatic acid; IC50 ¼92.8 mm) resulted
in a significant decrease in potency compared with 2a-OH function of 1 (IC50 ¼17 mm).
Introduction of hydrophilic groups into the side chain at C(28) was carried out in order

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 4
i) K2CO3, BnCl, DMF (95%). ii) 2,2-Dimethoxypropane/TsOH (86%). iii) Pyridinium chlorochromate
(PCC), CH2Cl2. iv) 1. NaBH4, THF; 2. 20% HCl (44%, over 2 steps). v) H2, 10% Pd/C, THF, r.t. (92%).
to improve H2O solubility of 1; however, the resulting compounds exhibited a markedly
decreased activity (i.e., 17) or no activity (i.e., 11–15, 18).
Conclusions. – In this study, 24 asiatic acid derivatives have been synthesized and
biologically evaluated as inhibitors of glycogen phosphorylase. Within this series of
compounds, asiatic acid benzyl ester (23; IC50 ¼3.8 mm) exhibited more potent activity
than its parent compound 1. SAR Analysis showed that asiatic acid (1) possessing a 2a-
OH function exhibited more potent GP inhibitory activity than eriantic acid B (27)
which possesses a 2b-OH function. Extensive lead optimization based on 1, aiming at
improvement of potency and pharmacokinetic properties, is ongoing in our laboratory,
and more results will be reported in due time.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
869
Table. Rabbit Muscle GPa Inhibition-Assay Results for Asiatic Acid Derivatives
Compound
IC50a) [mm]
Compound
IC50 a) [mm]
Compound
IC50 a) [mm]
1
2
3
4
5
6
7
8
9
17ꢁ1.2
33ꢁ2.9
n.d.c)
11
12
13
14
15
16
17
18
19
20
n.i.b)
n.i.
n.i.
n.i.
n.i.
21
22
23
24
25
26
27
n.i.
116ꢁ12.2
3.8ꢁ0.9
n.i.
36ꢁ3.6
n.i.
n.d.
101ꢁ12.2
112ꢁ17.3
n.i.
n.d.
55ꢁ3.2
93ꢁ7.1
114ꢁ14.5
132ꢁ15.3
173ꢁ27.4
n.i.
Caffeined)
104ꢁ11.6
10
n.i.
n.i.
a) Each value represents the meanꢁS.D. of three determinations. b) n.i.: No inhibition. c) n.d.: Not
determined. d) Positive control.
This program was financially supported by the National Natural Science Foundation of China (grants
30672523 and 90713037), research grants from the Chinese Ministry of Education (grants 706030 and
20050316008) and the program for New Century Excellent Talents in University (NCET-05-0495).
Experimental Part
General. Chemicals: Shanghai Chemical Reagent Company. Column chromatography (CC): silica gel
60 (200–300 mesh, Qingdao Ocean Chemical Company, China). TLC: 60 F254 silica-gel plates (250 mm,
Qingdao Ocean Chemical Company, China). M.p.: RY-1 melting-point tester; in cap. tube; uncorrected.
.
IR Spectra: Shimadzu FTIR-8400S spectrometer; ˜n in cmꢀ1 1H- and 13C-NMR spectra: ACF* 300Q
Bruker, CDCl3 unless otherwise indicated; d in ppm, J in Hz. LR-MS: Hewlett-Packard 1100 LC/MSD
spectrometer.
2a,3b,23-Triacetoxyurs-12-en-28-oic acid (2). To a soln. of asiatic acid (1; 0.20 g, 0.40 mmol) in 10 ml
of anh. pyridine, Ac2O (76 mg, 0.74 mmol) was slowly added while cooling and stirring. The mixture was
then stirred overnight at r.t. After the addition of AcOEt (20 ml), the mixture was worked up with 1n
HCl, sat. NaHCO3, and brine in sequence, dried (Na2SO4), and concentrated under reduced pressure.
The crude product was purified by CC (petroleum ether (PE)/AcOEt 10 :1) to give 2 (170 mg, 78%).
White solid. M.p. 93–958. IR (KBr): 3462, 2950, 1745, 1234. 1H-NMR (300 MHz): 0.78 (s, 3 H); 0.85 (d,
J¼6.4, 3 H; 0.88 (s, 3 H); 0.94 (d, J¼5.9, 3 H); 1.07, 1.11 (2s, each 3 H); 1.98, 2.02, 2.08 (3s, each 3 H);
2.19 (d, J ¼ 11.3, 1 H); 3.58 (d, J ¼ 11.8, 1 H); 3.85 (d, J ¼ 11.8, 1 H); 5.07 (d, J ¼ 10.3, 1 H); 5.12–5.17
(m, 1 H); 5.25 (br. s, 1 H). 13C-NMR (75 MHz): 182.9; 170.9; 170.5; 170.4; 138.0; 125.3; 77.4; 77.0; 76.6;
74.8; 69.9; 65.3; 52.5; 47.9; 47.6; 47.5; 43.7; 42.0; 41.9; 39.5; 39.0; 38.8; 37.8; 36.6; 32.4; 30,6; 27.9; 24.0; 23.4;
23.3; 21.2; 21.1; 20.9; 20.8; 17.9; 17.0; 17.0; 16.9; 13.9. ESI-MS: 637 ([MþNa]þ ).
