Synthesis and biological evaluation of oleanolic acid derivatives as inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1021/np100064m

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator in the process of insulin signaling and a promising drug target for diabetes and obesity. Derivatives of oleanolic acid were synthesized and evaluated as PTP1B inhibitors. Several derivatives exhibited moderate to good inhibitory activities against PTP1B, with 25f displaying the most promising inhibition (IC ₅₀ = 3.12 μM). Structure-activity relationship analyses of these derivatives demonstrated that the integrity of the A ring and 12-ene moieties was important in the retention of PTP1B enzyme inhibitory activities. In addition, hydrophilic and acidic groups as well as the distance between the oleanene and acid moieties were associated with PTP1B inhibitory activities. Possible binding modes of 25f were explored by molecular docking simulations.

Full text of DOI:10.1021/np100064m

J. Nat. Prod. 2010, 73, 1743–1750
1743
Synthesis and Biological Evaluation of Oleanolic Acid Derivatives As Inhibitors of Protein
Tyrosine Phosphatase 1B
Shan Qian,†,‡ Haijiao Li,‡ Yin Chen,§ Weiyu Zhang,§ Shengyong Yang,⊥ and Yong Wu*,‡
Bioengineering College, Xihua UniVersity, Chengdu 610039, People’s Republic of China, Key Laboratory of Drug Targeting and State Key
Laboratory of Biotherapy, West China School of Pharmacy, Sichuan UniVersity, Chengdu 610041, People’s Republic of China, and Drug
Screening Center, Di Ao Pharmaceutical Group, Chengdu 610041, People’s Republic of China
ReceiVed January 29, 2010
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator in the process of insulin signaling and a promising
drug target for diabetes and obesity. Derivatives of oleanolic acid were synthesized and evaluated as PTP1B inhibitors.
Several derivatives exhibited moderate to good inhibitory activities against PTP1B, with 25f displaying the most promising
inhibition (IC50 ) 3.12 µM). Structure-activity relationship analyses of these derivatives demonstrated that the integrity
of the A ring and 12-ene moieties was important in the retention of PTP1B enzyme inhibitory activities. In addition,
hydrophilic and acidic groups as well as the distance between the oleanene and acid moieties were associated with
PTP1B inhibitory activities. Possible binding modes of 25f were explored by molecular docking simulations.
Reversible phosphorylation of protein tyrosyl residues is one of
the key events that mediate the execution and regulation of many
cellular processes. A proper level of phosphorylation is critical for
these processes and is controlled by the opposing actions of protein
tyrosine kinases, which catalyze the transfer of a phosphoryl group
from adenosine 5′-triphosphate (ATP) to the p-hydroxy group of
tyrosine, and protein tyrosine phosphatases (PTPs), which hydrolyze
the phosphotyrosine (pY) back to tyrosine and inorganic phosphate.1
The human genome encodes a superfamily of approximately 100
types of PTPs, which share a highly conserved catalytic domain of
about 250 amino acids and consequently a common catalytic
mechanism.2 Protein tyrosine phosphatase 1B (PTP1B), first isolated
from human placenta, has been confirmed to play an important role
in insulin signaling.3 PTP1B represents an important but challenging
Results and Discussion
target class for drug discovery.4 The enzyme is highly specific for
OA (1) can be modified at the A ring, the C ring, and C-17,
the doubly charged pY residue, and consequently research seeking
while variations on the pentacyclic moiety were found to be
small-molecule inhibitors of PTP1B has been focused on finding
ineffective. Conformational analyses of PTP1B indicate that its
mimics of pY.5 However, the lack of drugs in clinical development
inhibitors should contain highly charged and polar functionalities,
highlights the need for potent and selective PTP1B inhibitors with
such as phosphonates and carboxylates, which occupy the active
desirable physicochemical properties and in vivo efficacies.
site of PTP1B.
Compounds derived from natural products with unique and
diverse chemical entities currently constitute a considerable resource
for developing novel medications. Oleanolic acid (OA, 1), a
pentacyclic triterpenoid, has been in active clinical use as an
antihepatitis drug in China for more than 20 years, while also
displaying hypoglycemic,6 anti-inflammatory,7 and antitumorigenic
effects,8 as well as protecting the liver against toxic injury.9
Baeyer-Villiger oxidation was utilized to modify the A ring of
OA (1) via introduction of highly charged and polar functionalities.
Jones oxidation of OA (1) furnished ketone 2, which was converted
to esters 3, 4, and 5 by esterification with Me,10 Et,11 and BnBr,12
respectively. Baeyer-Villiger oxidation of 5 was successfully
performed using 3-chloroperbenzoic acid (MCPBA) and Li2CO3,
forming the A-ring ε-lactone and preserving the C-12, C-13 double
bond. The structure of acid 6b (R ) H), formed by hydrogenolysis
of 6a (R ) Bn), was verified by elemental analyses, IR, 1H NMR,
13C NMR, and ESIMS data analysis. The 1H NMR data displayed
an olefinic hydrogen at δH 5.30. The 13C NMR spectrum displayed
resonances at δC 178.9 and 177.8, indicating the carbonyl groups
at C-28 and C-3, and resonances at δC 140.4 and 122.7, indicating
the C-12, C-13 double bond. The IR spectrum showed an ester
absorption band at 1724 cm-1, suggesting the formation of the
Given the significant biological importance and the potentially
new clinical utilities of OA (1) as a promising modulator of
glycogen metabolism, it would be of great interest to design OA
derivatives as potential PTP1B inhibitors. Therefore, we have
designed a variety of new OA derivatives with modified A and C
rings and C-17 moieties. In this article, we report the synthesis,
PTP1B inhibitory activities, and structure-activity relationships
(SAR) of these OA derivatives. The possible binding mode of 25f,
the most active compound, was established by molecular docking
simulation.
A-ring ε-lactone moiety, and an absorption band at 1699 cm-1
,
indicating the C-12, C-13 double bond. Elemental and ESIMS
analysis confirmed the structure of acid 6b. Finally, the A-ring
ε-lactone moiety of 6b (R ) H) was hydrolyzed to produce
diacid 7.
The synthesis and biological evaluation of 3,12,13-trihydroxy-
28,13-oleananolide indicated hypoglycemic activity.13 On the basis
of this result, we designed and synthesized the new OA derivative
10 (Scheme 1). Ozonolysis of ketone 213 produced 12R,13ꢀ-
* To whom correspondence should be addressed. Tel: +86-28-85503666.
Fax: +86-28-85503666. E-mail: wyong@scu.edu.cn.
† Bioengineering College, Xihua University.
‡ Key Laboratory of Drug Targeting.
§ Drug Screening Center.
⊥ State Key Laboratory of Biotherapy.
10.1021/np100064m  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/21/2010

1744 Journal of Natural Products, 2010, Vol. 73, No. 11
Scheme 1. Synthesis of 2-10a
Qian et al.
a Reagents and conditions: (a) 3 N Jones reagent, CH2Cl2/acetone, 0 °C; (b) RBr, K2CO3, DMF, rt; (c) MCPBA, Li2CO3, rt; (d) H2, Pd-C, EtOH; (e) KOH,
CH2Cl2/MeOH/H2O, rt; (f) O3, CH2Cl2, -70 °C.
