Protein tyrosine phosphatase 1B inhibitors isolated from Morus bombycis

Article abstract of DOI:10.1016/j.bmcl.2009.09.102

Bioassay-guided fractionation of the chloroform-soluble fraction of Morus bombycis, using an in vitro PTP1B inhibitory assay led to the identification of three 2-arylbenzofurans, albafuran A (1), mulberrofuran W (2) and mulberrofuran D (6), along with thr

Full text of DOI:10.1016/j.bmcl.2009.09.102

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6759–6761
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Protein tyrosine phosphatase 1B inhibitors isolated from Morus bombycis
Duc Manh Hoang b, , Tran Minh Ngoc a, , Nguyen Tien Dat c, Do Thi Ha a, Young Ho Kim a, Hoang Van Luong d,
a,
Jong Seog Ahn b, , KiHwan Bae
*
*
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Functional Metabolite Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea
c Institute of Natural Products Chemistry, Vietnamese Academic of Science and Technology, Hanoi, Viet Nam
d Vietnam Military Medical University, Hanoi, Viet Nam
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioassay-guided fractionation of the chloroform-soluble fraction of Morus bombycis, using an in vitro
PTP1B inhibitory assay led to the identification of three 2-arylbenzofurans, albafuran A (1), mulberrofu-
ran W (2) and mulberrofuran D (6), along with three chalcone-derived Diels–Alder products, kuwanon J
(3), kuwanon R (4), and kuwanon V (5). Compounds 1–6 showed remarkable inhibitory activity against
Received 15 June 2009
Revised 1 September 2009
Accepted 25 September 2009
Available online 7 October 2009
PTP1B with IC50 values ranging from 2.7 to 13.8 lM. Inhibition kinetics were analyzed by Lineweaver–
Burk plots, which suggested that compounds 1–6 inhibited PTP1B in a mixed-type manner. The present
results indicate that the respective lipophilic and hydroxyl groups of 2-arylbenzofurans and chalcone-
derived Diels–Alder products play an important role in inhibition of PTP1B.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Morus bombycis
2-Arylbenzofuran derivatives
Chalcone-derived Diels–Alder types
Protein tyrosine phosphatase 1B (PTP1B)
The world-wide incidence of type 2 diabetes mellitus (T2DM)
and obesity in the population is increasing at an alarming rate.
What was previously observed primarily in adult populations
and associated with a sedentary lifestyle has now also become a
medical problem in children.1,2 The relationship between obesity
and T2DM has a polygenetic component and is associated with
insulin resistance.3 Insulin resistance is evident in many tissues
that are important for glucose homeostasis, including muscle, liver
and, more recently, in fat and at the level of the central nervous
system. Metabolic insulin signal transduction occurs through acti-
vation of the insulin receptor (IR), including autophosphorylation
of tyrosine (Tyr) residues in the insulin receptor activation loop.4
Several protein tyrosine phosphatases (PTPs), such as receptor pro-
duce PTP1B activity or expression levels could not only be used for
the treatment of type 2 diabetes, but also obesity.
Mori Cortex Radicis, the root cortex of some Morus species, has
been used in traditional medicine for treating diabetes, as well as
in diuretic, expectorant, and laxative agents.7 Chemical and phar-
macological investigations of Morus sp. have resulted in the isola-
tion of a series of prenylated flavonoids, benzofurans, and other
phenolic compounds,8–10 which are cytotoxic agents that inhibit
COX-1, COX-2, NO production, and HIF1.11–13 During the course
of our program to screen for PTP1B inhibitors from plants, it was
found that the CHCl3-soluble fraction of the root cortexes of Morus
bombycis exhibited strong inhibitory activity (>80% inhibition at
30 lg/mL). Since it has been reported that PTP1B inhibitors may of-
tein tyrosine phosphatase (rPTP-
a
), leukocyte antigen-related
fer a potential treatment for obesity, diabetes, and related diseases,
a phytochemical investigation was carried out. This Letter de-
scribes the isolation and characterization of six compounds from
a CHCl3 extract of M. bombycis, as well as the evaluation of the
inhibitory activity of isolated compounds 1–6 against PTP1B.
