Synthesis of 3-deoxypentacyclic triterpene derivatives as inhibitors of glycogen phosphorylase

Article abstract of DOI:10.1021/np9002367

The 3-deoxy-2-keto derivatives 5 and 7 of oleanolic acid (1) and ursolic acid (2), respectively, served as precursors to the synthesis of 35 3-deoxy derivatives of pentacyclic triterpenes. The synthesized compounds were biologically assayed for their inhi

Full text of DOI:10.1021/np9002367

1414
J. Nat. Prod. 2009, 72, 1414–1418
Synthesis of 3-Deoxypentacyclic Triterpene Derivatives as Inhibitors of Glycogen Phosphorylase
Pu Zhang,† Jia Hao,† Jun Liu,‡ Qian Lu,‡ Huaming Sheng,† Luyong Zhang,*,‡ and Hongbin Sun*,†
Center for Drug DiscoVery, College of Pharmacy, China Pharmaceutical UniVersity, 24 Tongjia Xiang, Nanjing 210009, People’s Republic of China,
and Jiangsu Center for Drug Screening, China Pharmaceutical UniVersity, 1 Shennonglu, Nanjing 210038, People’s Republic of China
ReceiVed April 19, 2009
The 3-deoxy-2-keto derivatives 5 and 7 of oleanolic acid (1) and ursolic acid (2), respectively, served as precursors to
the synthesis of 35 3-deoxy derivatives of pentacyclic triterpenes. The synthesized compounds were biologically assayed
for their inhibitory activity against rabbit muscle glycogen phosphorylase a (GPa). Among this series of compounds,
2R-hydroxyurs-12-en-28-oic acid (18) (IC50 ) 1.2 µM) exhibited the most potent activity. Preliminary structure-activity
relationship analysis for the 3-deoxy triterpene derivatives as GP inhibitors is also discussed.
Oleanolic acid (1) and ursolic acid (2) (Figure 1) are two well-
known members of the family of pentacyclic triterpenes and are
the major effective components of many Traditional Chinese
Medicines (TCM). Pentacyclic triterpenes exhibit a variety of
biological activities such as anti-inflammatory, antibacterial, anti-
viral, antiparasitic, hepatoprotective, antitumor, wound healing,
antioxidant, antipruritic, antiangiogenic, antiallergic, and immuno-
modulatory activities.1-6 In previous studies, we reported that
pentacyclic triterpenes represented a new class of inhibitors of
glycogen phosphorylase (GP).7 GP catalyzes the process of
glycogenolysis, which is a key contributor to hepatic glucose output.
Previous studies showed that lowering hepatic glucose production
was effective in treatment of hyperglycemia in animals.8 A number
of synthetic GP inhibitors have been identified to evaluate their
therapeutic potential for the treatment of type 2 diabetes.9 We
previously reported the synthesis of 2-isooleanolic acid (3) and
2-isoursolic acid (4) as A-ring regioisomers of 1 and 2, respec-
tively.10 In structural comparison with most natural pentacyclic
triterpenes that possess 3-oxygenated functions, 3 and 4 represent
an interesting class of 3-deoxypentacyclic triterpenes. In continu-
ation of our efforts in structural modifications of pentacyclic
triterpenes, 3 and 4 were chosen as new lead compounds. In fact,
the result of initial GP assays for 3 and 4 showed that 3 and 4
were more potent GP inhibitors than 1 and 2, respectively. This
result motivated us to carry out further structural modifications on
3 and 4. In this paper, we present the synthesis and biological
evaluation of a series of 3-deoxy-2-functionalized pentacyclic
triterpene derivatives as GP inhibitors. Structure-activity relation-
ship analysis is also discussed.
Figure 1. Structures of oleanolic acid (1), ursolic acid (2),
2-isooleanolic acid (3), and 2-isoursolic acid (4).
borohydride produced alcohol 13 (76%), together with a small
amount of the 2R-isomer 14 (5%). Hydrogenolysis of 13 and 14
over palladium/carbon in THF furnished 2-isooleanolic acid (3)
(92%) and its 2R-isomer 15 (93%), respectively. In the same
fashion, 2-isoursolic acid (4) and its 2R-isomer 18 were synthesized
starting from ketone 7.10 2-O-Acyl derivatives 19, 21, 23, 25, and
27 were obtained in 78-89% yields by treatment of 13 with acyl
anhydride/pyridine or acyl chloride/pyridine. 2-O-Acyl triterpene
acids 20, 22, 24, 26, and 28 were produced in 83-98% yields via
hydrogenolysis of the corresponding benzyl esters, respectively.
Esterification of 3 with a bromide compound or methyl iodide in
the presence of potassium carbonate in DMF yielded C-28 ester
derivatives 29-37 in 73-95% yields.
The synthesized 3-deoxypentacyclic triterpenes were biologi-
cally evaluated for their inhibitory activities against rabbit muscle
glycogen phosphorylase a (RMGPa). As described previously,
the activity of RMGPa was measured through detecting the
released amount of phosphates from glucose-1-phosphates in the
direction of glycogen synthesis.11 The assay results are reported
in Table 1. Most of the newly synthesized compounds exhibited
inhibitory activity against RMGPa with IC50 values in the range
1.2-121.3 µM.