Scheme 2. Synthesis of 11-15a
C-12 carbonyl group. Esterification of 11 with phthalic anhydride
yielded mono- and diesters 14 and 15, respectively (Scheme 2).
Baeyer-Villiger oxidation of 3 (R ) CH3) and 5 (R ) Bn) using
excess amounts of MCPBA and NaHCO3 yielded 16a (R ) CH3)
and 16b (R ) Bn), respectively. Subsequent hydrolysis of 16b (R
) Bn) produced A-ring cleaved acid 17. The structures of 16a (R
) CH3) and 17 were verified by elemental, IR, 1H NMR, 13C NMR,
and ESIMS analyses.
During the semisynthesis of maslinic acid starting from OA,14
we obtained benzyl 2R-hydroxy-3-oxo-olean-12-en-28-oic acid,15
which was oxidized to the A-ring cleaved acid 18 and hydrolyzed
to produce diacid 19 (Scheme 3).
Modifications of the A and C rings of OA (1) provided 2-19;
however, the C-17 carboxylic group is also important from the
perspective of SAR. Acetalization of 4 with ethylene glycol,
followed by reduction with LiAlH4, afforded the alcohol 20 (Scheme
4). Deprotection of 20 yielded 3-oxo-erythrodiol 21,10 which was
converted to oleanolic trifluoromethanesulfonate 22 and bromide
23. Esterification of ketone 21 with dicarboxylic monobenzyl
esters16 followed by deprotection of the benzyl group afforded
25a-f.
The PTP1B inhibitory activities of OA (1) and its synthetic
derivatives 2-19 were evaluated (Table 1). The 3-keto derivative
2 showed less activity than OA (1). Esterification of the C-28
carboxylic acid group (3-5) diminished the inhibitory activities
compared to 1 and 2, indicating that C-3 hydroxy and C-28
carboxylic acid groups contribute to PTP1B inhibition. The
oleanolic hydroxy-olide derivatives 8, 11, and 12 displayed lower
PTP1B inhibitory activities than the olean-12-ene-28-acids 1 and
2. Introducing 2′-carboxybenzoyl groups at C-3 and/or C-12 of 11
increased the potency 8.5 and 2.9 times for 14 and 15, respectively,
compared to 11. The A-ring ε-lactone derivatives 6b (R ) H), 9,
and 16a (R ) CH3) and the A-ring cleaved acids 7, 10, 13, 17, 18,
and 19 displayed poor inhibitory activities. These results indicate
that the integrity of the A ring and 12-ene moieties was important
for the retention of PTP1B inhibitory activities. In addition,
a Reagents and conditions: (a) O3, CH2Cl2, -70 °C; (b) 3 N Jones reagent,
CH2Cl2/acetone, 0 °C; (c) MCPBA, Li2CO3, rt; (d) KOH, CH2Cl2/MeOH/H2O,
rt; (e) phthalic anhydride, DMAP, pyridine, 120 °C.
dihydroxy-3-oxo-28,13-oleananolide 8, which was converted to
the A-ring ε-lactone 9 via Baeyer-Villiger oxidation. As shown
1
in the H NMR spectrum, the formation of 9 could be explained
by the resonances of the olide-type bridges between C3 and C4,
and C-13 and C-28. 13C NMR data displayed resonances at δC 179.6
and 177.7, indicating the carbonyl groups at C-28 and C-3, and a
resonance at δC 74.2, indicating the hydroxy group at C-12. The
structure was further confirmed via the IR absorption bands at 1742
(νCdO) and 3484 (νOH) cm-1. Lactone 9 was hydrolyzed to yield
the A-ring cleaved hydroxy acid 10.
Ozonolysis of OA (1) followed by Jones oxidation13 yielded
diketone 12 (Scheme 2). Subsequent Baeyer-Villiger oxidation and
hydrolysis of 12 yielded hydroxy acid 13. The structure of 13 was
verified by elemental, IR, 1H NMR, 13C NMR, and ESIMS analyses.
13C NMR data displayed a resonance at δC 206.0, indicating the

Oleanolic Acid DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 11 1745
Scheme 3. Synthesis of 16-19a
a Reagents and conditions: (a) MCPBA, NaHCO3, rt; (b) KOH, CH2Cl2/MeOH/H2O, rt; (c) H2, Pd-C, EtOH; (d) MCPBA, CH2Cl2/MeOH, 0 °C; (e) Pb(OAc)4,
CH2Cl2/MeOH, 0 °C.
Scheme 4. Synthesis of 20-25a
Table 1. Inhibition Percent and IC50 Values for the PTP1B
Inhibition Assay
inhibition
IC50
inhibition
IC50
(µM)
a
a
compound
%
(µM)b
compounds
%
1
2
3
4
5
6b
7
8
9
10
11
12
13
14
15
16a
17
18
11.69
8.67
1.56
1.37
1.45
14.12
-4.12
0.69
0.49
14.69
6.78
4.42
4.33
57.63
19.92
1.76
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19
20
21
22
23
24b
24b
24c
24d
24e
24f
25a
25b
25c
25d
25e
25f
15.02
2.98
1.17
1.83
7.16
1.70
10.75
8.30
5.92
3.55
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4.11 ( 1.4
4.61 ( 2.9
6.39 ( 0.9
ND
2.98
98.44
89.92
71.13
55.56
12.00
88.82
96.36
ND
3.12 ( 1.7
ND
1.29
15.60
soduim
orthovanadatec
a The max inhibition concentration is 5 µg/mL. b Values are means of
three experiments. ND: not determined. c Soduim orthovanadate was
used as the positive control, of which the max inhibition concentration
is 100 µg/mL.
a Reagents and conditions: (a) ethylene glycol, toluene, 110 °C; (b) LiAlH4,
THF, 0 °C f rt; (c) 2 N HCl, THF, 100 °C; (d) trifluoromethanesulfonic acid
anhydride, CH2Cl2, rt; (e) (CH3)3SiBr, pyridine, CH2Cl2, rt; (f) HOOC(CH2)n-
COOBn, (COCl)2, DMF/CH2Cl2, rt f 0 °C; (g) 2-(benzyloxycarbonyl)benzoic
acid, (COCl)2, DMF/CH2Cl2, rt f 0 °C; (h) H2, Pd-C, EtOH.
groups of the signature motif and the phosphate with Arg-221. pTyr
was in close proximity to Cys-215, which played a crucial role in
the catalytic reaction. The catalytic site was highly hydrophilic:
when an affinity selection was carried out to identify high-affinity
peptides for PTP1B, a clear preference for acidic residues (Glu and
Asp) was observed.17 To explore the corresponding binding site
and mode of the derivatives of OA for PTP1B inhibition, 25f was
selected for subsequent molecular docking simulation.