2a,3b,23-Triacetoxyurs-12-en-28-amide (4). A mixture of 2 (60 mg, 0.098 mmol) and SOCl2 (2 ml)
was refluxed for 2 h, and excess reagent was removed under vacuum to give acyl chloride 3. Without
purification, a soln. of 3 in THF (10 ml) was added dropwise to a stirred soln. of conc. NH3 (5 ml) at 08,
and the mixture was stirred overnight. The mixture was concentrated under vacuum to remove the
solvent, and AcOEt was added. The org. layer was washed with 1n HCl, sat. NaHCO3, and brine in
sequence, dried (Na2SO4), and concentrated under reduced pressure. The crude product was purified by
CC (PE/AcOEt 3 :1) to afford 4 (49 mg, 83% for two steps). Pale yellow solid. M.p. 142–1468. IR (KBr):
2924, 1738, 1667, 1236. 1H-NMR (300 MHz): 0.86, 0.88, 0.89, 0.96 (4s, each 3 H); 1.11 (s, 6 H); 1.98, 2.02,
2.08 (3s, each 3 H); 3.58 (d, J ¼ 11.8, 1 H); 3.85 (d, J ¼ 11.8, 1 H); 5.08 (d, J ¼ 10.3, 1 H); 5.12–5.20 (m,
1 H); 5.32 (t, J ¼ 3.5, 1 H); 5.84 (br. s, 1 H). 13C-NMR (75 MHz): 180.8; 170.7; 170.4; 170.2; 140.0; 125.2;
74.9; 69.9; 65.4; 54.3; 47.8; 47.7; 43.9; 42.6; 42.0; 39.8; 39.6; 39.1; 37.9; 37.2; 32.4; 30.9; 29.7; 27.9; 24.9; 23.5;
23.2; 21.2; 21.0; 20.8; 20.7; 18.0; 17.2; 17.1; 13.9. ESI-MS: 636.5 ([MþNa]þ ).

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2a,3b,23-Trihydroxyurs-12-en-28-amide (5). To a soln. of 4 (100 mg, 0.16 mmol) in MeOH (2 ml) and
THF (3 ml), 4n NaOH (0.6 ml) was added dropwise, and the resulting mixture was stirred at r.t. for 2 h.
The mixture was acidified with aq. HCl and extracted with AcOEt. The org. layer was washed with sat.
NaHCO3 and brine in sequence, dried (Na2SO4), and concentrated under reduced pressure. The crude
product was purified by CC (CH2Cl2/MeOH 40 :1) to give 5 (68 mg, 86%). White solid. M.p. 248–2528.
IR (KBr): 3402, 2923, 1654, 1048. 1H-NMR (300 MHz, CD3OD): 0.69, 0.87 (2s, each 3 H); 0.90 (d, J¼6.4,
3 H); 0.97, 1.04, 1.14 (3s, each 3 H); 2.08 (d, J ¼ 11.3, 1 H); 3.25 (d, J ¼ 11.1, 1 H); 3.34 (d, J ¼ 9.6, 1 H);
3.49 (d, J ¼ 11.2, 1 H); 3.65–3.73 (m, 1 H); 5.33 (t, J ¼ 3.3, 1 H). 13C-NMR (75 MHz, CD3OD): 183.6;
140.2; 127.0; 78.5; 69.7; 66.8; 54.6; 49.8; 48.1; 48.0; 44.1; 43.5; 40.9; 40.8; 40.3; 39.0; 38.7; 33.6; 31.9; 29.0;
25.3; 24.5; 24.1; 21.5; 19.1; 17.9; 17.7; 13.8. ESI-MS: 488.4 ([MþH]þ ).
N-(2a,3b,23-Acetoxyurs-12-en-28-oyl)glycine Methyl Ester (6). A mixture of 2 (0.25 g, 0.40 mmol)
and SOCl2 (5 ml) was refluxed for 2 h, and the excess reagent was removed under vacuum to give 3 which
was re-dissolved in 10 ml of CH2Cl2. The soln. thus prepared was added dropwise to a mixture of glycine
methyl ester hydrochloride (0.15 g, 1.20 mmol) and DMAP (0.03 g, 0.22 mmol) in CH2Cl2 (10 ml) at 08.
