Oleanolic acid and its derivatives: New inhibitor of protein tyrosine phosphatase 1B with cellular activities

Article abstract of DOI:10.1016/j.bmc.2008.07.080

Protein tyrosine phosphatase 1B is a key factor in the negative regulation of insulin pathway and a promising target for treatment of diabetes and obesity. Herein, a series of competitive inhibitors were optimized from oleanolic acid, a natural triterpenoid identified against PTP1B by screening libraries of traditional Chinese medicinal herbs. Modifying at 3 and 28 positions, we obtained compound 13 with a K_i of 130 nM, which exhibited good selectivity between other phosphatases involved in insulin pathway except T-cell protein tyrosine phosphatase. Further evaluation in cell models illustrated that the derivatives enhanced insulin receptor phosphorylation in CHO/hIR cells and also stimulated glucose uptake in L6 myotubes with or addition of without insulin.

Full text of DOI:10.1016/j.bmc.2008.07.080

Bioorganic & Medicinal Chemistry 16 (2008) 8697–8705
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Oleanolic acid and its derivatives: New inhibitor of protein tyrosine phosphatase
1B with cellular activities
a,
Yi-Nan Zhang a, Wei Zhang b, Di Hong a, Lei Shi b, Qiang Shen b, Jing-Ya Li b, Jia Li b, , Li-Hong Hu
*
*
a Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Science,
Chinese Academy of Sciences, 199 Guo Shou Jing Road, Shanghai 201203, China
b National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, 189 Guo Shou Jing Road,
Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein tyrosine phosphatase 1B is a key factor in the negative regulation of insulin pathway and a prom-
ising target for treatment of diabetes and obesity. Herein, a series of competitive inhibitors were opti-
mized from oleanolic acid, a natural triterpenoid identified against PTP1B by screening libraries of
traditional Chinese medicinal herbs. Modifying at 3 and 28 positions, we obtained compound 13 with
a Ki of 130 nM, which exhibited good selectivity between other phosphatases involved in insulin pathway
except T-cell protein tyrosine phosphatase. Further evaluation in cell models illustrated that the deriva-
tives enhanced insulin receptor phosphorylation in CHO/hIR cells and also stimulated glucose uptake in
L6 myotubes with or addition of without insulin.
Received 8 July 2008
Revised 28 July 2008
Accepted 29 July 2008
Available online 3 August 2008
Keywords:
Protein tyrosine phosphatase 1B
Oleanolic acid
Ó 2008 Elsevier Ltd. All rights reserved.
Inhibitor
SAR (structure–activity relationship)
1. Introduction
emerged inhibitors14–16 were derived from natural products,17–21
which had been considered as a great diverse and drug-like library.
Protein tyrosine phosphatase 1B (PTP1B) has been identified as
an encouraging target against type 2 diabetes for about a decade.1
Anchored to endoplasmic reticulum, PTP1B is involved in the insu-
lin receptor (IR) dephosphorylation process, negatively regulating
the signal of insulin pathway. Two independent laboratories have
generated PTP1B knockout mice, which showed increased insulin
sensitivity in liver and muscle tissues and were resistant to weight
gain compared with normal ones. These results apparently con-
firmed the relationship between PTP1B and diabetes.2,3 Along with
the elucidation of its protein structure,4–6 many synthetic inhibi-
tors with submicro-, even nano-molar activity were discovered
through high-throughput screening (HTS) and structure-based de-
sign.7–10 However, their further development to clinical trials was
further restricted mainly for two reasons comparing with emer-
gence of several new drugs in kinases family.11,12 First, the selectiv-
ity between T-cell protein tyrosine phosphatase (TCPTP) and
PTP1B13 was still low because of their high conserved catalytic do-
main, despite the importance of which still need to be proven. Sec-
ond, low cell permeability quenched most competitive inhibitors
that always had strong negative chemical nature mimicking the
phosphate group in IR substrate. In addition, only several of recent
In the history of traditional Chinese medicine (TCM), medicinal
plants and their extracts were used to treat various diseases. Now-
adays, compounds derived from natural products with the unique
and diverse chemical entities still constituted a considerable re-
source for developing novel medicaments. Diabetes, referred as
‘‘thirsty disease” in ancient China, was recorded on different phar-
macopoeia and treated with different kinds of herbs. According to
the old prescription, we have established a TCM extracts library
and also a library of pure natural products isolated from TCM ex-
tracts since 2001. More than 200 anti-diabetic TCM extracts as well
as hundreds of isolated natural products were prepared for HTS
against PTP1B.22–24 As expected, several of them were found to
possess strong positive activities, such as ursolic acid,25 corosolic
acid,26 and oleanolic acid isolated from Cornus officinalis. A quanti-
tative structure–activity relationships (QSARs) on the inhibition
activities of oleanolic acid analogues against PTP1B were recently
studied27 based on our patent.28
In the work described herein, we focus on increasing the inhib-
itory activity of oleanolic acid, which was relatively easier to obtain
from plant extracts than ursolic acid and others. As shown in Fig-
ure 1, oleanolic acid has two positions which can be modified,
C-3 and C-28. Besides these two positions, variation of pentacyclic
moiety was ineffective according to the corresponding triterpene
4–9. Therefore, we reported the synthesis and PTP1B inhibitory
activity of oleanolic acid derivatives such as 10a–10p and
11a–11s at C-28 position (Scheme 1) and 12a–12m at C-3 position
* Corresponding authors. Tel./fax: +86 21 50800721 (J. Li); tel./fax: +86 21
50802221 (L.H. Hu).
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), simmhulh@mail.shcnc.ac.cn
(L.-H. Hu).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.080

8698
Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
Figure 1. Structures of compounds 1–9 and 14.
(Scheme 2), respectively. Moreover, selected compounds in the
series were evaluated in kinetics, selectivity and cell activities to
estimate if their biological properties were suitable for further
development.
was then converted to 12b–d via Corey–Bakshi–Shibata pyrroli-
dine catalystic reduction,34 Wolff-Kishner reduction,35 and Wittig
reaction, respectively. Esterification and etherification of 1 at 3-po-
sition were relatively more rigorous than common reaction oc-
curred on hydroxyl groups due to the steric hindrance around it.