The catalytic domains of PTP1B are extremely highly conserved
and are deeply buried in their binding cavities. This assumption
was proven by molecular docking simulations during which the
acidic residue of 25f was buried within a deep catalytic site cleft
on the protein’s molecular surface (Figure 1). The top-ranked
binding position of 25f was determined as a preferred docking
mode. The calculated dock score was 102.091 (PLP1 ) 97.18)
(Figure 2), which was slightly inferior to the docking result against
the pyridothiophene derivative crystallized conformation of 2B0718
(calculated dock score ) 122.826; PLP1 ) 120.65). The predicted
hydrophilic and acidic groups were favorable for retention of PTP1B
inhibitory activities.
Alcohol 21 and its derivatives 22 and 23 showed less activity
than the parent acid 2. Acids 25a-c exhibited moderate to good
inhibitory activities against PTP1B (25a: IC50 ) 4.11 µM, 25b:
IC50 ) 4.61 µM, 25c: IC50 ) 6.39 µM), with SAR analyses
indicating that the distance between the oleanene and acid moieties
determined PTP1B inhibitory potency (25a > 25b > 25c > 25d >
25e). Aromatic compound 25f also exhibited 7.6-fold more potent
PTP1B inhibitory activity than the parent compound OA (1).
The crystallographic structure of PTP1B with the substrate
showed that the pTyr was buried within a pocket formed by the
catalytic site. The base of the site was formed by the PTP “signature
motif” (H/V)C(X)RS/T with the pTyr complexed with the amide

1746 Journal of Natural Products, 2010, Vol. 73, No. 11
Qian et al.
group of the triterpene moiety and the hydroxy group of Tyr-46
plus the amino group of Lys-120, respectively, which enhanced
their contribution to the stability of complex and substrate recogni-
tion. Other interactions that contributed to the high affinity of 25f
included a network of hydrophobic interactions between 25f and
Arg-24, Tyr-46, Asp-48, Val-49, Phe-182, Ala-217, Ile-219, and
Gln-262, respectively. These hydrophobic networks were critical
in maintaining good inhibitory activities, and they highlighted the
contribution of the carboxylic group at C-17 in the triterpene moiety.
In summary, 34 oleanolic derivatives with modified A and C
rings and substitutions at C-17, including 25 new compounds, were
synthesized and biologically evaluated as inhibitors of PTP1B.
Within the three series of compounds, 25f (IC50 ) 3.12 µM,
inhibition % ) 88.82%) exhibited 7.6-fold more potent PTP1B
inhibitory activity than the parent compound 1 (inhibition % )
11.69%). SAR analysis shows that the integrity of the A ring and
12-ene moieties was important in the retention of PTP1B enzyme
inhibitory activities; hydrophilic and acidic groups as well as the
distance between the oleanene and acid moieties were associated
with PTP1B inhibitory activities. Possible binding modes of 25f
were explored by molecular docking simulations. On the basis of
these results, further design and biological evaluation of OA
derivatives as promising drugs are ongoing in our laboratories, and
results will be reported in due course.
Experimental Section
Figure 1. Enlarged surface view of binding site with the residues
around ligand 25f. The residues of the binding site are shown in
plane format in off-white, and 25f is shown as a stick structure in
green. All hydrogen atoms are omitted for clarity.
General Experimental Procedures. Melting points were measured
on a YRT-3 melting point apparatus. Optical rotations were determined
at room temperature with a Perkin-Elmer 141 polarimeter. IR spectra
were obtained on a Perkin-Elmer 983. NMR spectra were recorded on
a Varian INOVA400 instrument. Chemical shifts were expressed in δ
(ppm) with TMS as the internal reference and coupling constants (J)
expressed in Hz. Mass spectra were recorded on an Agilent 1946B
ESIMS instrument. Elemental analyses were performed by Atlantic
Microlab, Atlanta, GA. TLC was performed using precoated silica gel
GF254 (0.2 mm), and flash column chromatography was performed
using silica gel (200-300 mesh).
Oleanic acid (1) was purchased from Huakang Chemical Company
Inc. (Sichuan, P.R. China). PTP1B recombinant protein was supplied
by Di Ao Pharmaceutical Group. PTP’s substrate, p-nitrophenyl
phosphate disodium hexahydrate, was supplied by Aladdin Co. Ltd.
(Shanghai, P.R. China).
Olean-12-en-28-carboxy-3-oic acid ε-lactone [6b (R ) H)]. A
solution of benzyl ester 512 (1.6 g, 3.0 mmol), MCPBA (0.5 g, 3.0
mmol), and Li2CO3 (0.9 g, 12.0 mmol) in CH2Cl2 (45 mL) was stirred
at rt for 8 h, followed by quenching with Na2SO3. The reaction mixture
was diluted with CH2Cl2, washed successively with H2O and brine,
dried (MgSO4), filtered, and concentrated to yield a residue, 6a (R )
Bn) (1.3 g), which was used in the next step without purification.
The residue 6a (1.3 g) and 10% Pd-C (54.0 mg) were dispersed in
MeOH (5 mL) and stirred under H2 at 0.8 MPa for 24 h. The mixture
was filtered, the filtrate concentrated to dryness, and the residue purified
by column chromatography (PE/EtOAc, 6:1) to give 6b (0.9 g, 64% in
two steps) as white crystals: mp >250 °C; IR (KBr) νmax 3441, 2946,
1724, 1698, 1460, 1384, 1112, 1026 cm-1; 1H NMR (CDCl3, 400 MHz)
δ 5.30 (1H, s, H-12), 2.82 (1H, dd, J ) 13.2, 3.2 Hz, H-18), 2.60 (2H,
m, H-2), 1.52 (3H, s, H-26), 1.48 (3H, s, H-27), 1.41 (3H, s, H-25),
1.14, 1.13, 0.61, 0.60 (each 3H, s, H-23, H-24, H-29, H-30); 13C NMR
(CDCl3, 100 MHz) δ 178.9 (C, C-28), 177.8 (C, C-3), 140.4 (C,
C-13),122.7 (CH, C-12), 79.8 (C, C-4), 73.2 (CH, C-5), 57.8 (C, C-17),
50.8 (CH, C-9), 47.3 (CH2, C-19), 43.7 (C, C-14), 42.6 (C, C-10), 41.7
(C, C-8), 40.4 (CH, C-18), 37.6 (CH2, C-1), 34.8 (CH2, C-21),
33.3 (CH2, C-7), 32.1 (CH2, C-2), 31.2 (CH3, C-29), 31.0 (CH3, C-30),
30.5 (CH2, C-11), 30.1 (CH2, C-16), 29.8 (CH2, C-22), 27.4 (C, C-20),
25.2 (CH2, C-15), 22.8 (CH3, C-24), 22.2 (CH3, C-23), 22.0 (CH2, C-6),
20.8 (CH3, C-27), 15.6 (CH3, C-26), 15.1 (CH3, C-25); ESIMS m/z
471 [M + H]+; anal. C 76.49%, H 9.89%, calcd for C30H46O4, C
76.55%, H 9.85%.
Figure 2. Stereoview of the binding mode of 25f within PTP1B.
The PTP1B structure is presented in the chromatic color cartoon.