As shown in Table 1, migration of the 3-OH group to C-2
significantly improves the potency (3 vs 1; 15 vs 1; 4 vs 2; 18 vs
2). As for the compounds with a 28-O-benzyl ester group, the
compounds with 2R-OH groups are less potent than those with 2ꢀ-
OH groups (e.g., 14 vs 13; 17 vs 16). However, the potency trend
of the compounds with a 28-carboxyl group is not clear. For the
2-keto derivatives, the 28-O-benzyl esters are more potent than the
corresponding carboxylic acids (e.g., 5 vs 6; 7 vs 8). On the other
hand, there is a reverse trend among 2-oxime derivatives (e.g., 9
vs 10; 11 vs 12). The introduction of 2-keto and 2-oxime groups
in the oleanane skeleton enhanced GP inhibitory activity compared
with the parent compound 1 (e.g., 5, 6 vs 1; 9, 10 vs 1), while in
the ursane skeleton, the potency was slightly reduced compared
with the parent compound 2 (e.g., 7, 8 vs 2; 11 vs 2). Among the
Results and Discussion
The synthesis of 3-deoxypentacyclic triterpenes 3-37 is sum-
marized in Scheme 1. 2-Keto analogues 5 and 7 were readily
prepared in five steps starting from 1 and 2, respectively.10
Hydrogenolysis of 5 and 7 over palladium/carbon afforded keto
acids 6 (96%) and 8 (93%), respectively. Treatment of 5 with
hydroxylamine hydrochloride in pyridine at room temperature gave
oxime derivative 9 (93%), which was hydrogenolyzed over pal-
ladium/carbon to give carboxylic acid 10 (95%). In the same
fashion, carboxylic acid 12 was prepared from 7. All the oxime
derivatives were mixtures of almost equal amounts of E, Z isomers
that were extremely difficult to separate by common means. As
described in our previous report,10 reduction of 5 with sodium
* To whom correspondence should be addressed. Tel: +86-25-85327950.
Fax: +86-25-83271335. E-mail: hbsun2000@yahoo.com; drugscreen@
126.com.
† Center for Drug Discovery.
‡ Jiangsu Center for Drug Screening.
10.1021/np9002367 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/30/2009

3-Deoxypentacyclic Triterpene DeriVatiVes
Journal of Natural Products, 2009, Vol. 72, No. 8 1415
Scheme 1. Synthesis of 3-Deoxypentacyclic Triterpenes 3-37a
a (a) HONH2 · HCl, pyridine, rt; (b) 10% Pd-C, H2, THF, rt; (c) NaBH4, THF, EtOH, rt; (d) acyl chloride or anhydride, pyridine, rt; (e) a bromide compound or
methyl iodide, K2CO3, DMF, rt.
Table 1. Inhibition of RMGPa by Triterpenes 1-37
2-O-acyl triterpenes with a 28-O-benzyl group, the inhibitory
potency increased as the bulk of 2-O-substituents increased (e.g.,
19 < 21 < 23).
RMGPa
RMGPa
a
a
compound
IC50 (µM)
compound
IC50 (µM)
In summary, 35 3-deoxypentacyclic triterpene derivatives, in-
cluding 28 new compounds, have been synthesized and biologically
evaluated as inhibitors of rabbit muscle GPa. Within this series of
compounds, 18 (IC50 ) 1.2 µM) exhibited 13-fold more potent GP
inhibitory activity than the parent compound 2 (IC50 ) 15.3 µM).
SAR analysis shows that migration of the 3-OH group to C-2 of
pentacyclic triterpenes may enhance GP inhibition. On the other
hand, introduction of hydrophobic groups at C-2 and C-28 might
not be suitable for potency improvement regarding GP inhibition.
1
2
3
4
5
6
7
8
22.1 ( 1.9
15.3 ( 0.8
3.5 ( 0.3
5.5 ( 0.4
3.2 ( 0.5
14.9 ( 0.9
24.2 ( 2.2
28.9 ( 2.7
16.3 ( 1.2
13.1 ( 1.4
20.2 ( 2.0
14.7 ( 1.1
4.2 ( 0.3
5.6 ( 0.4
8.5 ( 0.6
22.3 ( 1.8
55.8 ( 4.5
1.2 ( 0.1
NI b
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
42.4 ( 3.7
121.3 ( 10.5
80.2 ( 7.8
107.9 ( 8.5
75.6 ( 5.1
48.4 ( 4.6
40.7 ( 2.3
80.9 ( 6.3
NI b
28.1 ( 2.5
15.3 ( 1.3
27.3 ( 2.5
13.8 ( 0.9
25.4 ( 2.6
37.2 ( 3.5
48.7 ( 3.9
22.5 ( 2.0
23.1 ( 1.9
75.3 ( 6.6
9
10
11
12
13
14
15
16
17
18
19
Experimental Section
General Experimental Procedures. All commercially available
solvents and reagents were used without further purification. Melting
points are uncorrected. 1H NMR and 13C NMR spectra were recorded
at 300 and 75 MHz, respectively. Chemical shifts are reported as values
from an internal tetramethylsilane standard. Low- and high-resolution
mass spectra (LRMS and HRMS) were recorded with electron impact
mode.
36
37
caffeinec
a Values are means of three experiments. b NI means no inhibition.
2-Oxoolean-12-en-28-oic acid (6). Ketone 510 (100 mg, 0.184 mmol)
was dissolved in THF (4 mL) and treated with 10% Pd/C (10 mg).
c Caffeine was used as a positive control.

1416 Journal of Natural Products, 2009, Vol. 72, No. 8
Zhang et al.
The mixture was stirred at room temperature under H2 atmospheric
pressure for 5 h. The reaction mixture was filtered through Celite, and
the insoluble substance was washed with THF. The filtrate was
155.5, 178.2; ESIMS m/z 468.3 [M - H]-; HRMS for C30H47NO3+H
calcd 470.36287, found 470.36389.
Compounds 3, 4, 13, 14, and 16 were prepared following the
literature procedues.10 2r-Hydroxyolean-12-en-28-oic acid (15).
Following the procedure described for preparation of 6, compound 15
was prepared from 145 as a white solid (93%): 1H NMR (pyridine-d5)
δ 0.87 (s, 3H), 0.94 (s, 3H), 0.95 (s, 3H), 0.96 (s, 3H), 1.01 (s, 6H),
1.02 (s, 3H), 1.27 (s, 3H), 3.30 (dd, J ) 4.0 Hz, 13.7 Hz, 1H),
4.12-4.21 (m, 1H), 5.48 (t, J ) 3.2 Hz, 1H); 13C NMR (pyridine-d5)
δ 16.7, 17.6, 18.8, 22.8, 22.9, 23.7, 23.8, 24.0, 26.2, 28.3, 31.0, 33.2,
33.3, 33.8, 34.3, 34.9, 39.2, 40.0, 42.1, 42.3, 46.5, 46.7, 48.3, 50.4,
52.2, 56.1, 63.9, 122.6, 144.9, 180.1; ESIMS m/z 479.4 [M + Na]+;
anal. calcd for C30H48O3 ·0.4CH3COOH C 76.95, H 10.40, found C
77.07, H 9.91.