12e was esterified with ethyl oxalyl chloride and then hydrolyzed
to get 12f. 12g–12i were conducted by adding the corresponding
acid anhydride to oleanolic acid in refluxing toluene overnight.
While 12j could be easily prepared with benzyl bromide and 10i
in DMF solution at room temperature, etherifying with different
carboxyl substituted benzyl bromides should be conducted under
refluxing dioxane with NaH overnight to prepare 12k–12m. Final-
ly, 12j–12m were achieved from their methyl ester using LiI in
refluxing DMF solution.
Though several reagents and different conditions were tried, the
synthetic process of compound 13 was blocked at the step of ether-
ification of 10l’s 3-position previously. Eventually, our target was
carried out through a reversal route (Scheme 3). Ether 13a was first
synthesized according to the procedure mentioned in synthesis of
12m, then the carboxyl group was protected to 2-alkyl-1,3-oxazo-
line 13b in two steps. Following the method gained 11a, 13b was
turned to elongated carboxyl acid 13c at 28-position via LiAlH4
reduction, Swern oxidation, and Wittig reaction successively. After
2. Results and discussion
2.1. Chemistry
The extracts of C. officinalis showed good inhibitory activity
against PTP1B in HTS assay, in which oleanolic acid 1, ursolic acid
2, and corosolic acid 3 were the main components. In our com-
pound library against diabetes, several other stored natural prod-
ucts with same scaffold were also selected to test, such as
oleanane 4 separated from Caesalpinia minax Hance, boswellic acid
5 and 11-keto boswellic acid 6 from Boswellia carteri, glycyrrhetinic
acid 7 from Glycyrrhiza yunnanensis, asiatic acid 8 and madecassic
acid 9 from Centella asiatica (L.) Urban.29–32
Compound 10a was achieved by reduction of 1 with LiAlH4, and
10b–10e were also synthesized from 1 via coupling the 3-acetyl
oleanolic acyl chloride with corresponding amino acid esters, then
hydrolyzing the protective groups. Esterification and amidization
of 1 with different alcohols and amines yielded 10f–10j. Com-
pounds 10k–10p were synthesized through repetitious Wittig
reaction between proper quaternary phosphonium salt or triethyl
phosphonoacetate and oleanolic aldehyde obtained from Swern
oxidation of protected 10b at C-3 position. Amide intermediates
of 11a–11s were gotten through coupling EE protected 10l with
different amino acid esters, among which the unnatural ones
(11f–11s) were synthesized following O’Donnell’s procedure.33
Deprotection were carried out with global method which used
pyridinium p-toluenesulfonate for acetals and aqueous base solu-
tion for esters.
coupling with L-phenylalanine, the oxazoline, and methyl ester
were deprotected to obtain carboxyl 13d. Finally, we achieved
compound 13 through selective hydrogenation in the presence of
benzyl group by adding Na2CO3 to poison palladium catalyst.
2.2. Biological evaluation
To examine the potential ability of the natural and artificial
pentacyclic triterpene derivatives, their PTP1B inhibitory activities
were assayed by the method of pNPP using compound 14 as refer-
ence compound.36 Moreover, their TCPTP inhibitory activities
were examined simultaneously by the same method for further
selectivity studying.
The starting material of 12a–12i was oleanolic acid, and of the
others (12j–12m) was oleanolic acid methyl ester. Adding 2-
iodoxybenzoic acid, oleanolic acid was oxidated to 12a, which

Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
8699
Scheme 1. Synthesis of 28-modified oleanolic acid. Reagents and conditions: (a) LiAIH4, THF, RT, 4 h; (b) Ac2O, pyridine, RT, overnight; (c) 1: (COCI)2; 2: different methyl
amino acid esters or amines, DCM, Et3N, 0 °C to RT, 2 h; 3: 5 N NaOH (aq.), THF, MeOH, RT; (d) MeOH or benzyl alcohol, DMAP, EDCI, reflux, overnight; (e) 1: ethyl vinyl ether,
PPTS, DCM, 2 h; 2: LiAIH4, THF, RT, 4 h; 3: (COCI)2, DMSO, DCM, À60 °C to RT, 1 h, then Et3N, 1 h; (f) 1: BrPh3P+(CH2)3COOH, KOtBu or BrPh3P+(CH2)5COOH, KOtBu or triethyl
phosphonoacetate, NaH; THF, RT, 2 h; 2: H2, EtOAc, Pd/C, RT, overnight; (g) PPTS, MeOH, 50 °C, 1 h; if n = 8, 10, 12, repeated LiAIH4 reduction and step f; (h) 1: different amino
acid esters, EDCI, HOBt, Et3N, DCM, 4A MS, RT, 8 h; 2: PPTS, MeOH, 50 °C, 1 h, then 5 N NaOH (aq.), THF, MeOH, RT.
Primarily, the core structure of pentacyclic triterpenoids shared
by 1–3 reminded us if the other compounds with the oleanane or
ursane scaffolds had the same effect. Compounds 4–9 from our li-
brary were tested for this purpose, but only 6 and 9 had weaker ef-
fect. After careful consideration of the unsatisfying results varied at
pentacyclic core, we selected oleanolic acid as the lead in the fol-
lowing optimization, because of its cheapness, commercial avail-
ability and safety. It is noticed that oleanolic acid has been used
as an over-the-counter drug for treatment of hepatitis in China
more than 20 years.37
the threshold value (>20 lM), which indicated that the presence of
carboxyl group was vital to the PTP1B inhibitory potency. It was
also proved by amide (10f–h) or ester (10i and 10j) substitution
compounds. Demonstrated by previous research,11 keeping proper
elongated phosphate mimics entering the catalytic domain with an
aryl group nearby was one important principle in designing com-
petitive PTP1B inhibitor. This rule was verified by 10b and 10k,
where compound 10c with a removed benzyl and replaced amide
was definitely less potent. So, compounds 10k–10p with the car-
bon chain length at 2, 4, 6, 8, 10, and 12 were synthesized, respec-
tively, to find the proper length. The corresponding results
suggested that the inhibitory potency of elongating series were
Simply changing the carboxyl group at C-28 position into
methyl (4) and hydroxyl group (10a) decreased the activity below

8700
Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
Scheme 2. Synthesis of 3-variated oleanolic acid. Reagents and conditions: (a) 2-iodoxybenzoic acid, toluene, DMSO, RT, 4 h; (b) 1: CH2N2, Et2O, 0 °C to RT; 2: (s)-(À)-2-
(diphenylhydroxymethyl)pyrrolidine, BH3 ÁSMe2, THF, RT, 1 h; (c) diethyl glycol, NH2NH2, KOH, 200 °C, 4 h; (d) BrPh3P+CH3, KOtBu, THF, RT, 2 h; (e) Lil, DMF, reflux, overnight;
(f) ethyl oxalyl chloride, Et3N, DCM, 0 °C to RT, 4 h; (g) NaHCO3 (aq), THF, MeOH, 1 h; (h) corresponding acid anhydride, toluene, pyridine, reflux, overnight; (i) if R2 = H, DMF,
NaH, RT, 4 h; if R2 = COOH, dioxane, NaH, reflux, overnight.