The mixture was then stirred overnight at r.t., and, after washing with 1n HCl, sat. NaHCO3, and brine in
sequence, dried (Na2SO4), and concentrated under reduced pressure. The crude product was purified by
CC (PE/AcOEt 4 :1) to give 6 (244 mg, 87%). White solid. M.p. 175–1788. IR (KBr): 2924, 2870, 1744,
1
1369, 1235. H-NMR (300 MHz): 0.73, 0.87, 0.89, 0.96 (4s, each 3 H); 1.10 (s, 6 H); 1.98, 2.02, 2.08 (3s,
each 3 H); 3.58 (d, J ¼ 11.8, 1 H); 3.76 (s, 3 H); 3.81–3.88 (m, 2 H); 4.05–4.13 (m, 1 H); 5.06–5.21 (m,
2 H); 5.41 (t, J ¼ 3.4, 1 H); 6.45 (t, J ¼ 4.2, 1 H). 13C-NMR (75 MHz): 177.9; 170.7; 170.6; 170.3; 170.2;
139.3; 125.8; 75.0; 69.9; 65.4; 53.8; 52.2; 47.9; 47.8; 47.7; 44.0; 42.5; 42.1; 41.6; 39.8; 39.1; 37.9; 37.1; 32.4;
30.9; 27.9; 24.9; 23.6; 23.3; 21.2; 21.0; 20.7; 18.0; 17.1; 17.0; 16.6; 13.9. ESI-MS: 686.4 ([MþH]þ ).
N-(2a,3b,23-Acetoxyurs-12-en-28-oyl)-l-alanine Methyl Ester (7). As described for the preparation
of 6, treatment of 2 (0.25 g, 0.40 mmol) with l-alanine methyl ester hydrochloride (0.17 g, 1.21 mmol)
afforded 0.15 g (54%) of 7. Pale yellow solid. M.p. 220–2228. IR (KBr): 2924, 1743, 1370, 1235. 1H-NMR
(300 MHz): 0.70 (s, 3 H); 0.88 (s, 6 H); 0.96 (s, 3 H); 1.09 (s, 9 H); 1.98, 2.02, 2.08 (3s, each 3 H); 3.57 (d,
J ¼ 11.8, 1 H); 3.75 (s, 3 H); 3.84 (d, J ¼ 11.8, 1 H); 4.45–4.49 (m, 1 H); 5.07 (d, J ¼ 10.3, 1 H); 5.12–5.17
(m, 1 H); 5.40 (br. s, 1 H); 6.57 (d, J ¼ 5.9, 1 H). 13C-NMR (75 MHz): 177.2; 173.7; 170.7; 170.3; 170.2;
138.7; 126.0; 75.0; 70.0; 65.4; 53.8; 52.3; 48.2; 47.8; 47.7; 44.0; 42.4; 42.1; 39.8; 39.7; 39.1; 37.9; 37.4; 32.5;
30.9; 29.7; 27.9; 24.7; 23.5; 23.3; 21.1; 21.0; 20.7; 20.7; 18.7; 18.0; 17.1; 16.7; 13.9. ESI-MS: 700.5 ([M þ H]þ ).
N-(2a,3b,23-Acetoxyurs-12-en-28-oyl)-l-valine Methyl Ester (8). As described for the preparation of
6, treatment of 2 (0.25 g, 0.40 mmol) with l-valine methyl ester hydrochloride (0.21 g, 1.25 mmol)
1
afforded 0.15 g (52%) of 8. White solid. M.p. 205–2088. IR (KBr): 2953, 1744, 1370, 1232. H-NMR
(300 MHz): 0.68 (s, 3 H); 0.88–0.97 (m, 15 H); 1.09 (s, 6 H); 1.98, 2.02, 2.08 (3s, each 3 H); 3.57 (d, J ¼
11.8, 1 H); 3.70 (s, 3 H); 3.84 (d, J ¼ 11.8, 1 H); 4.42–4.46 (m, 1 H); 5.07 (d, J ¼ 10.3, 1 H); 5.11–5.19 (m,
1 H); 5.38 (br. s, 1 H); 6.36 (d, J ¼ 7.4, 1 H). 13C-NMR (75 MHz): 177.4; 172.5; 170.6; 170.3; 170.2; 138.6;
126.0; 75.0; 70.0; 65.5; 57.2; 54.1; 51.8; 48.1; 47.8; 44.0; 42.5; 42.1; 39.8; 39.8; 39.2; 37.9; 37.8; 32.6; 32.0;
30.9; 27.9; 24.7; 23.5; 23.3; 21.1; 21.0; 20.7; 18.7; 18.3; 18.0; 17.1; 17.0; 16.8; 13.9. ESI-MS: 728.5 ([M þ H]þ ).