Benzyl 2r-Hydroxyurs-12-en-28-oate (17). Following the literature
procedures,5 compound 17 was prepared from 7 as a white solid (10%):
1H NMR (CDCl3) δ 0.64 (s, 3H), 0.86 (s, 3H), 0.87 (s, 3H), 0.94 (s,
6H), 0.95 (s, 3H), 1.08 (s, 3H), 2.27 (d, J ) 11.6 Hz, 1H), 3.89 (m,
1H), 4.98 (d, J ) 12.5 Hz, 1H), 5.10 (d, J ) 12.5 Hz, 1H), 5.25 (t, J
) 3.6 Hz, 1H), 7.26-7.36 (m, 5H); 13C NMR (CDCl3) δ 16.6, 17.0,
17.2, 18.4, 21.1, 22.7, 23.4, 23.6, 24.3, 28.0, 30.7, 33.0, 33.6, 34.9,
36.7, 38.9, 39.0, 39.2, 39.8, 42.2, 47.7, 48.2, 49.8, 51.3, 53.0, 55.7,
65.2, 66.0, 125.8, 127.9, 128.2, 128.4, 136.5, 138.3, 177.2; ESIMS m/z
569.4 [M + Na]+; HRMS for C37H54O3+H calcd 547.41457, found
547.41603.
1
concentrated in Vacuo to give 6 as a white solid (80 mg, 96%): H
NMR (pyridine-d5) δ 0.82 (s, 3H), 0.86 (s, 3H), 0.96 (s, 3H), 1.01 (s,
3H), 1.25 (s, 3H), 1.26 (s, 3H), 1.29 (s, 3H), 3.30 (d, J ) 12.6 Hz,
1H), 5.46 (s, 1H); 13C NMR (pyridine-d5) δ 16.5, 17.0, 19.3, 22.9,
23.4, 23.7, 26.1, 28.3, 29.6, 30.0, 31.0, 32.1, 32.8, 33.2, 33.3, 34.3,
39.1, 40.1, 42.0, 43.0, 46.5, 46.7, 47.5, 55.4, 55.6, 56.5, 122.1, 144.9,
180.1, 210.3; ESIMS m/z 453.3 [M - H]-; HRMS for C30H46O3 calcd
454.3447, found 454.3449.
2-Oxours-12-en-28-oic acid (8). Following the procedure described
for preparation of 6, compound 8 was prepared from 710 as a white
1
solid (93%): H NMR (pyridine-d5) δ 0.81 (s, 3H), 0.84 (s, 3H), 0.96
(s, 9H), 1.22 (s, 6H), 2.62 (d, J ) 10.7 Hz, 1H), 5.44 (s, 1H); 13C
NMR (pyridine-d5) δ 16.7, 17.1, 17.5, 19.3, 21.4, 23.4, 23.6, 23.9, 24.9,
28.7, 31.1, 33.1, 33.3, 37.4, 39.0, 39.4, 39.6, 40.4, 42.7, 42.9, 47.4,
48.1, 53.6, 55.5, 55.9, 56.5, 125.2, 139.4, 179.8, 210.4; ESIMS m/z
453.5 [M - H]-; HRMS for C30H46O3+H calcd 455.35197, found
455.35311.
Benzyl 2-hydroxyiminoolean-12-en-28-oate (9). To a solution of
5 (100 mg, 0.18 mmol) in pyridine (0.6 mL) was added hydroxylamine
hydrochloride (25 mg, 0.36 mmol). The reaction mixture was allowed
to stir at room temperature for 3 h, and the solution was acidified with
2 N HCl and extracted with EtOAc. The EtOAc layer was washed
with saturated NaHCO3 solution and brine, dried over anhydrous
Na2SO4, filtered, and evaporated to dryness. The residue was purified
by column chromatography on silica gel, eluted with an mixture of
petroleum ether/EtOAc (6:1 v:v), to give 9 as a white solid (95 mg,
2r-Hydroxyurs-12-en-28-oic acid (18). Following the procedure
described for preparation of 6, compound 18 was prepared from 17 as
1
a white solid (98%): H NMR (pyridine-d5) δ 0.85 (s, 3H), 0.91 (s,
3H), 0.94 (s, 9H), 0.97 (s, 3H), 1.15 (s, 3H), 2.43 (d, J ) 11.4 Hz,
1H), 3.99-4.03 (m, 1H), 5.37 (s, 1H); 13C NMR (pyridine-d5) δ 15.4,
16.0, 17.4, 20.1, 21.5, 22.3, 22.5, 23.4, 27.1, 29.7, 32.0, 32.5, 33.5,
35.9, 37.7, 37.9, 38.1, 38.7, 41.2, 46.6, 46.7, 48.8, 50.3, 52.0, 54.7,
62.7, 124.2, 137.7, 178.8; HRMS for C30H48O3+H calcd 457.36762,
found 457.36954.
1
93%): H NMR (CDCl3) δ 0.59 (s, 6H), 0.83 (s, 3H), 0.84 (s, 6H),
0.86 (s, 3H), 0.90 (s, 6H), 0.92 (s, 6H), 1.00 (s, 3H), 1.03 (s, 3H), 1.15
(s, 6H), 2.92 (dd, J ) 4.1 Hz, 13.4 Hz, 2H), 3.14 (d, J ) 13.6 Hz,
1H), 3.29 (d, J ) 12.7 Hz, 1H), 5.01-5.13 (m, 4H), 5.30 (s, 1H), 5.31
(s, 1H), 7.28-7.35 (m, 10H); 13C NMR (CDCl3) δ 15.5, 15.9, 16.6,
16.7, 18.7, 18.8, 22.3, 22.8, 23.1, 23.4, 23.56, 23.64, 25.8, 27.6, 30.7,
32.38, 32.45, 32.50, 32.8, 33.1, 33.9, 36.8, 38.7, 39.4, 39.6, 39.7, 40.7,
40.8, 41.42, 41.45, 41.80, 41.82, 45.9, 46.2, 46.8, 47.0, 47.2, 56.25,
56.36, 65.95, 66.00, 122.30, 122.32, 127.9, 128.0, 128.4, 136.4, 143.65,
143.70, 159.4, 159.5, 177.39, 177.41; ESIMS m/z 560.4 [M + H]+;
HRMS for C37H53NO3+H calcd 560.40982, found 560.41122.