low acceleration along with the increasing length gradually, which
suggested that the compounds with the length of 4–6 carbon chain
were suitable for further optimization due to their biological activ-
ities and synthetic facilities.
In further study, different substituted a-amino acids were cou-
pled with elongated oleanolic acid (10l) to examine the impact of
nuance in activities (IC50 = 0.44–0.82 lM) except 11c possessed
an electron-positive imidazole group, which might hardly enter
the catalytic domain surrounded by alkyl amino acids.8
Retaining the pentacyclic scaffold was also the principle of
exploration at C-3 position, because hydroxyl substitution (2, 8,
9) at neighbor positions did not get any valuable improvement.
Furthermore, activities did not ameliorate when we oxidating par-
ent compound 1 to carbonyl (12a), converting configuration (12b),
removing hydroxyl group (12c) and changing to hydrophobic
groups (12d, 12j). Stronger inhibitors 12e, 12f, and 12h suggested
that introducing carboxyl increased the activities. However, 12g
and 12i showed weaker effect than oleanolic acid. Fortunately,
benzyl group derivatives 12k–12m with different carboxyl
replacement gave us satisfied results, which displayed 7-fold in-
crease at para position, 6-fold at meta and retainment at ortho.
On the basis of preceding results, we combined 12m’s moiety at
vicinal aryl group. Primarily, similar activities shown by the dias-
teromers 11a and 11b linking with L- and D-phenylalanine, respec-
tively, meant that the stereochemistry of amino acids was less
important. Three essential amino acid derivatives (11c–11e) and
fourteen racemic artificial amino acid derivatives (11f–11s) were
obtained to enrich the complexity. Interestingly, piperonyl substi-
tuent (11p) had the best activity comparing with others, in which
the electron donating 1,3-benzodioxole may had the more affini-
tive
p-bond with Phe182 and less steric hindrance to rigid catalytic
domain.7 In general, all of these analogues showed only some

Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
8701
Scheme 3. Synthesis of compound 13. Reagents and conditions: (a) p-COOH benzyl bromide, dioxane, NaH, reflux, overnight; (b) 1: 2-amino-2-methylpropan-1-ol, EDCI,
HOBt, Et3N, DCM, 4 Å MS, RT, 8 h; 2: Ph3P, diisopropyl azodicarboxylate, THF, 0 °C to RT, 2 h; (c) 1: LiAIH4, THF, RT, 4 h; 2: (COCI)2, DMSO, DCM, À60 °C to RT, 1 h, then Et3N,
1 h; 3: BrÀPh3P+(CH2)3COOH, KOt-Bu, NaH; THF, RT, 2 h; (d) 1:
L-phenalanine methyl ester hydrochloride, EDCI, HOBt, Et3N, DCM, 4 Å MS, RT, 8 h; 2: 2 N HCI, EtOH, reflux, 2 h,
3: then 5 N NaOH (aq.), THF, MeOH, RT; (e) H2, EtOAc, Pd/C, Na2CO3, RT, overnight.
Table 1
C-3 position and 11a’s at C-28 position to design compound 13,
which showed the best activity against PTP1B in our derivatives.
Most of the compounds (except 10a–10j) were evaluated in
TCPTP inhibitory assay (Tables 1 and 2). Unfortunately, none of
them exhibited any obvious distinction (above 2-fold) between
these two homogeneous enzyme. The conclusion was similar to
most published PTP1B inhibitors, whereas the importance of selec-
tivity between PTP1B and TCPTP still need to be proved for their
remarkable differences in expressive amounts, endocellular
distribution, and biological functions.38
Inhibitory activity of compounds 1–10p against PTP1B and TCPTP
Compounds
IC50 (l
M)a
PTP1B
TCPTP
1
2
3
4
5
6
7
3.37 0.20
3.08 0.26
5.49 0.12
>20
>20
8.04 1.04
>20
3.40 0.26
3.33 0.19
11.31 0.19
>20
>20
9.45 0.77
>20
Besides TCPTP, there are several other critical protein tyrosine
phosphatases (PTPs) negatively regulating insulin dephosphoryla-
tion, such as src homology phosphatase-1 (SHP-1), leukocyte anti-
8
9
>20
12.38 3.19
>20
>20
>20
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
14c
NTb
3.31 1.11
15.44 0.80
16.35 1.20
3.19 0.08
4.76 0.63
9.20 0.90
8.10 1.08
4.44 0.05
8.61 0.50
2.10 0.36
1.33 0.23
0.88 0.06
0.78 0.08
0.72 0.07
0.59 0.07
5.73 0.32
NT
NT
NT
NT
NT
NT
NT
NT
gen-related phosphatase (LAR), protein tyrosine phosphatases
and (PTP and PTP
).39 So, we tested the inhibitory activity of
our derivatives on the purified recombinant human SHP1, LAR
D1, PTP D1 and PTP D1. As listed in Table 3, we concluded that
a
e
a
e
a
e
our inhibitors had no visible activities against receptor-like trans-
membrane PTPs and possessed about 2–10-folds selectivity
between PTP1B and SHP1.