N-(2a,3b,23-Acetoxyurs-12-en-28-oyl)-l-phenylalanine Methyl Ester (9). As described for the
preparation of 6, treatment of 2 (0.25 g, 0.40 mmol) with l-phenylalanine methyl ester hydrochloride
(0.26 g, 1.21 mmol) afforded 0.18 g (56%) of 9. White solid. M.p. 120–1248. IR (KBr): 3418, 2952, 1747,
1237. 1H-NMR (300 MHz): 0.60, 0.83, 0.88, 0.94, 1.05, 1.06 (6s, each 3 H); 1.97, 2.02, 2.08 (3s, each 3 H);
3.00–3.17 (m, 2 H); 3.56 (d, J ¼ 11.7, 1 H); 3.68 (s, 3 H); 3.84 (d, J ¼ 12.0, 1 H); 4.71 (dd, J ¼ 6.0, 12.0,
1 H); 5.06 (d, J ¼ 10.2, 1 H); 5.10–5.19 (m, 1 H); 5.25 (t, J ¼ 3.0, 1 H); 6.34 (d, J ¼ 6.0, 1 H); 7.08–7.31
(m, 5 H). 13C-NMR (75 MHz): 177.3; 172.2; 170.7; 170.3; 170.2; 138.5; 136.3; 129.4; 128.4; 127.0; 126.0;
75.0; 70.0; 65.4; 53.7; 53.6; 52.0; 47.8; 47.7; 47.7; 43.9; 42.4; 42.0; 39.7; 39.7; 39.1; 38.3; 37.9; 37.3; 32.4; 30.9;
29.7; 27.8; 24.8; 23.4; 23.3; 21.1; 21.0; 20.7; 17.9; 17.1; 16.6; 13.9. ESI-MS: 776.4 ([M þ H]þ ).
N-(2a,3b,23-Acetoxyurs-12-en-28-oyl)-l-serine Methyl Ester (10). As described for the preparation
of 6, treatment of 2 (0.25 g, 0.40 mmol) with l-serine methyl ester hydrochloride (0.14 g, 1.20 mmol)
1
afforded 0.19 g (68%) of 10. White solid. M.p. 217–2198. IR (KBr): 2924, 1744, 1369, 1235. H-NMR
(300 MHz): 0.70, 0.88, 0.88, 0.97, 1.09, 1.10 (6s, each 3 H); 1.98, 2.02, 2.08 (3s, each 3 H); 2.26 (d, J ¼ 17.1,
1 H); 3.57 (d, J ¼ 11.8, 1 H); 3.79 (s, 3 H); 3.82–3.86 (m, 2 H); 3.94–3.99 (m, 1 H); 4.52–4.53 (m, 1 H);
5.07 (d, J ¼ 10.3, 1 H); 5.12–5.16 (m, 1 H); 5.43 (t, J ¼ 3.0, 1 H); 6.85 (d, J ¼ 5.1, 1 H). 13C-NMR

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
871
(75 MHz): 179.1; 170.9; 170.7; 170.4; 170.3; 138.5; 126.2; 74.9; 69.9; 65.4; 64.4; 55.7; 53.8; 52.7; 48.0; 47.7;
47.7; 43.9; 42.4; 42.0; 39.8; 39.7; 39.0; 37.9; 37.6; 32.4; 30.9; 29.7; 27.9; 24.7; 23.5; 23.3; 21.1; 21.0; 20.8; 20.7;
17.9; 17.1; 16.6; 13.9. ESI-MS: 716.5 ([MþH]þ ).
N-(2a,3b,23-Hydroxyurs-12-en-28-oyl)glycine (11). As described for the preparation of 5, 11 was
obtained in 89% yield. White solid. M.p. 2698 (dec.). IR (KBr): 3403, 2924, 1633, 1048. 1H-NMR
(300 MHz, (D6)DMSOþD2O): 0.55, 0.65 (2s, each 3 H); 0.84 (d, J¼6.2, 3 H); 0.92 (s, 6 H); 1.04 (s, 3 H);
2.09 (d, J ¼ 10.7, 1 H); 3.06 (d, J ¼ 10.6, 1 H); 3.18 (d, J ¼ 9.5, 1 H); 3.30 (d, J ¼ 10.9, 1 H); 3.49–3.53 (m,
1 H); 3.59 (d, J ¼ 17.3, 1 H); 3.74 (d, J ¼ 17.5, 1 H); 5.24 (br. s, 1 H). 13C-NMR (75 MHz, (D6)DMSO):
176.4; 171.2; 138.3; 124.9; 75.9; 67.4; 64.3; 52.2; 47.1; 47.0; 46.6; 46.3; 42.5; 41.7; 41.1; 40.5; 38.8; 38.5; 37.3;
36.7; 32.2; 30.4; 27.3; 23.8; 23.3; 23.0; 21.1; 17.5; 17.1; 16.9; 16.6; 13.6. ESI-MS: 544.3 ([MꢀH]ꢀ ).
N-(2a,3b,23-Hydroxyurs-12-en-28-oyl)-l-alanine (12). As described for the preparation of 5, 12 was
obtained in 79% yield. White solid. M.p. 226–2308. IR (KBr): 3402, 2926, 1636, 1048. 1H-NMR
(300 MHz, (D6)DMSOþD2O): 0.51, 0.62 (2s, each 3 H); 0.80 (d, J¼5.3, 3 H); 0.88 (s, 6 H); 1.00 (s 3 H);
1.19 (d, J¼5.7, 3 H); 2.04 (d, J ¼ 10.5, 1 H); 3.02 (d, J ¼ 10.5, 1 H); 3.13 (d, J ¼ 9.1, 1 H); 3.26 (d, J ¼ 10.7,
1 H); 3.48 (t, J ¼10.5, 1 H); 4.06 (d, J ¼ 6.3, 1 H); 5.20 (br. s, 1 H). 13C-NMR (75 MHz, (D6)DMSO):
175.6; 174.0; 137.9; 125.1; 75.7; 67.3; 64.2; 52.3; 47.6; 47.0; 46.8; 46.4; 46.1; 42.3; 41.6; 40.3; 40.1; 38.9; 38.6;
38.3; 37.1; 36.6; 32.1; 30.2; 27.2; 23.5; 23.1; 22.9; 20.9; 17.6; 17.3; 16.9; 16.7; 16.5; 13.5. ESI-MS: 558.3 ([Mꢀ
H]ꢀ ).