Benzyl 2-hydroxyiminours-12-en-28-oate (11). Following the
procedure described for preparation of 9, compound 11 was prepared
from 7 as a white solid (92%): 1H NMR (CDCl3) δ 0.63 (s, 6H), 0.81
(s, 3H), 0.83 (s, 3H), 0.85 (s, 6H), 0.86 (s, 6H), 0.95 (s, 6H), 1.01 (s,
3H), 1.04 (s, 3H), 1.10 (s, 6H), 3.15 (dd, J ) 1.9 Hz, 13.5 Hz, 1H),
3.32 (d, J ) 13.0 Hz, 1H), 4.95-5.14 (m, 4H), 5.23-5.27 (m, 2H),
7.25-7.36 (m, 10H); 13C NMR (CDCl3) δ 15.6, 16.1, 16.7, 16.8, 16.9,
18.7, 18.8, 21.1, 22.3, 22.9, 23.3, 23.4, 23.5, 24.3, 28.0, 30.7, 32.73,
32.76, 32.82, 32.87, 36.6, 36.93, 36.96, 38.8, 39.0, 39.1, 39.5, 39.86,
39.95, 40.8, 41.0, 42.1, 42.2, 46.1, 46.6, 47.0, 47.1, 48.1, 52.9, 56.2,
56.3, 66.0, 125.4, 127.9, 128.2, 128.4, 136.3, 138.1, 138.2, 160.3, 177.1;
ESIMS m/z 560.4 [M + H]+; HRMS for C37H53NO3+H calcd
560.40982, found 560.41147.
General Procedure for the Acylation of 13. Compound 13 was
dissolved in pyridine and treated with an excess of anhydride or acyl
chloride. The reaction mixture was stirred at room temperature until
the substrate was consumed. Then, the solution was acidified with 2 N
HCl and extracted with EtOAc. The EtOAc layer was washed with
saturated NaHCO3 solution and brine, dried over anhydrous Na2SO4,
filtered, and evaporated to dryness. The residue was purified by column
chromatography on silica gel.
Benzyl 2ꢀ-acetoxyolean-12-en-28-oate (19). Following the general
procedure, compound 19 was prepared from 13 as a white solid (78%):
1H NMR (CDCl3) δ 0.65 (s, 3H), 0.92 (s, 3H), 0.94 (s, 6H), 1.01 (s,
3H), 1.13 (s, 3H), 1.15 (s, 3H), 2.04 (s, 3H), 2.93 (dd, J ) 4.1 Hz,
13.7 Hz, 1H), 5.04-5.15 (m, 3H), 5.31 (t, J ) 3.5 Hz, 1H), 7.32-7.38
(m, 5H); 13C NMR (CDCl3) δ 17.0, 17.1, 18.7, 21.5, 23.2, 23.5, 23.7,
23.8, 26.0, 27.7, 30.8, 32.5, 32.7, 33.1, 33.4, 34.1, 37.4, 39.7, 41.6,
42.1, 43.5, 46.0, 46.9, 48.2, 54.6, 66.0, 70.7, 122.7, 127.9, 128.1, 128.4,
136.6, 143.8, 170.4, 177.4; ESIMS m/z 611.5 [M + Na]+; HRMS for
C39H56O4+H calcd 589.42514, found 589.42701.
Benzyl 2ꢀ-(1-oxopropoxy)olean-12-en-28-oate (21). Following the
general procedure, compound 21 was prepared from 13 as a white solid
in a yield of 82%: 1H NMR (CDCl3) δ 0.63 (s, 3H), 0.90 (s, 3H), 0.92
(s, 6H), 0.99 (s, 3H), 1.11 (s, 3H), 1.12 (s, 3H), 1.13 (s, 3H), 2.26 (d,
J ) 7.6 Hz, 1H), 2.31 (d, J ) 7.6 Hz, 1H), 2.91 (dd, J ) 3.3 Hz, 13.5
Hz, 1H), 5.01-5.12 (m, 3H), 5.29 (t, J ) 3.1 Hz, 1H), 7.27-7.35 (m,
5H); 13C NMR (CDCl3) δ 9.1, 17.1, 18.7, 23.2, 23.5, 23.7, 26.0, 27.7,
28.3, 30.7, 32.5, 32.7, 33.1, 33.5, 34.0, 37.3, 39.7, 41.6, 42.1, 43.5,
43.6, 46.0, 46.9, 48.2, 54.7, 66.0, 70.5, 122.7, 127.9, 128.0, 128.4, 143.8;
ESIMS m/z 625.5 [M + Na]+; HRMS for C40H58O4+Na calcd
625.42273, found 625.42398.
2-Hydroxyiminoolean-12-en-28-oic acid (10). Following the pro-
cedure described for preparation of 6, compound 10 was prepared from
9 as a white solid (95%): 1H NMR (DMSO-d6) δ 0.71 (s, 3H), 0.72 (s,
3H), 0.76 (s, 6H), 0.81 (s, 3H), 0.88 (s,12H), 0.95 (s, 3H), 0.97 (s,
3H), 1.13 (s, 6H), 1.24 (s, 3H), 2.75 (d, J ) 10.4 Hz, 2H), 2.99 (d, J
) 13.0 Hz, 1H), 3.15 (d, J ) 12.3 Hz, 1H), 5.20 (s, 2H), 10.15 (s,
1H), 10.17 (s, 1H), 12.05 (s, 2H); 13C NMR (DMSO-d6) δ 15.0, 15.5,
16.4, 16.5, 18.1, 18.2, 21.9, 22.6, 22.7, 22.9, 23.2, 25.4, 27.1, 28.8,
30.2, 32.0, 32.3, 32.4, 32.7, 33.2, 35.9, 36.0, 37.8, 38.7, 38.8, 38.9,
39.1, 40.3, 40.7, 41.3, 45.4, 45.5, 45.6, 46.0, 46.3, 46.5, 55.36, 55.40,
121.2, 121.3, 143.6, 143.7, 155.3, 178.3; ESIMS m/z 468.3 [M - H]-;
HRMS for C30H47NO3+H calcd 470.36287, found 470.36391.