To elucidate the characteristics of our modified compounds, the
Ki values of three nodal compounds (11a, 12m, and 13) were at
NT
2.77 0.10
1.11 0.13
0.85 0.04
0.79 0.03
0.63 0.05
0.53 0.05
5.35 0.21
0.64, 0.97, and 0.13 lM, respectively, coinciding with the active
trend described in SAR study. As also elucidated in Supplementary
material, the increasing Km and retaining Vmax demonstrated that
our derivatives were competitive inhibitors, which probably had
good binding affinity at catalytic domain like other published com-
petitive inhibitors. And we also adopted a time-course inhibition
assay showed in supplementary material to verify that the inhibi-
tory effect was not related to irreversible binding, such as
oxidation mechanism.
a
The pNPP assay. IC50 values were determined by regression analyses and
expressed as means SD of three replications.
b
Not tested.
Positive control.
c
We tested the cellular effects of compounds 1, 11a, and 13 on
the phosphorylation level of IR and Akt in Chinese hamster ovary
cell line transfected with an expression plasmid encoding human
IR (CHO/hIR). As illustrated in Figure 2A–C, sodium vanadate
(1 mM) was used as a positive control after treating the cell with
10 nM insulin (vehicle, lane 1), while DMSO as negative control
(lane 2), compound 13 significantly increased the level of IR phos-
phorylation in a dose dependent manner (Fig. 2C). However,

8702
Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
Table 2
about 40% increment at 1 lM (Fig. 3A) compared with 75% under
only insulin-stimulated condition.40 Simultaneously, compound
13 was also tested in insulin-stimulated glucose uptake assay at
three different insulin concentrations (1–100 nM, Fig. 3B). Unfortu-
nately, our inhibitor had about 20% increment compared to sub-
maximal insulin concentrations, which was lower than the
condition without insulin cooperating. More experiment results
in vivo are going to support that if our inhibitors are
therapeutically meaningful.
Inhibitory activity of compounds 11a–13 against PTP1B and TCPTP
Compounds
IC50 (l
M)a
PTP1B
TCPTP
11a
11b
11c
11d
11e
11f
11g
11h
11i
0.57 0.15
0.74 0.07
4.62 0.20
0.59 0.10
0.55 0.07
0.56 0.07
0.51 0.04
0.61 0.20
0.57 0.02
0.45 0.01
0.55 0.33
0.65 0.04
0.53 0.07
0.52 0.08
0.60 0.10
0.44 0.02
0.66 0.25
0.82 0.20
0.63 0.17
5.32 0.09
5.05 0.69
2.61 0.52
2.85 0.47
2.89 0.36
2.86 0.23
5.97 0.32
2.33 0.24
4.58 0.58
2.67 0.20
2.72 0.24
0.62 0.04
0.54 0.09
0.15 0.02
5.73 0.32
0.63 0.09
0.68 0.03
4.31 0.21
0.59 0.15
0.75 0.12
0.55 0.07
0.52 0.04
0.48 0.05
0.65 0.05
0.36 0.03
0.50 0.03
0.66 0.05
0.45 0.07
0.52 0.04
0.55 0.07
0.60 0.05
0.75 0.15
0.70 0.19
0.85 0.20
5.63 0.31
5.10 0.31
3.00 0.24
2.53 0.35
3.66 0.25
4.55 0.42
5.36 0.57
1.38 0.15
5.00 0.80
2.75 0.30
2.22 0.21
0.49 0.04
0.47 0.04
0.16 0.01
5.35 0.21
The cell results demonstrated that our synthesized compounds
had well cell permeablity, due to their good lipophilic property
provided by pentacyclic core. Furthermore, showed in glucose up-
take assay in L6 myotubes the effect of 13 appeared significant be-
cause the skeletal muscles accounted for majority of glucose
uptake.40 In addition, the elevated tyrosine phosphorylation level
of IR and Akt indicated that our compounds stimulated the sensi-
tivity of cell to insulin through inhibition of PTP1B and other
phosphatases activities.
11j
11k
11l
11m
11n
11o
11p
11q
11r
11s
12a
12b
12c
12d
12e
12f
12g
12h
12i
3. Conclusion
Oleanolic acid, as far as we know, was first developed in SAR re-
search as PTP1B inhibitor derived from natural products. After
varying its structure at C-3 and C-28 positions, we found a series
of derivatives more potent than the primary hit, such as 13, 22-fold
more effective than the parent compound. Although their inhibi-
tory toward TCPTP and PTP1B still shared little difference, our com-
petitive and reversible inhibitors exhibited good selectivity against
other phosphatases involving in the insulin pathway. Moreover, 13
exhibited cell activities in enhancing IR and downstream Akt phos-
phorylation and in increasing the glucose uptake assay. The cellu-
lar results provided evidence that this type of inhibitor can be
potent and cell permeable. Further optimization will focus on
improving activity in vitro in order to choosing a PTP1B inhibitor
suitable for animal studies in diabetes and obesity.
12j
12k
12l
12m
13
14b
a
The pNPP assay. IC50 values were determined by regression analyses and
expressed as means SD of three replications.
b
Positive control.
4. Experimental
Table 3
4.1. General experimental procedure
Inhibitory activity of selected compounds against related PTPs
Compounds
IC50
(l
M)
PTP
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on
a Varian Mercury-VX300 Fourier transform spectrometer. The
chemical shifts were reported in d (ppm) using the d 7.26 signal
SHP-1
LAR
a
D1
PTPe D1
1
2
3
31.33 5.39
25.78 2.30
24.56 0.56
12.64 1.46
27.38 4.00
5.20 0.77
4.08 0.25
0.71 0.17
12.36 0.49
4.05 0.24
0.89 0.12
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
of CDCl3 (1H NMR) and the d 77.23 signal of CDCl3 13C NMR) as
(
internal standards. ESI-MS was run on a Bruker Esquire 3000 plus
spectrometer in MeOH and HR-ESI-MS was run on a Bruker Atex III
spectrometer in MeOH, respectively. All commercially available re-
agents were used without further purification. The solvents used
were all AR grade and were redistilled under positive pressure of
dry nitrogen atmosphere in the presence of proper desiccant when
necessary. The progress of the reactions was monitored by analyt-
ical thin-layer chromatography (TLC) on HSGF254 precoated silica
gel plates. Details of analytical HPLC profile of compounds in two
diverse systems were described in the Supplementary material.