N-(2a,3b,23-Hydroxyurs-12-en-28-oyl)-l-valine (13). As described for the preparation of 5, 13 was
obtained in 85% yield. White solid. M.p. 214–2188. IR (KBr): 3407, 2928, 1637, 1048. 1H-NMR
(300 MHz, (D6)DMSOþD2O): 0.54, 0.63 (2s, each 3 H); 0.83–0.91 (m, 15 H), 1.04 (s, 3 H); 2.09 (d, J ¼
10.5, 1 H); 3.03 (d, J ¼ 10.5, 1 H); 3.16 (d, J ¼ 9.3, 1 H); 3.29 (d, J ¼ 10.5, 2 H); 4.00 (t, J ¼ 6.6, 1 H); 5.20
(br. s, 1 H); 6.90 (d, J ¼ 7.5, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 176.3; 172.7; 137.8; 125.4; 75.7; 67.4;
64.1; 57.3; 52.6; 47.1; 47.0; 46.9; 46.2; 42.4; 41.8; 40.3; 38.8; 38.7; 38.4; 37.3; 37.0; 32.3; 30.5; 30.4; 27.3; 23.7;
23.2; 23.0; 20.9; 18.9; 18.5; 17.4; 17.0; 16.9; 16.7; 13.6. ESI-MS: 588.3 ([M þ H]þ ).
N-(2a,3b,23-Hydroxyurs-12-en-28-oyl)-l-phenylalanine (14). As described for the preparation of 5,
14 was obtained in 68% yield. White solid. M.p. 210–2148. IR (KBr): 3405, 2926, 1632, 1049. 1H-NMR
(300 MHz, (D6)DMSOþD2O): 0.52, 0.76, 0.78, 0.83, 0.87, 0.94 (6s, each 3 H); 2.88–2.91 (m, 1 H); 2.92–
2.96 (m, 2 H); 3.13 (d, J ¼ 9.3, 1 H); 3.27 (d, J ¼ 10.2, 1 H); 4.16 (br. s, 1 H); 5.01 (t, J ¼ 3.6, 1 H); 7.20 (s,
5 H). 13C-NMR (75 MHz, (D6)DMSO): 176.3; 173.0; 138.0; 129.4; 128.0; 126.2; 125.1; 75.8; 67.4; 64.3;
54.0; 52.5; 47.1; 46.9; 46.7; 46.3; 42.5; 41.7; 40.3; 40.1; 39.1; 38.9; 38.8; 38.7; 38.6; 37.3; 36.5; 32.0; 30.4; 27.3;
23.8; 23.3; 23.0; 21.1; 17.4; 17.1; 16.9; 16.2; 13.6. ESI-MS: 636.3 ([M þ H]þ ).
N-(2a,3b,23-Hydroxyurs-12-en-28-oyl)-l-serine (15). As described for the preparation of 5, 15 was
obtained in 82% yield. White solid. M.p. 224–2278. IR (KBr): 3403, 2925, 1631, 1047. 1H-NMR
(300 MHz, (D6)DMSOþD2O): 0.50, 0.60 (2s, each 3 H); 0.79 (d, J¼6.2, 3 H); 0.86, 0.87, 1.00 (3s, each
3 H); 2.99 (d, J ¼ 10.6, 1 H); 3.11 (d, J ¼ 9.3, 1 H); 3.24 (d, J ¼ 11.0, 1 H); 3.45–3.54 (m, 2 H); 4.10–4.11
(m, 1 H); 5.21 (br. s, 1 H); 6.96 (d, J ¼ 6.1, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 176.2; 172.1; 138.1;
125.4; 75.8; 67.6; 64.1; 61.7; 61.6; 54.7; 52.6; 47.2; 46.9; 46.2; 42.6; 41.9; 38.5; 37.4; 36.8; 32.3; 30.5; 27.5;
23.9; 23.4; 23.4; 23.1; 21.2; 17.5; 17.2; 17.0; 16.9; 16.6; 13.8. ESI-MS: 574.2 ([MꢀH]ꢀ ).