The dried root cortex of M. bombycis (2.5 kg) was collected in
August 2007, from Vinh Phuc province, Vietnam, and authenti-
cated by Prof. Pham Thanh Ky, Department of Pharmacognosy, Ha-
noi College of Pharmacy, Vietnam where the voucher specimens
were deposited.
tyrosine phosphatase (LAR), SH2-domain-containing phosphotyro-
sine phosphatase (SHP2), and protein tyrosine phosphatase 1B
(PTP1B) have been implicated in the dephosphorylation of the
IR.5 There is substantial evidence supporting PTP1B as the critical
PTP controlling insulin signaling pathway. PTP1B seems to be a
key regulator of insulin receptor activity, acting at the insulin
receptor and at downstream signaling insulin receptor substrate
proteins.6 Therefore, it has been suggested that compounds that re-
The dried root bark of M. bombycis (2.5 kg) was powdered and
extracted with MeOH (3 ꢀ 5 L) and then the MeOH extract was
concentrated. The residue (72 g) was suspended in H2O (1 L) and
partitioned with n-hexane (3 ꢀ 0.5 L) and CHCl3 (3 ꢀ 0.5 L), succes-
sively. The CHCl3-soluble extract was concentrated in vacuo to give
* Corresponding authors. Tel.: +82 42 860 4312; fax: +82 42 860 4595 (J.S.A.);
tel.: +82 42 821 5925; fax: +82 42 821 6566 (K.B.).
E-mail addresses: jsahn@kribb.re.kr (J.S. Ahn), baekh@cnu.ac.kr (K. Bae).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.102

6760
D. M. Hoang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6759–6761
a brown solid (15.5 g), which was then chromatographed on a sil-
ica gel column using a CHCl3–MeOH gradient as the mobile phase
to afford two fractions, C1 and C2. Fraction C2 was subjected to
preparative HPLC (YMC Pack Pro C18, 250 ꢀ 10 mm, 60% ACN in
H2O, flow rate 10 mL/min) to afford 1 (7.5 mg), 2 (7.2 mg), 3
(80 mg), and 4 (8.7 mg). Fraction C1 was chromatographed on a sil-
ica gel column eluted with an n-hexane–EtOAc gradient to give five
subfractions, C1.1–C1.5. Purification of fractions C1.4 and C1.5 by
preparative HPLC (YMC Pack Pro C18, 250 ꢀ 10 mm, 60% CH3CN
in H2O, flow rate 10 mL/min) gave 5 (41 mg) and 6 (160 mg),
respectively.
OH
R1
OH
HO
HO
7
HO
O
R1
1'
2'
R2
4'
5
3
OH
O
OH
O
R2
R1
H
H
R2
OH
1
2
6
geranyl
farnesyl
HO
geranyl prenyl
Compound 1 was obtained as yellow powder. The UV spectrum
exhibited maximal absorptions at kmax 219, 312, and 335 nm, indi-
cating a 2-arylbenzofuran-type skeleton.14 The 1H and 13C NMR
spectra showed characteristic signals of the 30,50,6-trihydroxy-2-
arylbenzofuran substructure along with the addition of a geranyl
moiety at C-7. The ESI-MS data displayed molecular ion peaks at
m/z 377.2 [MꢁH]ꢁ, 30,50,6-trihydroxy-2-arylbenzofuran fragment
at m/z 241.3, and m/z 136.3 of the geranyl fragment. The structure
of compound 1 was identified as albafuran A by comparison with
previous reported spectral data.15
R1
OH
H
R2
OH
OH
H
3
4
5
H
Figure 1. Structures of isolated compounds 1–6.
The nonenzymatic hydrolysis of 2 mM p-NPP was corrected by
measuring the increase in absorbance at 405 nm obtained in the
absence of PTP1B enzyme.19 The results are presented in Table 1.
It appears that isolated compounds 1–6 exhibited strong inhibition
of PTP1B activity with IC50 values ranging from 2.7 to 13.8
positive controls, RK-682 and ursolic acid (IC50 values of 4.5 and
3.9 M, respectively) are used as known phosphatase inhibitors.
Inhibition kinetics studies were carried out in the absence and
Compound 2 was obtained as yellow powder, and its UV spec-
trum also displayed a maximal absorption pattern characteristic
of a 2-arylbenzofuran skeleton. The 1H and 13C NMR data of 2 were
very similar to those of 1, with the exception of signals representa-
tive of a farnesyl group. ESI-MS data from 2 showed ion peaks at m/
z 445.4, 241.3, and 204.3 corresponding to the parent ion, the 2-
arylbenzofuran moiety, and the farnesyl fragment, respectively.