2-Hydroxyiminours-12-en-28-oic acid (12). Following the proce-
dure described for preparation of 6, compound 12 was prepared from
11 as a white solid (95%): 1H NMR (DMSO-d6) δ 0.75 (s, 12H), 0.82
(s, 12H), 0.92 (s, 6H), 0.95 (s, 3H), 0.96 (s, 3H), 1.08 (s, 6H), 2.99 (d,
J ) 12.0 Hz, 1H), 3.17 (d, J ) 12.3 Hz, 1H), 5.15 (s, 2H), 10.13 (s,
2H), 11.93 (brs, 2H); 13C NMR (DMSO-d6) δ 15.4, 15.8, 16.6, 16.7,
17.1, 18.3, 18.4, 21.1, 22.2, 22.8, 22.9, 23.0, 23.3, 23.9, 27.6, 30.3,
32.46, 32.53, 32.6, 36.1, 36.4, 38.1, 38.6, 38.9, 39.0, 39.5, 40.1, 40.5,
41.8, 45.8, 46.1, 46.4, 46.6, 46.9, 52.5, 55.5, 55.6, 124.5, 138.2, 138.3,
Benzyl 2ꢀ-(1-oxobutoxy)olean-12-en-28-oate (23). Following the
general procedure, compound 23 was prepared from 13 as a white solid
1
(87%): H NMR (CDCl3) δ 0.66 (s, 3H), 0.93 (s, 3H), 0.95 (s, 6H),
0.97 (s, 3H), 1.01 (s, 3H), 1.14 (s, 3H), 1.15 (s, 3H), 2.27 (t, J ) 7.5
Hz, 2H), 2.93 (dd, J ) 3.9 Hz, 13.8 Hz, 1H), 5.05-5.15 (m, 3H), 5.32
(t, J ) 3.5 Hz, 1H), 7.30-7.39 (m, 5H); 13C NMR (CDCl3) δ 13.7,
17.0, 17.1, 18.4, 18.7, 23.2, 23.5, 23.67, 23.75, 25.9, 27.6, 30.7, 32.5,
32.7, 33.1, 33.5, 34.0, 37.0, 37.3, 39.7, 41.6, 42.1, 43.56, 43.62, 46.0,
46.9, 48.2, 54.6, 66.0, 70.4, 122.7, 127.9, 128.0, 128.4, 136.6, 143.8,
173.0, 177.4; ESIMS m/z 639.4 [M + Na]+; HRMS for C41H60O4+Na
calcd 639.43838, found 639.44026.

3-Deoxypentacyclic Triterpene DeriVatiVes
Journal of Natural Products, 2009, Vol. 72, No. 8 1417
Benzyl 2ꢀ-benzoyloxyolean-12-en-28-oate (25). Following the
general procedure, compound 25 was prepared from 13 as a white solid
(79%): H NMR (CDCl3) δ 0.64 (s, 3H), 0.90 (s, 3H), 0.92 (s, 3H),
or methyl iodide. The reaction mixture was stirred at room temperature
until the substrate was consumed. Then, the mixture was filtered and
extracted with EtOAc. The EtOAc layer was washed with brine, dried
over anhydrous Na2SO4, filtered, and evaporated to dryness. The residue
was purified by column chromatography on silica gel.
1
0.96 (s, 3H), 1.09 (s, 3H), 1.15(s, 3H), 1.21 (s, 3H), 2.91 (d, J ) 11.0
Hz, 1H), 5.04 (d, J ) 12.6 Hz, 1H), 5.11 (d, J ) 12.7 Hz, 1H), 5.30
(s, 1H), 5.37 (t, J ) 4.0 Hz, 1H), 7.26-7.35 (m, 5H), 7.41-7.46 (m,
2H), 7.52-7.57 (m, 1H), 8.00-8.03 (m, 2H); 13C NMR (CDCl3) δ
17.0, 17.1, 18.7, 23.2, 23.5, 23.7, 23.8, 26.0, 27.6, 30.7, 32.5, 32.7,
33.1, 33.6, 34.0, 37.2, 39.7, 41.5, 42.0, 43.5, 43.8, 46.0, 46.9, 48.2,
54.9, 66.0, 71.5, 122.7, 127.9, 128.0, 128.34, 128.42, 129.5, 131.1,
132.7, 136.6, 143.8, 166.2, 177.4; HRMS for C44H58O4+Na calcd
673.42273, found 673.42546.
Methyl 2ꢀ-hydroxyolean-12-en-28-oate (29). Following the general
procedure, compound 29 was prepared from 3 as a white solid (95%):
1H NMR (CDCl3) δ 0.73 (s, 3H), 0.89 (s, 3H), 0.92 (s, 6H), 1.00 (s,
3H), 1.12 (s, 3H), 1.18 (s, 3H), 2.85 (dd, J ) 3.4 Hz, 14.0 Hz, 1H),
3.62 (s, 3H), 4.08 (t, J ) 4.8 Hz, 1H), 5.30 (s, 1H); 13C NMR (CDCl3)
δ 16.8, 18.5, 19.0, 23.1, 23.4, 23.6, 24.6, 25.9, 27.6, 30.7, 32.4, 32.5,
32.7, 33.08, 33.10, 33.9, 37.8, 39.6, 41.4, 41.9, 45.8, 46.6, 46.8, 47.2,
48.0, 51.5, 53.5, 67.7, 122.6, 143.7, 178.3; ESIMS m/z 493.4 [M +
Na]+; HRMS for C31H50O3+Na calcd 493.36522, found 493.36640.