The residues of the binding site are shown in line format, and 25f
is shown as a stick structure in green. All hydrogen atoms are
omitted for clarity.
binding position of the oleanolic acid moiety of 25f occupied a
similar location of the pyridothiophene inhibitor in the crystal
structure. The 18-residue PTP signature motif, which was close to
25f, was coordinated with 25f by many varieties of interactions
that contained the Arg-221 residue (Figure 2). The residue was
essential in maintaining conformation by optimizing salt bridge
interactions.
Olean-12-en-4-hydroxy-3,28-dioic acid (7). A solution of lactone
6 (100.0 mg, 0.2 mmol) and KOH (297.0 g, 5.3 mmol) in CH2Cl2/
MeOH/H2O (14 mL, 3:3:1) was stirred at rt for 4 h. The residue was
extracted with CH2Cl2, and the extract was washed successively with
In the binding mode of 25f with residues around the docked
position (Figure 3), hydrogen bonds formed between the carboxylic

Oleanolic Acid DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 11 1747
Figure 3. Binding mode of 25f with residues around the docked position.
H2O and HCl (1 M). The organic layer was washed with brine, dried
(MgSO4), filtered, and concentrated. The residue was purified by column
chromatography (CH2Cl2/MeOH, 10:1) to give colorless crystals of 7
76.8 (C, C-4), 74.2 (C, C-12), 57.6 (CH, C-5), 48.9 (CH, C-9), 45.1
(C, C-17), 42.9 (C, C-8), 42.7 (CH, C-18), 42.1 (C, C-14), 41.2 (C,
C-10), 36.3 (CH2, C-19), 34.1 (CH2, C-7), 34.0 (CH2, C-21), 33.2 (CH2,
C-2), 33.0 (CH2, C-1), 32.8 (CH3, C-29), 32.0 (CH3, C-30), 31.7 (C,
C-20), 31.8 (CH2, C-11), 25.6 (CH2, C-22), 25.2 (CH2, C-15), 24.5
(CH3, C-23), 24.1 (CH3, C-24), 23.3 (CH2, C-6),20.5 (CH3, C-27), 20.1
(CH2, C-16), 17.0 (CH3, C-26), 15.7 (CH3, C-25); ESIMS m/z 487 [M
+ H]+; anal. C 74.19%, H 9.59%, calcd for C30H46O5, C 74.04%, H
9.53%.
Olean-4,12r-dihydroxy-3-carboxy-28-oic acid γ-lactone (10).
Following the procedure described for the preparation of 7, 10 was
prepared from 9 as a white, amorphous solid (75%): 1H NMR (DMSO-
d6, 400 MHz) δ 11.60 (1H, s, COOH), 5.08 (1H, s, OH), 4.08 (1H, s,
OH), 3.65 (1H, s, H-12), 2.10-2.40 (3H, m, H-18, H-2), 1.25 (3H, s,
H-26), 1.16 (3H, s, H-27), 1.15 (3H, s, H-25), 1.06, 0.92, 0.91, 0.86
(each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z 505 [M + H]+;
anal. C 71.56%, H 9.63%, calcd for C30H48O6, C 71.39%, H 9.59%.
Olean-12-oxo-4-hydroxy-3-carboxy-28-oic acid γ-lactone (13).
Following the procedure described for the preparation of 10, 13 was
prepared from 1213 as white crystals (52% in two steps): mp 230-231
°C; IR (KBr) νmax 3449, 2961, 2879, 1739, 1702, 1465, 1386, 1269,
1196, 932 cm-1; 1H NMR (CDCl3, 400 MHz) δ 2.70 (1H, t, J ) 14.0
Hz, H-18), 2.40-2.58 (2H, m, H-2), 1.36 (3H, s, H-26), 1.32 (3H, s,
H-27), 1.28 (3H, s, H-25), 1.07, 0.98, 0.97, 0.96 (each 3H, s, H-23,
H-24, H-29, H-30); 13C NMR (CDCl3, 100 MHz) δ 206.0 (C, C-12),
178.9 (C, C-28), 178.0 (C, C-3), 77.3 (C, C-13), 77.0 (C, C-4), 76.6
(CH, C-5), 50.9 (CH, C-9), 44.3 (C, C-17), 43.9 (C, C-14), 43.7 (C,
C-8), 43.0 (CH, C-18), 42.2 (CH2, C-11), 41.2 (C, C-10), 37.3 (CH2,
C-19), 36.8 (CH2, C-1), 34.3 (CH2, C-7), 34.0 (CH2, C-21), 33.5 (CH2,
(76 mg, 73%): mp >250 °C; [R]25 +34.0 (c 0.5, MeOH); IR (KBr)
D
1
νmax 3439, 2948, 1703, 1462, 1383, 1271, 1191, 933 cm-1; H NMR
(DMSO-d6, 400 MHz) δ 11.90 (1H, s, COOH), 5.20 (1H, s, H-12),
2.78 (1H, dd, J ) 13.6, 4.0 Hz, H-18), 2.42 (1H, m, H-2a), 2.24 (1H,
m, H-2b), 1.15 (3H, s, H-26), 1.12 (3H, s, H-27), 1.10 (3H, s, H-25),
1.00, 0.89, 0.88, 0.68 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS
m/z 489 [M + H]+; anal. C 73.67%, H 9.99%, calcd for C30H48O5, C
73.73%, H 9.90%.
3-Oxo-olean-12r-hydroxy-28-oic acid γ-lactone (8). According to
the reported method,13 colorless crystals of 8 were obtained by
ozonolysis of 2 via chromatography (CH2Cl2/MeOH, 20:1): yield 62%;
mp >250 °C; [R]25D +35.6 (c 0.5, MeOH); 1H NMR (CDCl3, 400 MHz)
δ 3.92 (1H, s, H-12), 2.50-2.60 (1H, m, H-18), 2.40-2.50 (2H, m,
H-2), 1.31 (3H, s, H-26), 1.19 (3H, s, H-27), 1.10 (3H, s, H-25), 1.04,
0.99, 0.98, 0.90 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z
471 [M + H]+; anal. C 76.68%, H 9.90%, calcd for C30H46O4, C
76.55%, H 9.85%.
Olean-12r-hydroxy-3-ε-lacton-28-oic acid γ-lactone (9). Following
the procedure described for the preparation of 6, compound 9 was
prepared from 8 as white crystals (88%): mp 210-211 °C; [R]25D +35.6
(c 0.5, CH2Cl2); IR (KBr) νmax 3484, 2935, 2872, 1742, 1460, 1388,
1
1367, 1251, 1137, 1114, 946 cm-1; H NMR (CDCl3, 400 MHz) δ
3.92 (1H, s, H-12), 2.65 (1H, t, J ) 13.6 Hz, H-18), 2.40-2.50 (1H,
m, H-2), 1.48 (3H, s, H-26), 1.40 (3H, s, H-27), 1.28 (3H, s, H-25),
1.20, 1.10, 0.97, 0.89 (each 3H, s, H-23, H-24, H-29, H-30); 13C NMR
(CDCl3, 100 MHz,) δ 179.0 (C, C-28), 177.7 (C, C-3), 77.0 (C, C-13),

1748 Journal of Natural Products, 2010, Vol. 73, No. 11
Qian et al.