10b
10k
10l
11a
11p
12h
12m
13
compound 11a and oleanolic acid 1 only exhibited unclear effect in
the same assay, which successfully verified our SAR strategy on cell
model in qualitative analysis. The phosphor-Akt (Ser473), a down-
stream protein of IR, was also stimulated by treatment of com-
pound 13, which demonstrated that inhibition of PTP1B in CHO/
hIR cells enhanced insulin signal (Fig. 2C). In cytotoxicity experi-
ment, no changes in viability were observed when the CHO/hIR
4.2. Synthesis of compound 13
This synthetic procedure represented a typical method for the
variation at C-3 and C-28 positions, besides which, what was not
described herein was referred to the Supplementary material.
cells were incubated with 13 (20
l
M) for 48 h using MTT method
4.2.1. 3-(4-Carboxy-benzyloxy)-oleanolic methyl ester (13a)
NaH (800 mg, 20 mmol, 60% suspension in mineral oil) was
added to a solution of oleanolic methyl ester (940 mg, 2 mmol)
in dry dioxane (10 mL), then p-carboxy-benzyl bromide (430 mg,
2 mmol) was plunged into the mixture after stirring for 20 min
(data not shown). We also studied the effect of compound 13 on
glucose uptake in L6 myotubes either in the absence or presence
of insulin. In the absence of insulin, the rate of basal glucose uptake
increased in a dose-dependent manner and reached a flat roof with

Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
8703
Figure 2. Effect of compounds 1 (A), 11a (B) and 13 (C) on tyrosine phosphorylation of IRb and Akt in CHO/hlR celts. Sodium vanadate (1 mM) and DMSO were used as
postitive and negative controls (each lane 1 and 2). The levels of IRb and b-actin were used to normalization.
Figure 3. Effect of compound 13 on glucose uptake in L6 myotubes. L6 myotubes were starved for 2 h. Subsequently, the cell was treated with 250 nM, 500 nM, 1
lM and
2
l
M for 3 h in the absent of insulin (A); or incubated with 1.0 M compound 13 for 2.5 h and stimulated with a range of insulin concentrations for 30 min (B). The rate of
l
*
glucose uptake was expressed as the percentage of the basal value or cells treated with 0.2% DMSO. The figures showed the means standard error (SE) of triplet. ( P < 0.05,
**
P < 0.01).
at room temperature (RT). The mixture was heated to 105 °C over-
night, after which it was allowed to RT and ceased by adding satu-
rated NH4Cl solution. Extracting with ethyl acetate, the organic
phase was collected and washed with water and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
subjected to column chromatography (CC) on silica gel 200–300
mesh using petroleum ether 60 ꢀ 90 °C (PE) and ethyl acetate
(10:1) as eluant to get 13a (612 mg, 51%). 1H NMR (CDCl3,
300 MHz): d = 8.18 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 4.8 Hz, 2H), 5.25
(s, 1H), 4.71 (d, J = 12.9 Hz, 1H), 4.45 (d, J = 12.9 Hz, 1H), 3.59 (s,
3H), 2.91 (dd, J = 4.2, 11.4 Hz, 1H), 2.82 (dd, J = 4.5, 14.1 Hz, 1H),
2.00–0.70 (m, 43H); ESI-LRMS, [M+Na]+: 627.
J = 12.3 Hz, 1H), 4.44 (d, J = 12.3 Hz, 1H), 4.10 (s, 2H), 3.67 (s, 3H),
2.91 (dd, J = 4.2, 11.4 Hz, 1H), 2.85 (dd, J = 4.5, 14.1 Hz, 1H), 1.38
(s, 6H), 2.00–0.70 (m, 43H); ESI-LRMS, [M+H]+: 658.
4.2.3. 3-[4-(2,2-Dimethyl-oxazoline)-benzyloxy]-28-(1-ene-4-
butyric acid)-D
12-oleanene (13c)
To a suspension of LiAlH4 (100 mg, 2.5 mmol) in dry THF (3 mL)
at 0 °C, a solution of 13b (351 mg, 0.5 mmol) in dry THF (2 mL) was
added slowly. The reaction mixture was stirred at room tempera-
ture for 4 h and then quenched cautiously by the subsequent addi-
tion of water (0.1 mL), 5 N NaOH (aq) (0.1 mL) and water (0.3 mL).
The suspension was filtered through a fritted funnel and the filtrate
was concentrated in vacuo to give alcohol, which was used in the
next step without purification. A solution of DMSO (0.1 mL,
1.3 mmol) in dry DCM (1 mL) was added dropwise to a À60 °C
cooled solution of oxalyl chloride (0.05 mL, 0.55 mmol) in dry
DCM (2 mL). After 10 min of stirring at À60 °C, a solution of alcohol
obtained from previous step in dry DCM (2 mL) was added drop-
wise. After stirring for another 45 min at À60 °C, triethylamine
(0.35 mL, 2.5 mmol) was added. The mixture was allowed to RT
and stirred for 1 h, then diluted with DCM. The organic layer was
washed with water, brine, dried over anhydrous Na2SO4, and con-
centrated in vacuo to gain the colorless aldehyde. The aldehyde
dissolved in dry THF (2 mL) was syringed into the orange–red yield
suspension, prepared by adding 4-carboxybutyl triphenylphospho-
nium bromide (427 mg, 1 mmol) to the KOtBu (336 mg, 3 mmol) in
dry THF (3 mL). The mixture was stirred for 2 h, quenched by 1 N
HCl (5 mL) and diluted with ethyl acetate. The organic layer was
extracted, washed with water and brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The acid 13c (159 mg, 46%)
was purified by CC. (PE: ethyl acetate = 5:1) 1H NMR (CDCl3,
300 MHz): d = 7.85 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H),
5.30–5.20 (m, 2H), 5.19 (s, 1H), 4.69 (d, J = 12.3 Hz, 1H), 4.44 (d,
J = 12.3 Hz, 1H), 4.11 (s, 2H), 2.91 (dd, J = 3.9, 11.1 Hz, 1H), 2.45–
2.35 (m, 2H), 2.00–0.70 (m, 52H); ESI-LRMS, [M+H]+: 658.