Ethyl 2ꢀ-hydroxyolean-12-en-28-oate (30). Following the general
procedure, compound 30 was prepared from 3 as a white solid (94%):
1H NMR (CDCl3) δ 0.75 (s, 3H), 0.89 (s, 3H), 0.92 (s, 6H), 1.00 (s,
3H), 1.13 (s, 3H), 1.18 (s, 3H), 1.25 (s, 3H), 2.86 (dd, J ) 3.7 Hz,
13.8 Hz, 1H), 4.03-4.13 (m, 3H), 5.31 (t, J ) 3.3 Hz, 1H); 13C NMR
(CDCl3) δ 14.3, 16.9, 18.5, 19.0, 23.0, 23.5, 23.6, 24.7, 25.9, 27.6,
29.7, 30.7, 32.4, 32.6, 32.7, 33.07, 33.12, 34.0, 37.8, 39.7, 41.4, 41.9,
45.9, 46.6, 47.3, 48.0, 53.5, 60.1, 67.7, 122.6, 143.8, 177.7; ESIMS
m/z 507.5 [M + Na]+; HRMS for C32H52O3+H calcd 485.39892, found
485.39981.
Benzyl 2ꢀ-(3-carboxy-1-oxopropoxy)olean-12-en-28-oate (27).
Following the general procedure, compound 27 was prepared from 13
as a white solid (89%): 1H NMR (CDCl3) δ 0.63 (s, 3H), 0.90 (s, 3H),
0.92 (s, 6H), 0.98 (s, 3H), 1.10 (s, 3H), 1.12 (s, 3H), 2.57-2.71 (m,
4H), 2.88-2.92 (m, 1H), 5.02-5.14 (m, 3H), 5.29 (t, J ) 3.4 Hz, 1H),
7.28-7.36 (m, 5H); 13C NMR (CDCl3) δ 17.0, 17.2, 18.7, 23.2, 23.5,
23.7, 23.8, 25.9, 27.6, 28.6, 29.6, 30.7, 32.5, 32.7, 33.1, 33.4, 34.0,
37.3, 39.7, 41.6, 42.0, 43.4, 46.0, 46.9, 48.2, 54.5, 66.0, 71.4, 122.7,
127.9, 128.0, 128.4, 136.6, 143.8, 171.5, 177.4; ESIMS m/z 645.3 [M
- H]-; HRMS for C41H58O6+H calcd 647.43062, found 647.43359.
2ꢀ-Acetoxyolean-12-en-28-oic acid (20). Following the procedure
described for preparation of 6, compound 20 was prepared from 19 as
1
a white solid (97%): H NMR (CDCl3) δ 0.77 (s, 3H), 0.91 (s, 3H),
n-Propyl 2ꢀ-hydroxyolean-12-en-28-oate (31). Following the
0.92 (s, 3H), 0.93 (s, 3H), 0.98 (s, 3H), 1.13 (s, 3H), 1.14 (s, 3H), 2.00
(s, 3H), 2.83 (dd, J ) 4.4 Hz, 13.6 Hz, 1H), 5.06-5.10 (m, 1H), 5.29
(t, J ) 3.3 Hz, 1H); 13C NMR (CDCl3) δ 17.2, 18.7, 21.5, 23.1, 23.5,
23.6, 23.7, 26.0, 27.7, 30.7, 32.5, 32.6, 33.1, 33.4, 33.9, 37.4, 39.7,
41.3, 42.0, 43.5, 46.0, 46.6, 48.2, 54.6, 70.7, 122.9, 143.6, 170.4, 182.2;
ESIMS m/z 497.4 [M - H]-; HRMS for C32H50O4+H calcd 499.37819,
found 499.37929.
general procedure, compound 31 was prepared from 3 as a white solid
1
(92%): H NMR (CDCl3) δ 0.75 (s, 3H), 0.89 (s, 3H), 0.93 (s, 6H),
0.94 (s, 3H), 1.01 (s, 3H), 1.13 (s, 3H), 1.18 (s, 3H), 2.88 (dd, J ) 4.2
Hz, 13.7 Hz, 1H), 3.92-4.01 (m, 2H), 4.06-4.10 (m, 1H), 5.31 (t, J
) 3.5 Hz, 1H); 13C NMR (CDCl3) δ 10.6, 16.9, 18.5, 19.0, 22.0, 23.1,
23.5, 23.6, 24.7, 25.9, 27.6, 30.7, 32.5, 32.6, 32.7, 33.0, 33.1, 34.0,
37.8, 39.7, 41.5, 42.0, 45.9, 46.6, 46.8, 47.3, 48.0, 53.5, 65.8, 67.7,
122.6, 143.8, 177.7; ESIMS m/z 521.4 [M + Na]+; HRMS for
C33H54O3+H calcd 499.41457, found 499.41483.
2ꢀ-(1-Oxopropoxy)olean-12-en-28-oic acid (22). Following the
procedure described for preparation of 6, compound 22 was prepared
from 21 as a white solid (94%): 1H NMR (CDCl3) δ 0.78 (s, 3H), 0.93
(s, 3H), 0.94 (s, 3H), 0.95 (s, 3H), 1.00 (s, 3H), 1.15 (s, 6H), 1.17 (s,
3H), 2.31 (q, J ) 7.58 Hz, 2H), 2.85 (d, J ) 10.1 Hz, 1H), 5.14 (s,
1H), 5.31 (s, 1H); 13C NMR (CDCl3) δ 9.1, 17.0, 17.2, 18.7, 23.0,
23.5, 23.6, 26.0, 27.7, 28.3, 30.7, 32.5, 32.6, 33.1, 33.5, 33.9, 37.4,
39.7, 41.2, 41.9, 43.4, 43.6, 45.9, 46.7, 48.2, 54.7, 70.5, 122.8, 143.6,
173.8, 183.5; ESIMS m/z 511.5 [M - H]-; HRMS for C33H52O4+Na
calcd 535.37578, found 535.37770.
n-Butyl 2ꢀ-hydroxyolean-12-en-28-oate (32). Following the general
procedure, compound 32 was prepared from 3 as a white solid (86%):
1H NMR (CDCl3) δ 0.75 (s, 3H), 0.90 (s, 3H), 0.92 (s, 9H), 1.01 (s,
3H), 1.13 (s, 3H), 1.18 (s, 3H), 2.87 (d, J ) 12.8 Hz, 1H), 4.01-4.08
(m, 3H), 5.30 (s, 1H); 13C NMR (CDCl3) δ 13.7, 16.9, 18.5, 19.0, 19.2,
23.0, 23.5, 23.6, 24.6, 25.9, 27.5, 29.7, 30.7, 32.5, 32.6, 32.7, 33.0,
33.1, 33.9, 37.8, 39.6, 41.4, 41.9, 45.9, 46.6, 46.7, 47.3, 48.0, 53.4,
63.9, 67.7, 122.6, 143.8, 177.7; ESIMS m/z 535.4 [M + Na]+; HRMS
for C34H56O3+H calcd 513.43022, found 513.43176.