C-2), 33.1 (CH3, C-29), 32.1 (CH3, C-30), 31.5 (C, C-20), 28.6 (CH2,
C-22), 27.2 (CH3, C-23), 27.1 (CH3, C-24), 23.7 (CH2, C-15), 21.5
(CH3, C-27), 20.6 (CH2, C-6), 20.1 (CH2, C-16), 18.0 (CH3, C-26),
17.7 (CH3, C-25); ESIMS m/z 503 [M + H]+; anal. C 71.73%, H
9.28%, calcd for C30H46O6, C 71.68%, H 9.22%.
stirring for 50 min, the solution was filtered. The filtrate was evaporated
in vacuo to a residue. The residue was purified by column chroma-
tography (PE/EtOAc, 6:1) to give a colorless, amorphous solid, 18 (480
mg, 47%): IR (KBr) νmax 2959, 2928, 1724, 1460, 1381, 1255, 1148,
1030, 747, 699 cm-1; 1H NMR (CDCl3, 400 MHz) δ 9.82 (1H, s, CHO),
7.38 (5H, m, H-Ar), 5.25 (1H, t, J ) 3.6 Hz, H-12), 5.08 (1H, d, J )
12.8 Hz, PhCH2), 5.06 (1H, d, J ) 12.8 Hz, PhCH2), 3.58 (3H, s, CH3),
2.90 (1H, dd, J ) 13.6, 4.4 Hz, H-18), 1.25 (3H, s, H-26), 1.20 (3H,
s, H-27), 1.10 (3H, s, H-25), 0.98, 0.90, 0.89, 0.62 (each 3H, s, H-23,
H-24, H-29, H-30); ESIMS m/z 591 [M + H]+; anal. C 77.28%, H
9.28%, calcd for C38H54O5, C 77.25%, H 9.21%.
Following the procedure described for the preparation of 6,
compound 19 was prepared from 18 as a white, amorphous solid (91%):
IR (KBr) νmax 2926, 2860, 1723, 1460, 1380, 1259, 1142, 1028, 755
cm-1; 1H NMR (CDCl3, 400 MHz) δ 9.82 (1H, s, CHO), 5.30 (1H, s,
H-12), 3.58 (3H, s, CH3), 2.80 (1H, dd, J ) 13.6, 4.4 Hz, H-18), 1.25
(3H, s, H-26), 1.20 (3H, s, H-27), 1.10 (3H, s, H-25), 0.98, 0.90, 0.89,
0.62 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z 501 [M +
H]+; anal. C 74.48%, H 9.63%, calcd for C31H48O5, C 74.36%, H
9.66%.
3ꢀ-(2′-Carboxybenzoyl)-12r-hydroxy-28-oic acid γ-lactone (14)
and 3ꢀ,12r-(2′-Carboxybenzoyl)-28-oic acid γ-lactone (15). A solu-
tion of 1113 (300.0 mg, 0.6 mmol), phthalic anhydride (375.5 mg, 2.5
mmol), and DMAP (279.0 mg, 2.5 mmol) in dry pyridine (32 mL)
was stirred at 120 °C for 8 h, then concentrated. The residue was diluted
with CH2Cl2, and the extract was washed successively with HCl (1M),
H2O, and brine, dried (MgSO4), filtered, and concentrated. The residue
was purified by column chromatography (CH2Cl2/MeOH, 100:1) to give
colorless crystals of 14 (208.3 mg, 56%) and 15 (129.2 mg, 28%). 14:
mp >250 °C. [R]25D +28.5 (c 0.5, MeOH); IR (KBr) νmax 3525, 2950,
2873, 1735, 1465, 1396, 1289, 1136, 1073, 942 cm-1; 1H NMR (CDCl3,
400 MHz) δ 7.89 (1H, t, J ) 6.0, 2.4 Hz, H-Ar), 7.78 (1H, t, J ) 6.4,
2.0 Hz, H-Ar), 7.60 (2H, t, J ) 3.6 Hz, H-Ar), 4.79 (1H, dd, J ) 12.0,
4.8 Hz, H-3), 3.88 (1H, s, H-12), 1.32 (3H, s, H-26), 1.26 (3H, s, H-27),
1.15 (3H, s, H-25), 0.98, 0.97, 0.96, 0.90 (each 3H, s, H-23, H-24,
H-29, H-30); ESIMS m/z 622 [M + H]+; anal. C 73.59%, H 8.47%,
calcd for C38H52O7, C 73.52%, H 8.44%. 15: mp >250 °C; [R]25D +36.2
(c 0.5, CH2Cl2); IR (KBr) νmax 3462, 3071, 2955, 1775, 1724, 1467,
1288, 1131, 945 cm-1; 1H NMR (CDCl3, 400 MHz) δ 7.90 (1H, dd, J
) 7.6, 1.2 Hz, H-Ar), 7.78 (1H, d, J ) 6.8 Hz, H-Ar), 7.60 (6H, m,
H-Ar), 5.28 (1H, s, H-12), 4.79 (1H, dd, J ) 12.0, 4.8 Hz, H-3), 1.28
(3H, s, H-26), 1.20 (3H, s, H-27), 1.15 (3H, s, H-25), 0.92, 0.91, 0.88,
0.81 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z 790 [M +
H]+; anal. C 71.97%, H 7.39%, calcd for C46H56O10, C 71.85%, H
7.34%.
3-Spiro[1′,2′]dioxolanerythrodiol (20). The solution of 4 (2.3 g,
4.7 mmol), ethylene glycol (3 mL, 52.0 mmol), and pyridinium
p-toluenesulfonate (65.0 mg) in dry toluene (50 mL) was refluxed at
110 °C for 4 h by using the toluene constant distilling dehydrating
method. The reaction mixture was quenched cautiously by the
subsequent addition of saturated NaHCO3 and brine, dried (MgSO4),
filtered, and concentrated to give the crude ester, which was used in
the next step without purification.
LiAlH4 (0.3 g, 8.2 mmol) was suspended in THF (30 mL) and added
to a solution of the crude ester (2.1 g) in THF (30 mL) dropwise at 0
°C. The reaction mixture was left for a further 3 h at rt. The excess
reagent was destroyed by addition of H2O (2 mL) and 10% KOH (2
mL). The reaction mixture was then filtered and concentrated. The
residue was purified by column chromatography (PE/EtOAc, 5:1) to
give white crystals of 20 (1.8 g, 85% in two steps): mp 235-237 °C;
[R]25D +21.8 (c 0.5, CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 5.20 (1H,
t, J ) 3.6 Hz, H-12), 3.89-3.97 (4H, m, OCH2CH2O), 3.58 (1H, d, J
) 10.8 Hz, CH2OH), 3.21 (1H, d, J ) 10.8 Hz, CH2OH), 1.17 (3H, s,
H-26), 0.97 (3H, s, H-27), 0.93 (3H, s, H-25), 0.90, 0.89, 0.87, 0.85
(each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z 486 [M + H]+;
anal. C 9.44%, H 10.84%, calcd for C32H52O3: C 79.29%, H 10.81%.
3-Oxo-erythrodiol (21). To a solution of alcohol 20 (1.4 g, 3.0
mmol) in THF (50 mL) was added 2 N HCl (15 mL), and the reaction
mixture was stirred at 100 °C for 1 h. The organic layer was separated,
and the water layer was exacted with EtOAc. The collected organic
layer was washed with brine, dried (MgSO4), filtered, and concentrated.