Compound 2 was identified as mulberrofuran W by comparison
with previously published data.16
lM. The
l
presence of active compounds with various concentrations of p-
NNP (0.5, 1.0, 2.0, 5.0, and 10.0 mM) as substrate. The initial rate
was determined on the basis of the rate of increase in absorbance
at 405 nm. The Michaelis–Menten constant (Km) and maximal
velocity (Vmax) of PTP1B were determined by Lineweaver–Burk Plot
analysis for competitive inhibition and the intercept on vertical
axis for noncompetitive inhibition. Under the conditions employed
in this present investigation, the hydrolysis of p-NPP catalyzed by
PTP1B follows Michaelis–Menten kinetics. The kinetic parameters
Km and Vmax for hydrolysis obtained from an analysis of Linewe-
aver–Burk plots are listed in Table 1. As illustrated in Figure 2,
the reciprocal plots of the isolated compounds are intersected to
the left of the 1/V axis, suggesting compounds 1–6 inhibited PTP1B
in a mixed-type manner. This indicates that they may both dis-
place at the active site and an additional binding site of the PTP1B
enzyme.
The active site of PTP1B contains a structural motif common to
many PTPs. The base of the catalytic site is defined by the 214–221
PTP signature motif, a loop of eight amino-acid residues that forms
a rigid, cradle-like structure that coordinates to the aryl-phosphate
moiety of the substrate. The substrate-binding and catalytic mech-
anism of PTP1B has been explored using hydrophobic, salt-bridge,
hydrogen-bonding, and electrostatic interactions, as well as by
Compounds 3–5 were isolated as yellow powders, with ½a D25
ꢂ
+125.0, +65.8, and +110.0, respectively. Its UV spectrum showed
maximal absorption at kmax 225, 295, and 380 nm suggesting a
chalcone skeleton. The 1H NMR spectra of 3 revealed the chalcone
skeleton along with a methylcyclohexenyl moiety. The conforma-
tion of the methylcyclohexene ring was determined to be of the
cis–trans type based on the coupling constant around 5–6 Hz be-
tween H-300/H-400 and H-400/H-500. The 1H NMR spectra indicated
the location of the OH groups at C-2 and C-1600 for 3, and C-1600
for 4, were different from 5. The ESI-MS molecular ion peaks at
m/z 677.4, 661.5, and 645.5 [MꢁH]ꢁ of 3–5, respectively, con-
firmed the existence of different hydroxylated units. Structures of
3–5 were identified as chalcone-derived Diels–Alder-type com-
pounds as kuwanon J, R, and V, respectively, by comparison with
previously reported data for the Morus genus.17
Compound 6 was obtained as a yellow powder, and its UV spec-
trum showed maximal absorptions at kmax 245, 269, and 315 nm of
2-arylbenzofuran derivative. The 1H and 13C NMR data of 6 were
closely comparable with those of 1, except for the signals indicat-
ing the location of a prenyl group at C-20. This was supported by
the long-range correlations observed in the HMBC spectrum. The
structure of 6 was identified as mulberrofuran D by comparison
with previous literature data.16
Table 1
Inhibition effects of isolated compounds 1–6 against PTP1B
All isolated compounds 1–6 (Fig. 1), together with RK-682 (3-
hexadecanoyl-5-hydroxymethyl tetronic acid) and ursolic acid as
positive controls,18 were examined for their inhibitory effects on
PTP1B activity in an in vitro assay. PTP1B (human, recombinant)
was purchased from BIOMOLÒ International LP (USA) and the en-
zyme activity was measured using p-nitrophenyl phosphate (p-
NPP) as a substrate. To each well of a 96-well plate (final volume:
Compounds
Protein tyrosine phosphatasea
M) Km (mM) Vmax M/min) Type of inhibition
IC50
(l
(l
1
2
3
4
5
6
9.2 0.7
2.7 0.3
2.7 0.6
8.2 0.9
1.25 0.05 6.58 0.18
3.17 0.03 5.49 0.13
2.81 0.08 5.41 0.15
1.64 0.03 5.95 0.14
Mixture
Mixture
Mixture
Mixture
Mixture
Mixture
N.D.
13.8 1.1 1.19 0.04 6.71 0.20
4.3 0.5
4.7 0.4
200 lL), 2 mM p-NPP and PTP1B (0.1 lg) were added in a buffer
2.08 0.02 5.78 0.17
RK-682b
N.D.
N.D.
N.D.