2ꢀ-(1-Oxobutoxy)olean-12-en-28-oic acid (24). Following the
procedure described for preparation of 6, compound 24 was prepared
from 23 as a white solid (98%): 1H NMR (CDCl3) δ 0.75 (s, 3H), 0.90
(s, 3H), 0.91 (s, 3H), 0.92 (s, 3H), 0.93 (s, 3H), 0.99 (s, 3H), 1.12 (s,
3H), 1.14 (s, 3H), 2.23 (t, J ) 7.5 Hz, 2H), 2.82 (d, J ) 9.6 Hz, 1H),
5.11 (t, J ) 4.1 Hz, 1H), 5.28 (s, 1H); 13C NMR (CDCl3) δ 13.7, 17.1,
17.2, 18.4, 18.6, 23.0, 23.5, 23.6, 23.7, 26.0, 27.7, 30.7, 32.4, 32.6,
33.1, 33.5, 33.9, 37.0, 37.3, 39.6, 41.2, 41.9, 43.4, 43.6, 45.9, 46.6,
48.1, 54.6, 70.4, 122.8, 143.6, 173.0, 183.3; ESIMS m/z 525.5 [M -
H]-; HRMS for C34H54O4+H calcd 527.40949, found 527.41081.
2ꢀ-Benzoyloxyolean-12-en-28-oic acid (26). Following the proce-
dure described for preparation of 6, compound 26 was prepared from
(2-Bromoethyl) 2ꢀ-hydroxyolean-12-en-28-oate (33). Following
the general procedure, compound 33 was prepared from 3 as a white
1
solid (91%): H NMR (CDCl3) δ 0.75 (s, 3H), 0.90 (s, 3H), 0.93 (s,
6H), 1.01 (s, 3H), 1.14 (s, 3H), 1.18 (s, 3H), 2.88 (dd, J ) 3.5 Hz,
13.6 Hz, 1H), 3.50 (t, J ) 6.0 Hz, 2H), 4.06-4.10 (m, 1H), 4.28-4.39
(m, 2H), 5.33 (t, J ) 3.1 Hz, 1H); 13C NMR (CDCl3) δ 17.0, 18.5,
19.0, 23.0, 23.5, 23.6, 24.6, 25.9, 27.6, 29.0, 29.7, 30.7, 32.47, 32.55,
32.7, 33.1, 33.9, 37.8, 39.7, 41.4, 41.9, 45.8, 46.6, 46.9, 47.3, 48.0,
53.5, 63.6, 67.7, 122.9, 143.4, 177.3; ESIMS m/z 601.3 [M + K]+;
HRMS for C32H51BrO3+Na calcd 585.29138, found 585.29301.
(3-Bromopropyl) 2ꢀ-hydroxyolean-12-en-28-oate (34). Following
the general procedure, compound 34 was prepared from 3 as a white
1
25 as a white solid (95%): H NMR (CDCl3) δ 0.78 (s, 3H), 0.91 (s,
1
3H), 0.93 (s, 3H), 0.97 (s, 3H), 1.08 (s, 3H), 1.15 (s, 3H), 1.24 (s, 3H),
2.81-2.85 (m, 1H), 5.29 (s, 1H), 5.38 (t, J ) 3.7 Hz, 1H), 7.40-7.45
(m, 2H), 7.52-7.57 (m, 1H), 7.99-8.02 (m, 2H); 13C NMR (CDCl3)
δ 17.1, 17.2, 18.7, 23.1, 23.5, 23.6, 23.8, 26.0, 27.7, 30.7, 32.5, 32.6,
32.7, 33.0, 33.6, 34.0, 37.3, 39.7, 41.3, 42.0, 43.5, 43.8, 46.0, 46.6,
48.2, 54.9, 71.5, 122.9, 128.4, 129.5, 131.1, 132.6, 143.6, 166.2, 182.0;
ESIMS m/z 583.3 [M + Na]+; HRMS for C37H52O4+Na calcd
583.37578.40949, found 583.37677.
solid (82%): H NMR (CDCl3) δ 0.75 (s, 3H), 0.90 (s, 3H), 0.92 (s,
6H), 1.00 (s, 3H), 1.13 (s, 3H), 1.18 (s, 3H), 2.11-2.20 (m, 2H), 2.85
(d, J ) 10.0 Hz, 1H), 3.46 (t, J ) 6.6 Hz, 2H), 4.09-4.17 (m, 3H),
5.30 (s, 1H); 13C NMR (CDCl3) δ 17.1, 18.5, 19.0, 23.1, 23.5, 23.6,
24.6, 25.9, 27.5, 29.5, 29.7, 30.7, 31.8, 32.51, 32.56, 32.7, 33.1, 33.9,
37.8, 39.7, 41.5, 42.0, 45.8, 46.6, 46.9, 47.3, 48.0, 53.5, 61.8, 67.6,
122.8, 143.7, 177.5; ESIMS m/z 615.3 [M + K]+; HRMS for
C33H53BrO3+Na calcd 599.30703, found 599.30758.