2-Bromoethyl 2a,3b,23-Trihydroxyurs-12-en-28-oate (16). According to the literature methods
[11][12], alkylation of 1 with 1,2-dibromoethane in the presence of K2CO3 in DMF gave 16 (82%). White
solid. M.p. 183–1858. IR (KBr): 3442, 2923, 1724, 1639. 1H-NMR (300 MHz): 0.77 (s, 3 H); 0.86 (d, J¼
6.4, 3 H); 0.90 (s, 3 H); 0.96 (d, J¼10.4, 3 H); 1.04, 1.09 (2s, each 3 H); 2.24 (d, J¼11.2, 1 H); 3.40–3.50
(m, 4 H); 3.69 (d, J¼10.4, 1 H); 3.73–3.79 (m, 1 H); 4.31 (t, J ¼ 6.0, 2 H); 5.28 (t, J ¼ 3.3, 1 H). ESI-MS:
595 ([M þ H]þ ).
2-(Piperidin-1-yl)ethyl 2a,3b,23-Trihydroxyurs-12-en-28-oate (17). According to the literature
methods [11][12], reaction of 16 with piperidine gave 17 (77%). M.p. 178–1808. IR (KBr): 3417, 2929,
1726, 1047. 1H-NMR (300 MHz): 0.75 (s, 3 H); 0.84 (d, J¼6.4, 3 H); 0.90 (s, 3 H); 0.95 (d, J¼4.7, 3 H);
1.04, 1.07 (2s, each 3 H); 2.22 (d, J¼11.2, 1 H); 2.47 (br. s, 4 H); 2.60 (t, J ¼ 5.9, 2 H); 3.42 (t, J ¼ 7.9, 2 H);
3.68 (d, J¼10.5, 1 H); 3.71–3.79 (m, 1 H); 4.14 (t, J ¼ 6.1, 2 H); 5.23 (t, J ¼ 3.4, 1 H). ESI-MS: 600
([M þ H]þ ).
2-(Morpholin-4-yl)ethyl 2a,3b,23-Trihydroxyurs-12-en-28-oate (18). According to the literature
methods [11][12], reaction of 16 with morpholine gave 18 (72%). M.p. 173–1758. IR (KBr): 3427, 2923,

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1724, 1116. 1H-NMR (300 MHz): 0.76 (s, 3 H); 0.85 (d, J¼6.4, 3 H); 0.90 (s, 3 H); 0.94 (d, J¼4.6, 3 H);
1.04, 1.08 (2s, each 3 H); 2.53 (br. s, 4 H); 2.63 (br. s, 2 H); 2.22 (d, J¼11.1, 1 H); 3.43 (m, 2 H); 3.60–3.80
(m, 6 H); 4.16 (br. s, 2 H); 5.24 (t, J ¼ 3.3, 1 H). ESI-MS: 602 ([M þ H]þ ).
Asiatic Acid Ethyl Ester (19). According to the literature methods [11][12], reaction of 1 with EtBr
gave 19 (82%). M.p. 182–1848. IR (KBr): 3444, 2925, 1724, 1035. 1H-NMR (300 MHz): 0.76 (s, 3 H); 0.85
(d, J¼6.4, 3 H); 0.90 (s, 3 H); 0.93 (d, J¼4.6, 3 H); 1.04, 1.08 (2s, each 3 H); 1.21 (t, J ¼ 7.1, 3 H); 2.23 (d,
J¼11.2, 1 H); 3.40–3.46 (m, 2 H); 3.68 (d, J¼10.4, 1 H); 3.73–3.80 (m, 1 H); 4.05 (q, J ¼ 7.1, 2 H); 5.25
(t, J ¼ 3.4, 1 H). ESI-MS: 539 ([M þ Na]þ ).
(Ethoxycarbonyl)methyl 2a,3b,23-Trihydroxyurs-12-en-28-oate (20). According to the literature
methods [11][12], reaction of 1 with BrCH2COOEt gave 20 (92%) as a white solid. M.p. 208–2108. IR
1
(KBr): 3444, 2925, 1762, 1737, 1047. H-NMR (300 MHz): 0.74 (s, 3 H); 0.86 (d, J¼6.4, 3 H); 0.91 (s,
3 H); 0.95 (d, J¼5.9, 3 H); 1.04, 1.09 (2s, each 3 H); 1.26 (t, J¼7.1, 3 H); 2.26 (d, J¼11.5, 1 H); 3.42 (m,
2 H); 3.69 (d, J¼10.4, 1 H); 3.75 (m, 1 H); 4.19 (q, J¼7.1, 2 H); 4.46 (d, J¼15.7, 1 H); 4.56 (d, J¼15.7,
1 H); 5.26 (t, J ¼ 3.3, 1 H). ESI-MS: 597 ([M þ Na]þ ).
(Benzyloxycarbonyl)methyl 2a,3b,23-Trihydroxyurs-12-en-28-oate (21). According to the literature
method [11][12], reaction of 1 with BrCH2COOBn gave 21 (89%). White solid. M.p. 215–2178. IR
1
(KBr): 3407, 2923, 1762, 1733, 1049. H-NMR (300 MHz): 0.72 (s, 3 H); 0.85 (d, J¼6.4, 3 H); 0.90 (s,
3 H); 0.94 (d, J¼5.0, 3 H); 1.03, 1.08 (2s, each 3 H); 2.25 (d, J¼11.1, 1 H); 3.42 (m, 2 H); 3.76 (m, 1 H);
3.69 (d, J¼10.5, 1 H); 4.52 (d, J¼15.8, 1 H); 4.63 (d, J¼15.8, 1 H); 5.17 (s, 2 H); 5.25 (t, J ¼ 3.5, 1 H);
7.30–7.39 (m, 5 H). ESI-MS: 659 ([M þ Na]þ ).