The residue was purified by column chromatography (PE/EtOAc, 5:1)
to give colorless crystals (1.3 g, 96%): mp 160-161 °C; [R]25D +50.7
Olean-12-oxo-28-methoxycarbonyl-3-oic acid ε-lactone [16a (R
) CH3)]. A solution of methyl ester 310 (60 mg, 0.1 mmol), MCPBA
(62.2 mg, 0.3 mmol), and NaHCO3 (83.4 mg, 1.0 mmol) in CH2Cl2
(16 mL) was stirred at 40 °C for 24 h, and the reaction was quenched
with Na2SO3. The residue was diluted with CH2Cl2, and the extract
was washed successively with H2O and brine, dried (MgSO4), filtered,
and concentrated. The residue was purified by column chromatography
(PE/EtOAc, 10:1) to give a colorless, amorphous solid, 16a (28 mg,
1
47%): H NMR (CDCl3, 400 MHz) δ 3.70 (3H, s, OCH3), 2.80 (1H,
m, H-2a), 2.58-2.66 (3H, m, H-2b, H-11), 1.48 (3H, s, H-26), 1.44
(3H, s, H-27), 1.18 (3H, s, H-25), 1.12, 0.98, 0.96, 0.90 (each 3H, s,
H-23, H-24, H-29, H-30); 13C NMR (CDCl3, 100 MHz) δ 210.7 (C,
C-12), 77.3 (C, C-4), 77.0 (C, C-13), 76.6 (CH, C-5), 54.0 (CH3, OCH3),
51.8 (CH, C-9), 51.7 (C, C-17), 50.7 (C, C-14), 50.7 (C, C-8), 49.6(C,
C-10), 38.9 (CH2, C-19), 37.5 (CH2, C-11), 36.1(CH2, C-1), 34.4 (CH,
C-18), 33.3 (CH2, C-7), 32.8 (CH2, C-21), 32.1 (CH2, C-2), 31.9 (CH3,
C-29), 31.8 (CH3, C-30), 30.8 (CH2, C-16), 30.6 (CH2, C-22), 27.4 (C,
C-20), 25.8 (CH2, C-15), 23.0 (CH3, C-24), 22.9 (CH3, C-23), 22.7
(CH2, C-6), 20.2 (CH3, C-27), 16.4 (CH3, C-26), 15.5 (CH3, C-25);
ESIMS m/z 501 [M + H]+; anal. C 74.45%, H 9.70%, calcd for
C31H48O5, C 74.36%, H 9.66%.
1
(c 0.5, CH2Cl2); IR (KBr) νmax 3500, 1705 cm-1; H NMR (CDCl3,
400 MHz) δ 5.22 (1H, t, J ) 4.0 Hz, H-12), 3.58 (1H, d, J ) 10.8 Hz,
CH2OH), 3.21 (1H, d, J ) 10.8 Hz, CH2OH), 2.56 (1H, m, H-2a),
2.38 (1H, m, H-2b), 1.78 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H,
s, H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30);
ESIMS m/z 442 [M + H]+; anal. C 81.89%, H 11.05%, calcd for
C30H48O2, C 81.76%, H 10.98%.
Olean-12-oxo-4-hydroxy-3,28-dioic acid (17). Following the pro-
cedure described for the preparation of 16a (R ) CH3), benzyl ester
16b (R ) Bn) was prepared from 5 as a white solid. Following the
procedure described for the preparation of 7, 17 was prepared from
benzyl ester as white crystals (31% in three steps): mp 190-192 °C;
3-Oxo-12-enolean-28-oic acid trifluoromethanesulfonate (22). A
solution of alcohol 21 (1.0 g, 2.0 mmol) in anhydrous CH2Cl2 (50 mL)
was added to the solution of trifluoromethanesulfonic acid anhydride
(564 mg, 2.0 mmol) in CH2Cl2 (10 mL), and the reaction mixture was
stirred at rt for 2 h under an N2 layer. The suspension was filtered, and
the filtrate was washed with H2O and brine, dried (MgSO4), filtered,
and concentrated. The residue was purified by column chromatography
1
IR (KBr) νmax 3444, 2951, 1697, 1465, 1387, 1192, 1108 cm-1; H
NMR (DMSO-d6, 400 MHz) δ 12.0 (1H, s, COOH), 4.05 (1H, s, OH),
2.60 (2H, m, H-2), 1.18 (3H, s, H-26), 1.16 (3H, s, H-27), 1.05 (3H,
s, H-25), 1.03, 1.00, 0.98, 0.96 (each 3H, s, H-23, H-24, H-29, H-30);
13C NMR (DMSO-d6, 100 MHz) δ 210.7 (C, C-12), 179.2 (C, C-28),
175.5 (C, C-3), 74.1 (C, C-13), 55.1 (C, C-4), 51.1 (CH, C-9), 46.2
(CH, C-5), 41.7 (C, C-17), 41.2 (C, C-14), 40.7 (C, C-8), 40.4 (C,
C-10), 40.3 (CH2, C-19), 40.1 (CH2, C-11), 39.9 (CH2, C-1), 39.7 (CH,
C-18), 39.5 (CH2, C-7), 39.2 (CH2, C-21), 39.0 (CH2, C-2), 37.7 (CH3,
C-29), 34.1 (CH3, C-30), 33.7 (CH2, C-16), 33.5 (CH2, C-22), 31.6 (C,
C-20), 30.6 (CH2, C-15), 30.5 (CH3, C-24), 28.5 (CH3, C-23), 27.9
(CH2, C-6), 23.3 (CH3, C-27), 19.7 (CH3, C-26), 15.6 (CH3, C-25);
ESIMS m/z 505 [M + H]+; anal. C 71.43%, H 9.64%, calcd for
C30H48O6, C 71.39%, H 9.59%.
(PE/EtOAc, 100:1) to give colorless semisolid 22 (0.9 g, 70%): [R]25
D
1
+32.8 (c 0.5, CH2Cl2); H NMR (CDCl3, 400 MHz) δ 5.22 (1H, t, J
) 3.6 Hz, H-12), 4.50 (1H, d, J ) 9.6 Hz, CH2SO3CF3), 3.34 (1H, d,
J ) 9.6 Hz, CH2SO3CF3), 2.58 (1H, m, H-2a), 2.38 (1H, m, H-2b),
1.20 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H, s, H-25), 1.05, 1.00,
0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z 573 [M
+ H]+; anal. C 65.12%, H 8.22%, S 5.54%, calcd for C31H47F3O4S, C
65.01%, H 8.27%, S 5.60%.
3-Oxo-12-ene-28-bromo-oleanane (23). To a stirred solution of
alcohol 21 (1.5 g, 3.4 mmol) in a dry mixture of CH2Cl2 (10 mL) and
pyridine (2 mL) was added (CH3)3SiBr (1.8 mL, 13.6 mmol) at 0 °C,
and the reaction mixture was left for a further 2 h at rt. The reaction
mixture was quenched cautiously by the addition of H2O. The organic
Olean-12-ene-2-formyl-28-carboxy-3-oic acid methyl ester (19).