2ꢀ-(3-Carboxy-1-oxopropoxy)olean-12-en-28-oic acid (28). Fol-
lowing the procedure described for preparation of 6, compound 28 was
prepared from 27 as a white solid (83%): 1H NMR (C5D5N) δ 0.85 (s,
3H), 0.93 (s, 3H), 0.98 (s, 3H), 0.99 (s, 3H), 1.01 (s, 3H), 1.15 (s, 3H),
1.23 (s, 3H), 2.81-2.90 (m, 4H), 3.27 (d, J ) 10.4 Hz, 1H), 5.29 (s,
1H), 5.46 (s, 1H); 13C NMR (C5D5N) δ 17.4, 18.9, 23.8, 24.0, 26.2,
28.2, 29.9, 30.5, 30.9, 32.5, 33.0, 33.2, 33.4, 34.3, 37.5, 40.0, 42.1,
42.4, 43.6, 46.5, 46.7, 48.5, 54.7, 70.9, 122.5, 144.9, 172.0, 174.7, 180.0;
ESIMS m/z 579.3 [M + Na]+; HRMS for C34H52O6+Na calcd
579.36561, found 579.36777.
(4-Bromobutyl) 2ꢀ-hydroxyolean-12-en-28-oate (35). Following
the general procedure, compound 35 was prepared from 3 as a white
1
solid (85%): H NMR (CDCl3) δ 0.74 (s, 3H), 0.90 (s, 3H), 0.93 (s,
6H), 1.01 (s, 3H), 1.13 (s, 3H), 1.18 (s, 3H), 2.86 (dd, J ) 3.9 Hz,
13.5 Hz, 1H), 3.43 (t, J ) 6.7 Hz, 2H), 4.03-4.10 (m, 3H), 5.31 (t, J
) 3.5 Hz, 1H); 13C NMR (CDCl3) δ 17.0, 18.5, 19.0, 23.1, 23.5, 23.6,
24.6, 25.9, 27.4, 27.6, 29.6, 29.7, 30.7, 32.5, 32.6, 32.7, 33.0, 33.1,
33.9, 37.8, 39.7, 41.5, 42.0, 45.9, 46.6, 46.8, 47.3, 48.0, 53.5, 63.2,
67.7, 122.7, 143.7, 177.7; ESIMS m/z 613.4 [M + Na]+; HRMS for
C34H55BrO3+H calcd 591.34073, found 591.34282.
General Procedure for the Esterification of 3. Compound 35 was
dissolved in DMF and treated with 1.2 equiv of a bromide compound
(2-Ethoxy-2-oxoethyl) 2ꢀ-hydroxyolean-12-en-28-oate (36). Fol-
lowing the general procedure, compound 36 was prepared from 3 as a

1418 Journal of Natural Products, 2009, Vol. 72, No. 8
Zhang et al.
white solid (84%): 1H NMR (CDCl3) δ 0.74 (s, 3H), 0.90 (s, 3H), 0.92
(s, 3H), 0.93 (s, 3H), 1.01 (s, 3H), 1.14 (s, 3H), 1.18 (s, 3H), 1.27 (t,
J ) 7.1 Hz, 3H), 2.88 (dd, J ) 4.0 Hz, 13.6 Hz, 1H), 4.08 (brs, 1H),
4.20 (q, J ) 7.1 Hz, 2H), 4.49 (d, J ) 15.7 Hz, 1H), 4.61 (d, J ) 15.7
Hz, 1H), 5.32 (t, J ) 3.4 Hz, 1H); 13C NMR (CDCl3) δ 14.1, 16.8,
18.5, 19.0, 23.2, 23.5, 23.6, 24.7, 25.8, 27.6, 29.7, 30.7, 32.2, 32.5,
32.7, 33.1, 33.9, 37.8, 39.7, 41.4, 42.0, 46.0, 46.6, 46.8, 47.3, 48.1,
53.5, 60.5, 61.2, 67.7, 122.8, 143.5, 168.1, 177.0; ESIMS m/z 565.3
[M + Na]+; HRMS for C34H54O5+Na calcd 565.38635, found
565.38817.
research grants from the Ministry of Education (grants 706030 and
20050316008), and the program for New Century Excellent Talents in
University (NCET-05-0495). This work is partially supported by the
“111 Project” from the Ministry of Education of China and the State
Administration of Foreign Expert Affairs of China (No. 111-2-07).
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.
References and Notes
Allyl 2ꢀ-hydroxyolean-12-en-28-oate (37). Following the general
procedure, compound 37 was prepared from 3 as a white solid (73%):
1H NMR (CDCl3) δ 0.74 (s, 3H), 0.90 (s, 3H), 0.93 (s, 6H), 1.01 (s,
3H), 1.13 (s, 3H), 1.18 (s, 3H), 2.89 (dd, J ) 4.1 Hz, 13.8 Hz, 1H),
4.05-4.10 (m, 1H), 4.48-4.54 (m, 2H), 5.18-5.22 (m, 1H), 5.28-5.35
(m, 1H), 5.85-5.94 (m, 1H); 13C NMR (CDCl3) δ 16.9, 18.5, 19.0,
23.1, 23.5, 23.6, 24.6, 25.9, 27.6, 29.6, 30.7, 32.5, 32.6, 32.7, 33.1,
33.9, 37.8, 39.7, 41.5, 41.9, 45.9, 46.6, 46.8, 47.3, 48.0, 53.5, 64.8,
67.7, 117.7, 122.7, 132.6, 143.7, 177.3; ESIMS m/z 519.5 [M + Na]+;
HRMS for C33H52O3+H calcd 497.39892, found 497.40056.
Enzyme Assay. The inhibitory activity of the compounds against
rabbit muscle GPa was monitored using a microplate reader (BIO-RAD)
based on the published method.7 In brief, GPa activity was measured
in the direction of glycogen synthesis by the release of phosphate from
glucose-1-phosphate. Each test compound was dissolved in DMSO and
diluted at different concentrations for IC50 determination. The enzyme
was added into 100 µL of buffer containing 50 mM Hepes (pH 7.2),
100 mM KCl, 2.5 mM MgCl2, 0.5 mM glucose-1-phosphate, 1 mg/
mL glycogen, and the test compound in 96-well microplates (Costar).
After the addition of 150 µL of 1 M HCl containing 10 mg/mL
ammonium molybdate and 0.38 mg/mL malachite green, reactions were
run at 22 °C for 25 min, and then the phosphate 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.
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Acknowledgment. This program is financially supported by the
National Natural Science Foundation (grants 30672523 and 90713037),
NP9002367