Carboxymethyl 2a,3b,23-Trihydroxyurs-12-en-28-oate (22). According to the literature method
[11][12], reaction of 20 with aq. NaOH gave 22 (59%). White solid. M.p. 252–2548. IR (KBr): 3414,
2932, 1611, 1047. 1H-NMR (300 MHz, CD3OD): 0.70, 0.78 (2s, each 3 H); 0.89 (d, J¼6.4, 3 H); 0.95, 1.04,
1.13 (3s, each 3 H); 2.31 (d, J ¼ 10.9, 1 H); 3.26 (d, J ¼ 11.0, 1 H); 3.35 (d, J ¼ 9.6, 1 H); 3.50 (d, J ¼ 11.0,
1 H); 3.65–3.74 (m, 1 H); 4.33 (dd, J ¼ 15.0, 34.0, 2 H); 5.25 (t, J ¼ 3.6, 1 H). 13C-NMR (75 MHz,
CD3OD): 178.3; 171.1; 139.4; 126.9; 78.3; 69.6; 66.6; 61.3; 54.2; 49.6; 48.4; 48.3; 48.0; 44.0; 43.3; 40.8; 40.3;
40.2; 38.9; 37.6; 33.6; 31.7; 29.0; 25.3; 24.4; 24.2; 21.6; 19.1; 17.8; 17.7; 17.6; 13.9. ESI-MS: 545.3 ([MꢀH]ꢀ ).
Asiatic Acid Benzyl Ester (23). To a mixture of asiatic acid (1; 1.00 g, 2.05 mmol) and K2CO3 (0.57 g,
5.4 mmol) in DMF (8 ml) was added BnCl (0.25 ml, 2.18 mmol). The mixture was stirred at 608 for 2 h
and then cooled, and ice H2O (20 ml) was added. The resulting solid was filtered, washed with H2O, and
dried to yield 23 (1.13 g, 95%). White solid. M.p. 189–1908. IR (KBr): 3418, 2925, 1723, 1049. 1H-NMR
(300 MHz): 0.63, 0.84, 0.85, 0.94, 1.00, 1.07 (6s, each 3 H); 2.26 (d, J ¼ 11.2, 1 H); 2.92 (br. s, 1 H); 3.38–
3.42 (m, 3 H); 3.62 (d, J ¼ 10.6, 1 H); 3.71–3.80 (m, 1 H); 4.97 (d, J ¼ 12.5, 1 H); 5.09 (d, J ¼ 12.5, 3 H);
5.23 (br. s, 1 H); 7.33 (s, 5 H). 13C-NMR (75 MHz): 177.2; 138.2; 136.4; 128.4; 128.1; 127.9; 125.4; 80.4;
77.4; 70.4; 68.8; 66.0; 52.9; 49.1; 48.1; 47.5; 46.3; 42.5; 42.1; 39.6; 39.1; 38.8; 38.1; 36.6; 32.7; 30.7; 27.9; 24.2;
23.6; 23.3; 21.1; 18.3; 17.1; 17.0; 17.0; 12.8. ESI-MS: 577.5 ([MꢀH]ꢀ ).
Benzyl 2a-Hydroxy-3b,23-(isopropylidenedioxy)urs-12-en-28-oate (24). To a mixture of 23 (1.00 g,
1.73 mmol) and TsOH (35 mg, 0.20 mmol) in anh. DMF (20 ml), 2,2-dimethoxypropane (0.7 ml,
5.71 mmol) was added by injection. The mixture was stirred at r.t. for 7 h. After neutralization with 5%
aq. NaOH to pH 7–8, the mixture was extracted with AcOEt and washed with brine, dried (Na2SO4),
and concentrated under reduced pressure. The crude product was purified by CC (PE/AcOEt 8 :1) to
give 24 (0.92 g, 86%). White solid. M.p. 88–918. IR (KBr): 3487, 2924, 1722, 1454. 1H-NMR (300 MHz):
0.61, 0.86, 0.93, 1.00, 1.06, 1.08 (6s, each 3 H); 1.44 (s, 6 H); 2.27 (d, J ¼ 11.3, 1 H); 3.28 (d, J ¼ 8.7, 1 H);
3.43 (d, J ¼ 10.6, 1 H); 3.49 (d, J ¼ 10.6, 1 H); 3.73–3.81 (m, 1 H); 4.97 (d, J ¼ 12.5, 1 H); 5.08 (d, J ¼
12.5, 1 H); 5.23 (t, J ¼ 3.1, 1 H); 7.33 (s, 5 H). 13C-NMR (75 MHz): 177.2; 138.0; 136.3; 128.4; 128.1; 127.9;
125.4; 99.5; 82.1; 76.6; 72.7; 65.9; 65.2; 52.8; 51.4; 48.0; 47.6; 46.5; 42.0; 39.6; 39.1; 38.8; 37.9; 36.9; 36.6;
32.4; 30.6; 29.7; 27.9; 24.2; 23.6; 23.1; 21.1; 19.4; 17.9; 17.5; 17.0; 16.9; 13.5. ESI-MS: 641.4 ([M þ Na]þ ).