4.2.2. 3-[4-(2,2-Dimethyl-oxazoline)-benzyloxy]-oleanolic
methyl ester (13b)
A solution of 13a (612 mg, 1 mmol), 1-hydroxybenzotriazole
(HOBt, 148 mg, 1.1 mmol), 2-amino-2-methylpropan-1-ol (89 mg,
1.1 mmol), triethylamine, (0.28 mL, 2 mmol), 4 Å molecular sieves
(200 mg) in dry CH2Cl2 (10 mL) was treated with 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 210 mg,
1.1 mmol) and the resulting reaction mixture was stirred at RT
for 8 h. After that the mixture was partitioned by dichloromethane
(DCM) and water, washed by 1 N HCl (5 mL), saturated NaHCO3
and brine, dried over anhydrous Na2SO4, and concentrated in va-
cuo. Without further purification, the residue dissolved in dry
THF (10 mL) with triphenyl phosphine (268 mg, 1 mmol), and
diisopropyl azodicarboxylate (0.2 mL, 1 mmol) was added to the
mixture under ice bath. The muddy solution was gradually clarified
along with stirring for 2 h at RT. The reaction mixture was
quenched by the addition of water and diluted with ethyl acetate.
The organic layer was separated, washed with water and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The res-
idue was purified by CC (PE:ethyl acetate = 15:1) to give the prod-
uct 13b (351 mg, 53%). 1H NMR (CDCl3, 300 MHz): d = 7.89 (d,
J = 8.1 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 5.27 (s, 1H), 4.69 (d,

8704
Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
4.2.4. 3-(4-Carboxy-benzyloxy)-28-[1-en-4-butyric ((s)-1-
carboxy-phenylethyl)-amide]-
12-oleanene (13d)
solution of 13c (159 mg, 0.23 mmol), HOBt (33 mg,
different concentrations of inhibitor. In the presence of the com-
petitive inhibitor, the Michaelis–Menton equation is described as
1/v = [Km/(Vmax Á[S])](1 + [I]/Ki) + 1/Vmax, where v is the initial rate,
Vmax is the maximum rate, and [S] is the substrate concentration.
Ki value was obtained by linear re-plot of apparent Km/Vmax (slope)
from primary reciprocal plot vs. inhibitor concentration [I] accord-
ing to the equation Km/Vmax = 1 + [I]/Ki.
D
A
0.25 mmol), L-phenalanine methyl ester hydrochloride (54 mg,
0.25 mmol), triethylamine, (0.07 mL, 0.5 mmol), 4 Å molecular
sieves (50 mg) in dry CH2Cl2 (2 mL) was treated with EDCI
(48 mg, 0.25 mmol) and the resulting reaction mixture was stirred
at RT for 8 h. After that the mixture was partitioned by DCM and
water, washed by 1 N HCl (1 mL), saturated NaHCO3 and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The
crude product dissovled in EtOH (2 mL) containing 2 N HCl
(2 mL), and the solution was heated at 90 °C for 2 h. After the reac-
tant disappearing monitored by TLC, the mixture was allowed to
RT and separated between ethyl acetate and water. The organic
layer was washed with water and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. Without further purification,
the residue was also directly hydrolyzed in the solution of 5 N
NaOH (1 mL), MeOH (1 mL), and THF (2 mL) at RT overnight. After
neutralizing the superfluous base with 2 N HCl, the mixture was
partitioned between EtOAc and water, dried over anhydrous
Na2SO4, and evaporated to afford the crude product, in which the
diacid 13d (79 mg, 43%) was purified through CC (CHCl3:
MeOH = 10:1). 1H NMR (CDCl3, 300 MHz): d = 8.05 (d, J = 8.1 Hz,
2H), 7.44 (d, J = 8.1 Hz, 2H), 7.40–7.15 (m, 5H), 6.09 (d, J = 7.2 Hz,
1H), 5.30–5.20 (m, 2H), 5.19 (s, 1H), 5.10–4.90 (m, 1H), 4.73 (d,
J = 13.2 Hz, 1H), 4.47 (d, J = 13.2 Hz, 1H), 3.20 (ddd, J = 5.7, 14.1,
20.1 Hz, 2H), 2.93 (dd, J = 3.9, 11.1 Hz, 1H), 2.45–2.35 (m, 2H),
2.00–0.70 (m, 46H); ESI-LRMS, [M+Na]+: 815.
4.3.3. Insulin receptor and Akt phosphorylation cellular assay
The Chinese hamster ovary cell line transfected with an expres-
sion plasmid encoding human IR (CHO/hIR) were cultured in six-
well plates. Nearly confluent CHO/hIR cells were starved 2 h with
Ham’s F-12 medium without serum. Cells were incubated with
compound for 3 h, followed by stimulation with 10 nM insulin
for 10 min. Cells were scraped and lysed with loading buffer. The
samples were resolved by 8% SDS-PAGE, eletrotransferred to nitro-
cellulose membranes, and probed with anti-pIR (Tyr1162/1163)
antibody from Biosource, anti-pAKT (Thr473) antibody from Cell
Signaling Technology and anti-b-actin antibodies from Santa Cruz
Biotechnology. The bolts were developed using the ECL chemilumi-
nescence detection system.