To a stirred solution of olean-2R-hydroxy-3-oxo-12-en-28-oic acid15
(1.0 g, 1.8 mmol) in a mixture of MeOH (30 mL) and CH2Cl2 (10
mL), cooled to 0 °C, was added Pb(OAc)4 (1.6 g, 3.6 mmol). After

Oleanolic Acid DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 11 1749
layer was separated and washed with H2O and brine, dried (MgSO4),
filtered, and concentrated. The residue was purified by column
chromatography (PE/EtOAc, 100:1) to give an amorphous solid, 23
(1.2 g, 71%): [R]25D +34.8 (c 0.5, CH2Cl2); 1H NMR (CDCl3, 400 MHz)
δ 5.20 (1H, t, J ) 3.6 Hz, H-12), 3.21 (1H, d, J ) 10.0 Hz, CH2Br),
3.05 (1H, d, J ) 9.6 Hz, CH2Br), 2.58 (1H, m, H-2a), 2.38 (1H, m,
H-2b), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H, s, H-25), 1.05,
1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z
505 [M + H]+; anal. C 71.62%, H 9.47%, calcd for C30H47BrO, C
71.55%, H 9.41%.
3-Oxo-28-(succinyl-4′)erythrane (25b). Following the procedure
described for the preparation of 19, compound 25b was prepared from
25b as white crystals (98.3 mg, 91%): mp 138-140 °C; [R]25D +34.0
(c 1.3, CH2Cl2); IR (KBr) νmax 3279, 2945, 2865, 1743, 1703, 1462,
1387, 1161, 1001, 820 cm-1; 1H NMR (CDCl3, 400 MHz) δ 5.21 (1H,
t, J ) 3.6 Hz, H-12), 4.04 (1H, d, J ) 11.2 Hz, OCH2), 3.78 (1H, d,
J ) 11.6 Hz, OCH2), 2.68 (4H, m, H-2′, H-3′), 2.58 (1H, m, H-2a),
2.38 (1H, m, H-2b), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H,
s, H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30);
ESIMS m/z 541 [M + H]+; anal. C 75.64%, H 9.72%, calcd for
C34H52O5, C 75.51%, H 9.69%.
3-Oxo-28-(benzylsuccinyl-4′-)erythrane (24b). To a solution of
succinic acid monobenzyl ester16 (280.8 mg, 1.4 mmol) in dry CH2Cl2
(10 mL) was added dropwise a solution of oxalyl chloride (173 µL,
2.0 mmol) in dry CH2Cl2 (10 mL) and DMF (one drop) at 0 °C. After
the excess oxalyl chloride was removed in vacuo, the residual oil was
dissolved in CH2Cl2 (10 mL). A solution of alcohol 21 (0.6 g, 1.4 mmol)
obtained from the previous step in dry CH2Cl2 (10 mL) and Et3N (0.4
mL) was added dropwise at 0 °C. After stirring for another 1 h at rt,
the mixture was diluted with CH2Cl2. The organic layer was washed
with H2O and brine, dried over anhydrous Na2SO4, and concentrated
in vacuo. The residue was purified by column chromatography (PE/
EtOAc, 20:1) to give colorless crystals of 24b (0.8 g, 86%): mp 66-68
Compounds 25a-25e were prepared according to the same procedure
described for 25b.
3-Oxo-28-(malonyl-4′)erythrane (25a): colorless crystals: mp
78-80 °C; [R]25D +34.6 (c 0.5, CH2Cl2); 1H NMR (CDCl3, 400 MHz)
δ 5.22 (1H, s, H-12), 4.18 (1H, d, J ) 10.8 Hz, OCH2), 3.78 (1H, d,
J ) 10.8 Hz, OCH2), 3.46 (2H, s, H-2′), 2.58 (1H, m, H-2a), 2.38 (1H,
m, H-2b), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H, s, H-25),
1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS
m/z 527 [M + H]+; anal. C 75.29%, H 9.61%, calcd for C33H50O5, C
75.25%, H 9.57%.
3-Oxo-28-(glutaryl-4′)erythrane (25c): amorphous solid; 1H NMR
(CDCl3, 400 MHz) δ 5.21 (1H, t, J ) 3.6 Hz, H-12), 4.02 (1H, d, J )
10.8 Hz, OCH2), 3.78 (1H, d, J ) 11.2 Hz, OCH2), 2.58 (1H, m, H-2a),
2.44 (4H, m, H-2′, H-4′), 2.38 (1H, m, H-2b), 1.16 (3H, s, H-26), 1.10
(3H, s, H-27), 1.07 (3H, s, H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s,
H-23, H-24, H-29, H-30); ESIMS m/z 555 [M + H]+; anal. C 75.85%,
H 9.86%, calcd for C35H54O5, C 75.77%, H 9.81%.
°C; [R]25 +31.7 (c 0.5, CH2Cl2); IR (KBr) νmax 2948, 2866, 1739,
D
1
1703, 1460, 1386, 1154, 999 cm-1; H NMR (CDCl3, 400 MHz) δ
7.39 (5H, m, H-Ar), 5.21 (1H, t, J ) 3.6 Hz, H-12), 5.19 (2H, s,
PhCH2), 4.04 (1H, d, J ) 10.8 Hz, OCH2), 3.67 (1H, d, J ) 10.8 Hz,
OCH2), 2.70 (4H, m, H-2′, H-3′), 2.58 (1H, m, H-2a), 2.38 (1H, m,
H-2b), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H, s, H-25), 1.05,
1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z
632 [M + H]+; anal. C 78.12%, H 9.34%, calcd for C41H58O5, C
78.05%, H 9.27%.
3-Oxo-28-(adipoyl-4′)erythrane (25d): amorphous solid; 1H NMR
(CDCl3, 400 MHz) δ 5.21 (1H, s, H-12), 4.02 (1H, d, J ) 11.2 Hz,
OCH2), 3.77 (1H, d, J ) 11.2 Hz, OCH2), 2.59 (1H, m, H-2a), 2.39
(5H, m, H-2b, H-2′, H-5′), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07
(3H, s, H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29,
H-30); ESIMS m/z 570 [M + H]+; anal. C 76.18%, H 9.96%, calcd
for C36H56O5, C 76.01%, H 9.92%.
Compounds 24a-24e were prepared according to the same procedure
described for 24b.
3-Oxo-28-(benzylmalonyl-4′)erythrane (24a): amorphous solid;
[R]25D +28.4 (c 0.5, CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 7.38 (5H,
m, H-Ar), 5.21 (1H, t, J ) 3.6 Hz, H-12), 5.19 (2H, s, PhCH2), 4.05
(1H, d, J ) 10.8 Hz, OCH2), 3.68 (1H, d, J ) 10.8 Hz, OCH2), 3.46
(2H, s, H-2′), 2.58 (1H, m, H-2a), 2.38 (1H, m, H-2b), 1.16 (3H, s,
H-26), 1.10 (3H, s, H-27), 1.07 (3H, s, H-25), 1.05, 1.00, 0.89, 0.87
(each 3H, s, H-23, H-24, H-29, H-30); ESIMS m/z 617 [M + H]+;
anal. C 77.92%, H 9.12%, calcd for C40H56O5, C 77.88%, H 9.15%.