Benzyl 2b,3b,23-Trihydroxyurs-12-en-28-oate (26). To a soln. of 24 (0.55 g, 0.89 mmol) in CH2Cl2
(7 ml) was added PCC (0.21 g) at 08, and the mixture was stirred at r.t. for 12 h. The mixture was then
filtered through Celite, and the filtrate was evaporated under vacuum to give ketone 25, which was used
for next reaction without further purification. Ketone 25 was dissolved in THF (15 ml), and NaBH4
(41 mg, 1.07 mmol) was added at 08. After stirring at r.t. for 7 h, 20% aq. HCl was added to the mixture,
which was then extracted with AcOEt. After washing with sat. NaHCO3 and brine, the org. layer was

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
873
dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by CC (PE/
AcOEt 3 :1) to give 26 (0.23 g, 44% for two steps). White solid. M.p. 117–1198. IR (KBr): 3395, 2923,
1
1725, 1107. H-NMR (300 MHz): 0.64 (s, 3 H); 0.83–0.88 (m, 6 H); 0.94 (s, 3 H); 1.06 (s, 3 H); 1.11 (s,
3 H); 3.41 (d, J ¼ 10.0, 1 H); 3.61 (d, J ¼ 3.9, 1 H); 3.71 (d, J ¼ 10.2, 1 H); 4.10–4.11 (m, 1 H); 4.97 (d,
J ¼ 12.4, 1 H); 5.10 (d, J ¼ 12.5, 1 H); 5.24 (t, J ¼ 3.0, 1 H); 7.34 (s, 5 H). 13C-NMR (75 MHz): 177.3;
138.1; 136.4; 128.4; 128.2; 128.0; 125.8; 76.2; 73.3; 70.8; 66.0; 52.9; 49.80; 48.0; 43.7; 42.2; 41.1; 39.8; 39.1;
38.8; 36.6; 36.5; 32.8; 31.9; 30.7; 29.7; 29.4; 27.8; 24.2; 23.6; 23.3; 22.7; 21.2; 18.2; 17.0; 16.9; 14.1; 13.1. ESI-
MS: 601.3 ([M þ Na]þ ).
Eriantic Acid B (27). A mixture of 26 (64 mg, 0.11 mmol) and 10% Pd/C (8 mg) in THF (2 ml) was
stirred at r.t. under H2 for 12 h. The mixture was filtered through Celite, and the insoluble substance was
washed with THF. The filtrate was concentrated under reduced pressure to give 27 (51 mg, 92%). White
solid. M.p. 259–2638. IR (KBr): 3462, 2920, 1678, 1048. 1H-NMR (300 MHz, (D5)Pyridine): 0.73 (s, 3 H);
0.78 (d, J ¼ 6.3, 3 H); 0.91 (s, 3 H); 0.98 (s, 3 H); 1.14 (s, 3 H); 1.37 (s, 3 H); 2.61 (d, J ¼ 10.9, 1 H); 3.70
(d, J ¼ 10.4, 1 H); 4.15 (d, J ¼ 10.4, 1 H); 4.25 (d, J ¼ 4.1, 1 H); 4.31–4.32 (m, 1 H); 5.48 (t, J ¼ 3.6, 1 H).
13C-NMR (75 MHz, (D5)Pyridine): 179.9; 139.3; 125.9; 73.2; 71.6; 68.0; 53.7; 48.5; 48.2; 48.1; 45.1; 42.8;
42.4; 40.2; 39.5; 39.5; 37.5; 37.2; 33.4; 31.2; 30.0; 28.7; 25.0; 24.0; 24.0; 21.4; 18.4; 17.6; 17.5; 14.6. ESI-MS:
487.4 ([MꢀH]ꢀ ).
Enzymatic Activity Assays. The inhibitory activity of the test compounds against rabbit muscle
glycogen phosphorylase was monitored using microplate reader (BIORAD) based on the method
described in [16]. 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 6
different concentrations (100, 50, 25, 12.5, 6.25, 3.125 mm) for IC50 determination. The enzyme (GPa) was
added into 100 ml 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 ml of 1m HCl containing 10 mg/ml ammonium molybdate and 0.38 mg/ml malachite
green, reactions were run at 228 for 25 min. 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. The results are listed in the Table. Values were expressed as meanꢁstandard error of the mean.
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Received March 3, 2008