4.3.4. Glucose uptake assay
L6 myoblast cells were grown and maintained in alpha
Dulbeco’s modified Eagle’ medium (a-DMEM) containing 10% FBS
under 5% CO2 environment at 37 °C. For differentiation of L6 myo-
blasts, cells were seeded in appropriate culture plates, and after the
cells had reached subconfluency, the medium was changed to a-
DMEM containing 2% FBS. The medium was then changed every
2 days until the cells were fully differentiated. Glucose uptake on
L6 myotubes was measured on the 5th day. L6 myotubes were
4.2.5. 3-(4-Carboxy-benzyloxy)-28-[4-butyric ((s)-1-carboxy-
phenylethyl)-amide]-D
12-oleanene (13)
A mixture of 13d (79 mg, 0.1 mmol), Na2CO3 (31 mg, 0.3 mmol),
10% Pd on carbon (20 mg) in EtOAc (1 mL) was stirred under an
atmosphere of H2 overnight. After adding several drops of formic
acid (0.2 mL) for neutralizing, the mixture was filtered through a
fritted funnel and the filtrate was concentrated in vacuo. The resi-
due was subject to CC (CHCl3:MeOH = 10:1) to furnish 3b-(p-
carboxy-benzyloxy)-28-[4-butyric((s)-1-carboxy-phenylethyl)-
starved for 2 h with a-DMEM containing 0.2% BSA without serum.
For the effect of inhibitors on basal glucose uptake, the cell was
treated with differrent concentrations of inhibitors for 3 h in the
absence of insulin. In the presence of insulin, L6 myotubes were
treated with 1.0 lM inhibitors (2.5 h) and stimulated with 1, 10,
and 100 nM of insulin concentrations for 30 min, respectively. Cells
were then treated with 2-deoxy [1,2-3H] glucose for 10 min (final
amide]-
D
12-oleanene 13 (40 mg, 51%) as white amorphous solid.
concentration of 0.5 lCi and 0.1 mM glucose) in HEPES-buffered
1H NMR (CDCl3, 300 MHz): d = 8.05 (d, J = 8.1 Hz, 2 H), 7.44 (d,
J = 8.1 Hz, 2H), 7.40–7.15 (m, 5H), 6.03 (m, 1H), 5.16 (s, 1H),
4.95–4.92 (m, 1H), 4.74 (d, J = 13.2 Hz, 1H), 4.48 (d, J = 13.2 Hz,
1H), 3.20 (ddd, J = 5.7, 15.0, 20.1 Hz, 2H), 2.95 (dd, J = 3.9,
11.1 Hz, 1H), 2.20 (t, J = 7.2 Hz, 2H), 2.00–0.70 (m, 50H); 13C NMR
(CDCl3, 75 MHz): d = 176.1, 174.0, 171.9, 146.2, 145.2, 135.9,
130.4 Â 2, 129.6 Â 2, 128.8 Â 2, 128.2, 127.4, 127.2 Â 2, 122.2,
87.4, 70.9, 55.8, 53.3, 47.8, 47.5, 46.8, 41.8, 40.1, 39.9, 39.1, 38.6,
37.5, 37.1, 36.7, 34.7, 33.5, 33.4, 32.7, 31.2, 29.9, 28.5, 26.8, 26.3,
25.9, 23.9, 23.9, 23.2, 22.9, 22.3, 18.5, 17.0, 16.9, 15.7; ESI-HRMS:
saline solution [20 mM HEPES (pH 7.4), 136 mM NaCl, 4.7 mM
KCl, 1.25 mM MgSO4, 1.2 mM CaCl2, 0.2% BSA]. Transport was ter-
minated by aspirating the radioactive solution and followed by
three rapid washes with cold PBS. Radioactivity inside the cells
was measured after cells were lysed with 0.1% Triton and
normalized with protein concentration levels.
Acknowledgments
Financial support by the Chinese National Natural Science
Foundation of China (NNSF; No. 30572241, 30623008) and Shang-
hai Science and Technology Commission (04QMH1409) was
gratefully acknowledged.
calcd for
C
51H71NO6 [M+Na]+: 816.5179, founded: 816.5185.
Scanned 1H and 13C NMR figures were shown in the Supplemen-
tary material.
4.3. Biological assay
Supplementary data
4.3.1. PTP1B and other phosphatases expression and biological
assay
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.07.080.
The enzymatic assays of PTP1B, TC-PTP, LAR, PTP
a and PTPe
were referred.25 SHP1 (742–1710 according to BC002523) assay
References and notes
was carried in 50 mM Tris at pH 8.0 with 10
substrates.
lM OMFP as
1. Barford, D.; Flint, A. J.; Tonks, N. K. Science 1994, 263, 1397.
2. Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Colins, S.; Loy, A. L.;
Normandin, D.; Chenng, A.; Himms-Hagen, J. C.; Chan, C. Science 1999, 283,
1544.
3. Klaman, L. D.; Boss, O.; Peroni, O. D.; Kim, J. K.; Zabotny, J. M.; Moghal, N.;
Lubkin, M.; Kim, Y. B.; Sharpe, A. H.; Stricker-Krongrad, A.; Shulman, G. I.; Neel,
B. G.; Kahn, B. B. Mol. Cell. Biol. 2000, 20, 5479.
4.3.2. Characterization of the inhibitors’ enzyme kinetics
To determine the inhibition mode of inhibitors, assay was car-
ried out in a 100
lL assay mixture contained 50 mM MOPS at pH
6.5, 30 nM PTP1B, pNPP in 2-fold dilution up to 80 mM, and

Y.-N. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8697–8705
8705
4. Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawrence, D. S.; Almo, S. C.; Zhang, Z. Y. Proc.
Natl. Acad. Sci. 1997, 94, 13420.
18. Na, M.; Jang, J.; Njamen, D.; Mbafor, J. T.; Fomum, Z. T.; Kim, B. Y.; Oh, W. K.;
Ahn, J. S. J. Nat. Prod. 2006, 69, 1572.
5. Iversen, L. F.; Andersen, H. S.; Branner, S.; Mortensen, S. B.; Peters, G. H.; Norris,
K.; Olsen, O. H.; Jeppesen, C. B.; Lundt, B. F.; Ripka, W.; Møller, K. B.; Møller, N.
P. H. J. Biol. Chem. 2000, 275, 10300.
6. Wiesmann, C.; Barr, K. J.; Kung, J.; Zhu, J.; Erlanson, D. A.; Shen, W.; Fahr, B. J.;
Zhong, M.; Taylor, L.; Randal, M.; McDowell, R. S.; Hansen, S. K. Nat. Struct. Mol.