N.D.
containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and
1 mM dithiothreitol (DTT) with or without test compounds. Fol-
lowing incubation at 37 °C for 30 min, the reaction was terminated
by the addition of 10 M NaOH. The amount of produced p-nitro
phenol was estimated by measuring the absorbance at 405 nm.
Ursolic acidb 3.8 0.3
N.D.
N.D.: Not determined.
a
Values present mean SD of triplicate experiments.
Positive control.
b

D. M. Hoang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6759–6761
6761
tively. Interestingly, compounds 3–5 contain the same basic skele-
ton, only differing in the number of OH function groups. Compound
5, which possesses OH groups at C-4, C-20, C-40, C-1100, C1300, and C-
1800, exhibited strong dose-independent inhibition with an IC50 va-
lue of 13.8
l
M, but was less potent than 4, which contained one
M. Compound
more OH group at C-1600, with the IC50 value of 9.2
l
3 contained a very similar structure 4, except with an additional
OH group at C-2. This additional functionality increased the po-
tency of 3 3.4-fold with respect to 4. Hence, the order of inhibitory
activity of tested chalcone-derived Diels–Alder-type compounds
was 3 > 4 > 5. The hydroxyl groups not only provided the needed
penetration into the active site and likely produced a hydrogen-
bonding interaction with the amide backbone of the active-site
loop, but also could have protonated or donated a hydrogen bond
to the more acidic of inhibitors that extend into the adjacent
aryl-phosphate binding site.1,20 The above results suggest that
increasing the number of OH groups in chalcone-derived Diels–Al-
der-type compound improves potential inhibitory effects against
PTP1B.
Previous investigations have shown that a variety of biological
activities such as antioxidant, hepatoprotective, anticancer, and
as well as inhibitory effects against on tyrosinase, glucosidase,
and PTP1B have been reported for the constituents of Morus spe-
cies. Bioassay-guide fractionation of a MeOH extract of Morus sp.
revealed a class of flavonoids that act as PTP1B inhibitors.14 This re-
port describes the 2-arylbenzofuran- and chalcone-derived Diels–
Alder-type compounds isolated from a CHCl3 fraction of the root
bark of M. bombycis as new mixed-type inhibitors of PTP1B and
thus offers an additional natural PTP1B inhibitor resource for the
treatment of diabetes and obesity.
Acknowledgments
This work was supported by a grant from the Korea Food and
Drug Administration (2009) and a grant from the Brain Korea 21.
Figure 2. The Lineweaver–Burk plots of PTP1B inhibitory activity with p-NPP as
substrate, in the presence of 1–6. Symbols: (A)—1, (j) 5 M; 2, (ꢀ) 5 M; 6, (N)
M and (B)—3, (e) 5 M, 4 ( ) 5 M, 5 (h) 5 M, and DMSO (d) as control. The
l
l
References and notes
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l
l
D
l
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data represent the mean SD of triplicate difference experiment.
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several N-termini that are capable of binding to an acidic site.1 The
need to have compounds with improved physico-chemical proper-
ties that are closely related to PTP1B has become a central issue
that needs to be addressed in the development of PTP inhibitors.
The isolated compounds 1–6 showed significantly differences in
with respect to their biological activities and chemical structures.
The 30,50,6-trihydroxy-2-arylbenzofuran derivatives, albafuran A
(1), which possesses a geranyl group at C-20, showed a strong
inhibitory effect against PTP1B. Mulberrofuran W (6), which con-
tains an additional prenyl unit at C-20, was more inhibitory against
PTP1B activity than 1. The former had an IC50 value of 4.3 lM,
while the latter had an IC50 value of 9.2 lM. On the other hand,
the farnesyl unit at C-20 in mulberrofuran W (2) seemed to impart
the most inhibitory activity against PTP1B, with a low IC50 value of
2.7 lM. These results suggest that increasing the lipophilicity and
decreasing the charge of aliphatic side chain leads to stronger bind-
ing. The prenyl, geranyl, and/or farnesyl moieties found in 1, 2 and
6 increase cellular permeability, and thus make 1–6 potential
treatments for PTP1B and obesity. It appears that the lipophilic
group plays a role in direct modulation consisted of Meth258
and Phe52,20 but the precise reason for this effect remains unclear.
The methyl cyclohexene substituted chalcone-derived Diels–Al-
der-type compounds 3–5 displayed strong inhibition effects
against PTP1B with IC50 values of 2.7, 9.2, and 13.8 lM, respec-