3-Oxo-28-(benzylglutaryl-4′)erythrane (24c): amorphous solid;
[R]25D +28.2 (c 0.5, CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 7.38 (5H,
m, H-Ar), 5.21 (1H, t, J ) 3.6 Hz, H-12), 5.19 (2H, s, PhCH2), 4.05
(1H, d, J ) 10.8 Hz, OCH2), 3.68 (1H, d, J ) 10.8 Hz, OCH2), 2.58
(1H, m, H-2a), 2.35-2.45 (5H, m, H-2b, H-2′, H-4′), 1.16 (3H, s, H-26),
1.10 (3H, s, H-27), 1.07 (3H, s, H-25), 1.05, 1.00, 0.89, 0.87 (each
3H, s, H-23, H-24, H-29, H-30); ESIMS m/z 645 [M + H]+; anal. C
78.34%, H 9.41%, calcd for C42H60O5, C 78.22%, H 9.38%.
3-Oxo-28-(benzyladipoyl-4′)erythrane (24d): amorphous solid; 1H
NMR (CDCl3, 400 MHz) δ 7.38 (5H, m, H-Ar), 5.21 (1H, t, J ) 3.6
Hz, H-12), 5.19 (2H, s, PhCH2), 4.05 (1H, d, J ) 10.8 Hz, OCH2),
3.68 (1H, d, J ) 10.8 Hz, OCH2), 2.58 (1H, m, H-2a), 2.39 (5H, m,
H-2b, H-2′, H-5′), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H, s,
H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30);
ESIMS m/z 659 [M + H]+; anal. C 78.52%, H 9.41%, calcd for
C43H62O5, C 78.38%, H 9.48%.
3-Oxo-28-(sebacoyl-4′)erythrane (25e): amorphous solid; 1H NMR
(CDCl3, 400 MHz) δ 5.22 (1H, t, J ) 3.2 Hz, H-12), 4.39 (1H, d, J )
10.8 Hz, OCH2), 3.95 (1H, d, J ) 11.2 Hz, OCH2), 2.58 (1H, m, H-2a),
2.30-2.40 (5H, m, H-2b, H-2′, H-9′), 1.16 (3H, s, H-26), 1.10 (3H, s,
H-27), 1.07 (3H, s, H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23,
H-24, H-29, H-30); ESIMS m/z 626 [M + H]+; anal. C 76.92%, H
10.36%, calcd for C40H64O5, C 76.88%, H 10.32%.
3-Oxo-28-(phthaloyl-4′)-erythrane (25f): colorless crystals; mp
92-94 °C; [R]25D +30.7 (c 0.5, CH2Cl2); 1H NMR (CDCl3, 400 MHz)
δ 7.97 (1H, dd, J ) 6.4, 0.8 Hz, H-Ar), 7.74 (1H, d, J ) 7.6 Hz,
H-Ar), 7.60 (2H, m, H-Ar), 5.22 (1H, t, J ) 3.2 Hz, H-12), 4.39 (1H,
d, J ) 10.8 Hz, OCH2), 3.95 (1H, d, J ) 11.2 Hz, OCH2), 2.59 (1H,
m, H-2a), 2.39 (1H, m, H-2b), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27),
1.07 (3H, s, H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24,
H-29, H-30); ESIMS m/z 590 [M + H]+; anal. C 77.68%, H 8.94%,
calcd for C38H52O5, C 77.51%, H 8.90%.
Enzymatic-Activity Assays. With a spectrophotometer, the inhibi-
tory activities of all samples against PTP1B (recombinant protein
obtained from Escherichia coli BL21 expression system) were measured
at 37 °C with 0.2 units/mL enzyme in a buffer (25 mM HEPES, 50
mM NaCl, 2.5 mM EDTA, 0.1% BSA, pH 7.2). Sodium orthovanadate
(100 µg/mL) was used as positive control. The reaction system was
stirred for 10 min at 37 °C, and the substrate of PTP (p-nitrophenyl
phosphate disodium hexahydrate) was added. After 30 min, 2 mol/mL
Na2CO3 was added to terminate the reaction. The OD value was
monitored continuously with a spectrophotometer at 405 nm. The assay
result was provided by the drug screening center of Di Ao Pharmaceuti-
cal Group.
Molecular Docking Simulation. To further identify the putative
binding site and corresponding binding comformation of the com-
pounds, molecular docking simulation was performed against the
PTP1B protein with Accelrys Discovery 1.7 (Accelrys Software Inc.,
USA). The high-resolution crystal structure of PTP1B complexed with
pyridothiophene inhibitor was obtained from the Protein Data Bank
(PDB code: 2B0718). All cocrystallized ligands and water were
removed. The starting 3D conformations of 25f were prepared with
Dock Ligands Fit. The calculated dock score and PLP1 of all docking
positions were evaluated. The best ranked position from each of the
binding sites was determined by the highest calculated dock score and
PLP1.
3-Oxo-28-(benzylsebacoyl-4′)erythrane (24e): amorphous solid; 1H
NMR (CDCl3, 400 MHz) δ 7.38 (5H, m, H-Ar), 5.21 (1H, t, J ) 3.6
Hz, H-12), 5.19 (2H, s, PhCH2), 4.05 (1H, d, J ) 10.8 Hz, OCH2),
3.68 (1H, d, J ) 10.8 Hz, OCH2), 2.58 (1H, m, H-2a), 2.39 (5H, m,
H-2b, H-2′, H-9′), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H, s,
H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30);
ESIMS m/z 715 [M + H]+; anal. C 79.08%, H 9.90%, calcd for
C47H70O5, C 78.95%, H 9.87%.
3-Oxo-28-(benzylphthaloyl-4′)erythrane (24f): amorphous solid;
[R]25D +31.3 (c 0.5, CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 7.78 (2H,
m, H-Ar), 7.58 (2H, m, H-Ar), 7.38 (4H, m, H-Ar), 5.60 (2H, m,
PhCH2), 5.21 (1H, t, J ) 3.6 Hz, H-12), 4.05 (1H, d, J ) 10.8 Hz,
OCH2), 3.68 (1H, d, J ) 10.8 Hz, OCH2), 2.58 (1H, m, H-2a), 2.39
(1H, m, H-2b), 1.16 (3H, s, H-26), 1.10 (3H, s, H-27), 1.07 (3H, s,
H-25), 1.05, 1.00, 0.89, 0.87 (each 3H, s, H-23, H-24, H-29, H-30);
ESIMS m/z 679 [M + H]+; anal. C 79.69%, H 8.69%, calcd for
C45H58O5, C 79.61%, H 8.61%.

1750 Journal of Natural Products, 2010, Vol. 73, No. 11
Qian et al.
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Acknowledgment. This work was supported by the Key Scientific
Research Foundation of Xihua University, Scientific Research Fund
of Sichuan Provincial Education Department, and Science and Technol-
ogy Planning Project of Sichuan Province, China (05SG011-021).
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