Biol. 2004, 11, 730.
19. Wang, Y.; Shang, X. Y.; Wang, S. J.; Mo, S. Y.; Li, S.; Yang, Y. C.; Ye, F.; Shi, J. G.;
He, L. J. Nat. Prod. 2007, 70, 296.
20. Cui, L.; Ndinteh, D. T.; Na, M.; Thuong, P. T.; Silike-Muruumu, J.; Njamen, D.;
Mbafor, J. T.; Fomum, Z. T.; Ahn, J. S.; Oh, W. K. J. Nat. Prod. 2007, 70, 1039.
21. Gong, J. X.; Shen, X.; Yao, L. G.; Jiang, H.; Krohn, K.; Guo, Y. W. Org. Lett. 2007, 9,
1715.
7. Johnson, T. O.; Ermolieff, J.; Jirousek, M. R. Nat. Rev. Drug Disc. 2002, 1, 696.
8. Liu, G. Curr. Med. Chem. 2003, 10, 1407.
22. Chen, R. M.; Hu, L. H.; Shen, Q.; Li, J. Bioorg. Med. Chem. Lett. 2002, 12, 3387.
23. Li, Y. F.; Hu, L. H.; Lou, F. C.; Shen, Q.; Li, J. J. Asian Nat. Prod. Res. 2005, 7, 13.
24. Li, Y. F.; Shen, Q.; Li, J.; Hu, L. H. Chem. Biodiv. 2007, 4, 961.
25. Zhang, W.; Hong, D.; Zhou, Y. Y.; Zhang, Y. N.; Shen, Q.; Li, J. Y.; Hu, L. H.;
Li, J. Biochim. Biophys. Acta 2006, 1760, 1505.
26. Shi, L.; Zhang, W.; Zhou, Y. Y.; Zhang, Y. N.; Li, J. Y.; Hu, L. H.; Li, J. Eur. J.
Pharmacol. 2008, 584, 21.
27. Chung, Y.-H.; Jang, S.-C.; Kim, S.-J.; Sung, N.-D. J. Appl. Biol. Chem. 2007, 50, 52.
28. Lihong, Hu et al. PCT CN2005/000172, 2005.
29. Matsunaga, S.; Tanaka, R.; Akagi, M. Phytochemistry 1988, 27, 255.
30. Pardhy, R. S.; Bhattacharyya, S. C. Ind. J. Chem. 1978, 16B, 176.
31. Shim, S. B.; Kim, N. J.; Kim, D. H. Planta Medica 2000, 66, 40.
32. Bonte, F.; Dumas, M.; Chaudagne, C.; Meybeck, A. Planta Medica 1994, 60, 133.
33. O’Donnell, M. J.; Boniece, J. M.; Earp, S. E. Tetrahedron Lett. 1978, 19, 2641.
34. Corey, E. J.; Shibata, S.; Bakshi, R. K. J. Org. Chem. 1988, 53, 2861.
35. Karliner, J.; Djerassi, C. J. Org. Chem. 1966, 31, 1945.
9. Bialy, L.; Waldmann, H. Angew. Chem., Int. Ed. 2005, 44, 3814.
10. Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007, 12, 373.
11. Goldman, J. M.; Melo, J. V. N. Engl. J. Med. 2001, 344, 1084.
12. Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.; Smith, R. A.;
Schwartz, B.; Simantov, R.; Kelley, S. Nat. Rev. Drug Disc. 2006, 5, 835.
13. Iverson, L. F.; Møller, K. B.; Pedersen, A. K.; Peters, G. H.; Petersen, A. S.;
Andersen, H. S.; Branner, S.; Mortensen, S. B.; Møller, N. P. H. J. Biol. Chem. 2002,
277, 19982.
14. Combs, A. P.; Zhu, W.; Crawley, M. L.; Glass, B.; Polam, P.; Sparks, R. B.; Modi,
D.; Takvorian, A.; McLaughlin, E.; Yue, E. W.; Wasserman, Z.; Bower, M.; Wei,
M.; Rupar, M.; Ala, P. J.; Reid, B. M.; Ellis, D.; Gonneville, L.; Emm, T.; Taylor, N.;
Yeleswaram, S.; Li, Y.; Wynn, R.; Burn, T. C.; Hollis, G.; Liu, P. C. C.; Metcalf, B. J.
Med. Chem. 2006, 49, 3774.
15. Boutselis, I. G.; Yu, X.; Zhang, Z. Y.; Borch, R. F. J. Med. Chem. 2007, 50, 856.
16. Wilson, D. P.; Wan, Z. K.; Xu, W. X.; Kirincich, S. J.; Follows, B. C.; Joseph-
McCarthy, D.; Foreman, K.; Moretto, A.; Wu, J.; Zhu, M.; Binnun, E.; Zhang, Y. L.;
Tam, M.; Erbe, D. V.; Tobin, J.; Xu, X.; Leung, L.; Shilling, A.; Tam, S. Y.; Mansour,
T. S.; Lee, J. J. Med. Chem. 2007, 50, 4681.
36. Chen, Y. T.; Seto, C. T. J. Med. Chem. 2002, 45, 3946.
37. Liu, J.; Liu, Y.; Parkinson, A. J. J. Pharmacol. Exp. Ther. 1995, 275, 768.
38. Bourdeau, A.; Dube, N.; Tremblay, M. L. Curr. Opin. Cel. Biol. 2005, 17, 203.
39. Cheng, A.; Dube, N.; Gu, F.; Tremblay, M. L. Eur. J. Biochem. 2002, 269, 1050.
40. DeFronzo, R. A.; Jacot, E.; Jequier, E.; Maeder, E.; Wahren, J.; Felber, J. P. Diabetes
1981, 30, 1000.
17. Cui, L.; Na, M.; Oh, H.; Bae, E. Y.; Jeong, D. G.; Ryu, S. E.; Kim, S.; Kim, B. Y.; Ahn,
J. S.; Oh, W. K. Bioorg. Med. Chem. Lett. 2006, 16, 1426.